Human AZU-1 gene, variants thereof and expressed gene products

Abstract

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Description

BACKGROUND OF THE INVENTION

Field of the Invention

This invention concerns a novel human AZU-1 gene, mutants, variants and fragments thereof, and protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. In particular, this invention concerns identifying, isolation and characterization of novel AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. Additionally, the invention concerns findings that AZU-1 and AZU-2 genes exhibit tissue-specific expression profiles and that AZU-1 gene expression in tumor cells is low or absent. The invention further concerns recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes. The invention also concerns monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

BACKGROUND AND RELATED DISCLOSURE

The evolvement of breast cancer is a multistep and cumulative process and understanding of the genetic and phenotypic alterations in successive steps is essential for designing therapeutic interventions and diagnostic assays.

In the human body, the epithelial component of the breast is embedded in the stroma and forms a branching ductal structure that emanates from the nipple, repeatedly bifurcates, and terminates in lobules, alveoli, and end buds. Although stroma accounts for >80% of the breast volume, approximately 95% of the cancers produced in the breast are of epithelial origin. To elucidate the advancement of human breast cancer, a functionally relevant cell culture model is required. The differences in breast tissue compartmentalization, phenotypic characteristics, and the mutagenic frequency between human and rodents underline the need to develop a human breast cell model.

An unconventional spontaneously-transformed HMT-3522 cell lines was described recently in Cancer Research , 56:2039 (1996) where the immortalized human mammary epithelial cells (S1) established from fibrocystic breast tissue was propagated in chemically defined medium described in In Vitro Call. Dev. Biol ., 23:181 (1987). S1 cells were near-diploid and expressed luminal epithelial cell differentiation markers cytokeratin-18 and sialomucin. Genetic changes such as p53 point mutation ( Exp. Cell. Res ., 215:380 (1994)) and c-myc amplification ( Cancer Research , 52:1210 (1992)) have already been noted in later passages, i.e., >50 of the nonmalignant S1 cells. In passage 118, cells were adapted to grow in epidermal growth factor (EGF)-free medium and a new EGF-independent subline (S2, premalignant) was isolated (ibid).

Alterations in gene expression of EGF receptor, transforming growth factor (TGF)-α and c-erb-B2 were seen in S2 cells. The established S2 cell line underwent cytogenetic evolution and exhibited genomic instability and heterogeneity ( Cancer Genetics and Cytogenetics , 78:189 (1994)). Nevertheless, S2 cells remained nontumorigenic until passage 238. At that time, it was able to induce tumor growth in nude mice. Following second round of mouse transplantation, another subline (T4-2, tumorigenic) existed as a relatively homogenous malignantly-transformed cell population. One extra copy of chromosome 7p which harbors EGF receptor gene was found in the T4-2 cells ( Cancer Res ., 56:2039 (1996)).

Detection and suppression of cancerous tissue growth is of extreme interest and importance. It is, therefore, a primary objective of this invention to provide means for identification, detection and suppression of cancerous growth in breast tissue.

All patents, patent applications and publications cited herein are hereby incorporated by reference.

SUMMARY

One aspect of the current invention is a human AZU-1 gene or a variant, mutant, or fragment thereof.

Another aspect of the current invention is a nucleotide sequence of AZU-1 and AZU-2 genes, identified as SEQ ID NO: 1 and SEQ ID NO: 2.

Another aspect of the current invention is a DNA sequence identified as SEQ ID NO: 1 encoding a protein comprising the amino acid sequence identified as SEQ ID NO: 3.

Still another aspect of the current invention is a protein of the amino acid sequence identified as SEQ ID NO: 3.

Still yet another aspect of the current invention is a protein encoded by AZU-1 gene, or by a variant, mutant or fragment thereof, or any protein containing said protein encoded by AZU-1 gene, variant, mutant, or fragment thereof, or any protein which shares homology with AZU-1 encoded protein or AZU-2 encoded protein, variant, mutant or fragment thereof.

Still another aspect of the current invention is a protein encoded by the AZU-1 gene, variant, mutant, analog or fragment thereof acting as a tumor suppressor or a marker of malignancy progression and tumorigenic reversion.

Still yet another aspect of the current invention is a method for diagnosis and detection of progression of human breast cancer by detecting presence and quantity of a protein identified as SEQ ID NO: 3 or a homolog thereof in human breast cells or tissue or by detecting expression of AZU-1 gene, mutant, variant, fragment thereof by in situ hybridization or RT-PCR.

Still yet another aspect of the current invention is a diagnostic method for detection of the presence of AZU-1 protein in human breast cancer by treating a biopsy sample of a subject patient with a polyclonal or monoclonal AZU-1 antibodies or detection of the level of AZU-1 message.

Yet another aspect of the current invention is a method for prevention or treatment of human breast cancer by providing a subject in need thereof a therapeutically effective amount of a protein identified as SEQ ID NO: 3 or homolog thereof, able to act as a tumor suppressor of human breast cancer cells.

Still yet another aspect of the current invention is a tissue targeted gene therapy for treatment of human breast tumor.

Still another aspect of the current invention is an ELISA kit for detection of expression of AZU-1 gene or a variant thereof by detecting presence or absence of AZU-1 encoded protein with AZU-1 monoclonal or polyclonal antibodies.

Still another aspect of the current invention is a message detection kit for detection of expression of AZU-1 gene or a variant thereof by bDNA technology (Quantigene Gene Expression Assay, Chiron Corp., Emeryville, Calif., in situ hybridization or RT-PCR by detecting presence or absence of AZU-1 message.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of necessary fee.

FIG. 1 is a schematic diagram of malignant transformation of HMT3522 human breast epithelial cells.

FIG. 2 shows phenotypic recapitulation of tissue morphology in 3-D reconstituted basement membrane (rBM) culture assay for S1 and T4-2 cells.

FIG. 3 shows characterization of HMT-3522 progression series. FIG. 3A shows comparative genomic hybridization (CGH) performed to determine genomic content and alteration in cells throughout the HMT-3522 progression series. FIG. 3B are phase contrast micrographs showing the morphology of mammary epithelial cells (MECs) recapitulated in 3-D rBM assay. FIG. 3C shows immunostaining with Ki-67 marker. FIG. 3D shows organized cortical F-actin in S-1 acini and disorganized F-actin filaments in S-2 and T4-2 tumor colonies. FIG. 3E shows E-cadherin staining pattern in S1, S2 and T4-2 cells.

FIGS. 4A-4B is the cDNA sequence (SEQ ID NO: 1) of AZU-1 gene showing a divergent site between nucleotides 429-430.

FIGS. 5A-5B is the cDNA sequence (SEQ ID NO: 2) of AZU-2 gene showing a divergent site between nucleotide 764-765.

FIG. 6 is an amino acid sequence (SEQ ID NO: 3) of the protein encoded by AZU-1 gene. FIG. 6A shows SPAZI (SEQ ID NO: 7), REGION I (SEQ ID NO: 13), REGION II (SEQUENCE ID NO: 14) and Coiled-Coil (CCD) (SEQ ID NO: 8) domains. FIG. 6B is a schematic diagram of all AZU-1 and TACC1 domains. FIG. 6C shows SPAZI domain homology for AZU-1 (SEQ ID NO: 3), TACC1 (A) (SEQ ID NO: 9), TACC2 (B) (SEQ ID NO: 15), TACC3 (SEQ ID NO: 16), and BCK1 (SEQ ID NO: 10) protein. FIG. 6D shows homology regions for coiled-coil domain (CCD) in AZU-1 (SEQ ID NO: 8), TACC1 (SEQ ID NO: 11), TACC3 (SEQ ID NO: 17) and SB1.8 (SEQ ID NO: 12) genes.

FIG. 7 shows Western blot analysis of AZU-1 recombinant proteins.

FIG. 8 is a Western blot illustrating recognition of a 64 kD protein by AZU-1 antibody.

FIG. 9 illustrates differential expression of AZU-1 gene in premalignant and breast tumor cell lines. FIG. 9A shows differential display analysis in premalignant S2 and T4 tumor cells. FIG. 9B shows Northern blot analysis in premalignant S2 and T4 tumor cells.

FIG. 10 shows downregulation of AZU-1 in breast tumor cell lines and biopsies. FIG. 10A shows expression of AZU-1 in S1 and MCF10A nonmalignant cell lines and in luminal epithelial and myoepithelial nonmalignant primary cells. FIG. 10B shows downregulation of AZU-1 gene in premalignant S2 and malignant T4, HMT 3909, MCF-7, CAMA-1, NDA-MB 468, SKBR-3, T47D, MDA-MB 231, Hs578T and BT 549 cells. FIG. 10C shows downregulation of AZU-1 gene in in situ carcinoma.

FIG. 11 shows tissue specific expression of AZU-1 in various normal human tissues.

FIG. 12 is a color image showing localization of AZU-1 gene to chromosome 10q26.

FIG. 13 is a color image which shows the association of AZU-1 with cytoskeletal complexes in nonmalignant breast cells. FIG. 13A is the image taken before, and FIG. 13B is the image taken after, treatment with triton X-100 to remove soluble proteins.

FIG. 14 is a color image of in situ staining of AZU-1 in normal breast acinus ( FIG. 14A ) and breast duct (FIG. 14 B).

FIG. 15 illustrates the presence of E-cadherin and β-catenin in AZU-1 immunocomplexes.

FIG. 16 shows that ectopically-expressed AZU-1 gene reduces tumorigenicity and invasiveness. FIG. 16A shows Northern blot of AZU-1 and Western blot of AZU-1 in S1, T4-2 (mock) and T4-2+AZU-1 cells. FIG. 16B shows the number of colonies of S1, T4-2, T4-2 (mock) cells and reduction in number of colonies in T4-2 cells in the presence of AZU-1 (T4-2+AZU-1) grown in soft agar. FIG. 16C shows in vitro invasiveness in S1, T4-2, T4-2 (mock) and T4-2+AZU-1 cells.

FIG. 17 shows ectopically-expressed AZU-1 induces tissue morphogenesis in three-dimensional cultures. FIG. 17A shows phase contrast images for S1, T4-2 (mock) and T4-2+AZU-1 cells. FIG. 1B shows basement membrane staining of T4-2 (mock) and T4-2+AZU-1 cells. FIG. 17C shows the colony size in μm for S1, T4-2 (mock) and T4-2+AZU-1 cells.

FIG. 18 illustrates upregulation of AZU-1 in morphologically reorganized breast tumor cells. FIG. 18A is a schematic diagram of morphology of T4, S1, T4β1 and T4tyr, phase contrast microscopy (FIG. 18 B), AZU-1 message ( FIG. 18C ) and GADPH message (FIG. 18 D).

FIGS. 19A-19B shows a sequence alignment of AZU-1 and its variant TACC2 cDNAs. Sequence insertions are indicated by dots.

FIGS. 20A-20B shows an amino acid sequence alignment of AZU-1 and its variant TACC2 proteins. Sequence insertions are indicated by dots.

DEFINITIONS

As used herein,

“CCD” means coiled-coil domain.

“ER” means estrogen receptor.

“RACE” means rapid amplification of cDNA ends.

“AZU-1” or “AZU-1 gene” means antizuai-1 gene. AZU-1 refers to the entire AZU-1 genomic sequence, including enhancers, promoter sequences, introns and exons.

“AZU-1 mutant”, “AZU-1 variant” or “AZU-1 fragment” mean all AZU-1 gene products, such as all potential splice variants derived from the gene and their protein products. Their associated 5′ and 3′ untranslated regions are also included. Also included are all mutant forms of AZU-1, whether spontaneously arising or specifically engineered. All known AZU-1-related genes, such as TACC-1, TACC-2 and TACC-3 are also included under this definition as long as they share about 25% of homology.

“AZU-1 gene encoded protein” means and includes all protein coding sequences encoded by AZU-1 gene, variant or mutant thereof, as well as 5′ and 3′ untranslated sequences identified here and in other AZU-1 splice variants.

“HMT-3522” means human mammary tumor cell line 3522.

“Ki-67” means a marker for proliferating cells.

“Cadherin” means cell-adherens junction protein.

“E-cadherin” means epithelial cadherin.

“β-catenin” means an adherens junction protein.

“GAPDH” means a metabolic protein GAPDH (glyceraldehyde phosphate dehydrogenase) expressed by a common gene in metabolic pathway. In this application, the message level of GADPH is used as control for RNA loading in Northern blot.

“S1” or “S-1” means a nonmalignant breast cell line.

“S2” or “S-2” means a premalignant breast cell line.

“MCF10A” means a nonmalignant breast cell line.

“Luminal epithelial cells” means normal cells present in the breast tissue.

“Myoepithelial cells” means normal cells present in the breast tissue.

“T4”, “MCF-7”, “CAMA-1”, “BT-20”, “MDA-MB 468”, “SKBR-3”, “T47D”, “MDA-MB 231”, “Hs578T”, and “BT 549” means breast tumor cell lines specifically so identified.

“HMT 3909” means a breast tumor cell line contaminated with normal myoepithelial cells.

“T4-2 (mock)” means breast tumor cells transfected with empty expression vector.

“Ectopically expressed AZU-1” means artificially expressed or overexpressed AZU-1 gene.

“Upregulation of AZU-1 gene” means observed increased expression of AZU-1 gene.

“Variant” means any variant derived from splicing, exon shuffling, deletion or insertion causing frame shifting. Variant is exemplarized by AZU-2 gene and TACC2 gene where TACC2 gene is a variant of AZU-1 gene.

“Mutant” means AZU-1 gene containing a point mutation, deletion, insertion, or truncation.

“Fragment” means a functional domain, such as SPAZI or coiled-coil domain involved in protein-protein interaction.

“Homolog” means any homologous protein sharing a substantial sequence similarity (about 25% or more) with AZU-1 expressed protein. Exemplary proteins are proteins expressed by TACC1 or TACC3 or a variant thereof expressed by TACC2.

“TACC1” means embryonically expressed TACC1 gene from the 8p11 breast cancer amplicon.

“TACC2” means expressed TACC2 gene from the 10q25-q26 locus.

“TACC3” means expressed TACC3 gene from the 4p16.3 locus.

“SPAZI” means serine-proline rich AZU-1 domain.

“Coiled-coil” means heptad repeats participating in protein-protein interactions.

“BCK1” means a Saccharomyces cerevisiae yeast gene.

“BLAST” means basic local alignment sequence tool. BLAST is a service available from the National Center for Biotechnology Information which compares and matches a nucleotide or protein sequence against databases at the NCBI.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a cancer-related gene AZU-1. AZU-1 gene is novel and has never before been described or disclosed. AZU-1 gene and its encoded protein have been found to be present in normal breast cells. The expression of AZU-1 gene in tumor cells, however, has been found to be significantly decreased in ten human breast cancer cell lines and carcinoma cells in situ. The level of the AZU-1 encoded protein is significantly decreased in these cell lines.

A protein encoded by AZU-1 gene is believed to act as a protective agent against cancer by suppressing a tumor growth and the detection of its level in breast cells is useful as a marker of malignancy progression and tumorigenic reversion. The invention is useful for diagnosis, prognosis and treatment of breast cancer.

The invention also relates to identification, isolation and sequencing of the human AZU-1 gene and its variant AZU-2 gene encoding proteins acting as tumor suppressors and markers for tumor progression and tumorigenicity reversion.

AZU-1 gene was isolated and sequenced (SEQ ID NO: 1) and the amino acid sequence of AZU-1 encoded protein was deduced (SEQ ID NO: 3). The protein was additionally identified by specific AZU-1 antibodies. Functional significance of the loss of AZU-1 expression in tumor cells has been investigated in vitro and in vivo and linked to tumorigenic and invasion suppressive roles.

Additionally, the invention relates to a method for treatment, detection and prognosis of breast cancer by providing a patient in need of such treatment a therapeutically effective amount of a recombinant protein, by detecting the level of native protein encoded by AZU-1 gene in the breast tissue biopsy sample or by determining a degree of expression of AZU-1 gene and/or levels of expressed AZU-1 protein.

I. Tumorigenic Cell Line Progression Model

The current invention was developed and tested on various models of normal or breast tumor cell lines which were developed and are described below. Progression model for tumorigenic cell line T4-2 was developed by malignant transformation of human breast epithelial cells.

Presence or absence of AZU-1 gene expression was tested in normal epithelial or myoepithelial cells, nonmalignant S1 cell line, premalignant S2 cell line and in T4-2 breast tumor cell line.

Malignant T4-2 breast tumor cell line was derived from nonmalignant fibrocystic breast cells. Malignant transformation of nonmalignant HMT3522-S1 cell line into malignant T4-2 cells is illustrated in FIG. 1 .

The nonmalignant cells were obtained as a cell mixture from a patient suffering from fibrocystic disease. The cells were cultured to specifically promote the growth of epithelial cells. The epithelial cells were then immortalized as a nonmalignant HMT 3522-S1 cell line. The HMT 3522-S1 cells were repeatedly passaged 20 (S-1 20), 50 (S-1 50), 110 (S-1 110), 175 (S-1 175) up to 400 passages (S-1 400) in the presence of EGF (+EGF) and were found to be nonmalignant. When, after 110 passages, epidermal growth factor (EGF) was removed from the culture, at about 150 passages, the EGF deprived cells turned into premalignant S-2 cell line. After the S-2 cell line was further propagated up to S-2 215, the S-2 cells at every 10-passage intervals from 150 passages up were injected into nude mice. None of the S-2 cells up to passage 238 were found to produce tumors. After about 8 weeks, approximately 50% of the S2-238 injected nude mice were found to produce tumors. Their tumor nodules were then removed and the cells were further propagated and these cells were again injected into the nude mice. After 8 weeks, more than 90% of nude mice were found to have tumors. The tumor nodules were removed again and the cells were propagated as T4-2 cells. T4-2 cells were tested for AZU-1 gene expression and/or for the presence or absence of AZU-1 encoded protein in assays described below.

Normal cells, cell lines and malignant cell lines were then tested in a series of morphological, structural and adhesion studies and differences between normal S-1 and malignant T4-2 cell lines observed in these studies are seen in FIG. 2 . FIG. 2 is a comparative phenotypic recapitulation of normal S-1 cell line and malignant T4-2 cell line in 3-D basement membrane culture assay.

As seen in FIG. 2 , a schematic diagram of S-1 and T4-2 cell lines (extreme left) shows S-1 cells are organized in a sphere, which corresponds to its morphological organization seen in morphology subset (middle left). In morphology subset, normal S-1 cell are seen to be organized in spherical manner. The malignant T4-2 cells on the other hand, are shown to form disorganized colonies, seen in both schematic and morphology subsets.

The normal S-1 cells were seen having a smooth spherical shape basement membrane when tested in a 3-D laminin-rich reconstituted basement membrane culture assay and immunostained with human type IV collagen (middle right). When the malignant cells were analyzed by human type IV collagen immunostaining, they revealed a disorganized pattern. Staining with E-cadherin (E-cadherin subset, extreme right) shows intact cell-cell adhesion network in S1 cells and disrupted organization in T4-2 cells.

Differences between the normal nonmalignant, premalignant and malignant cells which were obtained in progression series of HMT 3522, seen in FIG. 1 , are further illustrated in FIG. 3 , which shows characterization of the HMT 3522 progression series. All types of cells, that is the normal S-1 (S1-50, S1-110 and 51-175) cells, premalignant 82 (S2-215) cells and malignant T4-2 (T4-2-25) cells obtained as described in FIG. 1 were analyzed by comparative genomic hybridization, phase contrast microscopy, and immunostaining with F-actin phalloidin staining, and with E-cadherin.

The HMT-3522 human breast tumor progression series is comprised of a continuum of genetically related cell populations that range in phenotypic behavior from non-malignant to tumorigenic. In order to identify genes that play a crucial role in the final stages of breast tumor progression, a differential display strategy was utilized to compare the gene expression patterns of the model's tumorigenic cell population (called T4-2) with that of its pre-malignant progenitor (called S2). One of the genes identified using this approach is abundantly expressed in non-malignant luminal epithelial cells but is expressed at significantly lower levels in a variety of breast tumor cell types. Because of this expression pattern, which is commonly observed with tumor suppressors of the Type II class, this gene is referred to as anti-zuai-1 (or AZU-1, with “zuai” meaning breast cancer in Chinese).

To determine genomic content and alteration in cells throughout the HMT-3522 progression series, comparative genomic hybridization was performed. Results are seen in FIG. 3 A. As shown in FIG. 3A , genetic alteration, e.g., chromosomal amplification and deletion, were accumulated in HMT 3522 cells during tumorigenic progression.

FIG. 3B are phase contrast micrographs showing the morphology of mammary epithelial cells (MECs) as identified at the top of the FIG. 3 , i.e., nonmalignant S1 cells (S1-50, S1-110 and S1-175), premalignant S-2 cells (S2-215) and malignant T4-2 passage 25. In this study, cells were grown embedded in a (3-D) laminin-rich basement membrane (BM) for 10 days. At that time, S1 cells formed growth-arrested structures reminiscent of true acini and S2 premalignant and T4-2 tumor cells formed large, irregular colonies.

FIG. 3C shows immunostaining of tested cells for Ki-67, a marker of cell cycle entry. S1 passages 50 and 110 were negative, that is, they did not show any Ki-67 immunostaining, while the percentage of Ki-67 positive cells increased from S1-175 to the T4-2 tumor cells. These results show a loss of growth-arrest control in response to the BM occurs in progression to malignancy. S1-175 cells remained organized, but the acini were larger.

FIG. 3D shows results of staining the cells with F-actin. F-actin phalloidin staining shows organized cortical F-actin in the S1 cells while both S2 premalignant and T4-2 tumor colonies are seen to contain only disorganized actin filaments.

FIG. 3E illustrates E-cadherin studies. E-cadherin immunofluoresence, seen in FIG. 3E , shows organized cell-cell contact staining in the S1 cells, while in both the S2 premalignant and T4-2 tumor colonies, the cells had punctate, dispersed membrane and intracellular stainings.

II. AZU-1 Gene and AZU-1 Encoded Protein

AZU-1 gene has been discovered to be present and expressed in abundance in normal nonmalignant breast cells.

Functional studies indicate that overexpression of the AZU-1 message in tumorigenic T4-2 cells is sufficient to reduce their tumorigenic phenotype as measured by growth in soft agar, invasion assays and by tumor formation in nude mice. Moreover, overexpression of AZU-1 restores T4-2 cells with a normal polarized phenotype when cultured in a 3-dimensional reconstituted basement membrane.

Collectively, these results indicate that AZU-1 gene and/or its protein product is a candidate breast tumor suppressor that may play a role in attenuating cell growth controlling disorganized malignant growth and maintaining appropriate tissue architecture.

A. Isolation, Identification and Sequencing of AZU-1 Gene

The AZU-1 gene has been now identified, isolated, sequenced (SEQ ID NO: 1) and its variant AZU-2 gene (SEQ ID NO: 2) nucleotide sequence has been determined. The sequence of AZU-1 gene is shown in FIG. 4 as SEQ ID NO: 1. The sequence of AZU-2 gene is shown in FIG. 5 . Sequences of the related gene TACC2 gene is identified as SEQ ID NO: 5. Other homologs of AZU-1 protein, TACC1 (A), TACC1 (B), TACC3, BCK1 and SB1.8 are identified as SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 10 and SEQ ID NO: 12, respectively.

1. Sequences of AZU-1 Gene and Variants

AZU-1 gene is localized to a tumor suppressive locus at chromosome 10q 26 genomic locus. AZU-1 is a novel gene whose sequence gives rise to an AZU-1 protein product (SEQ ID NO: 3). The AZU-1 protein comprises 571 amino acids and is comprised of 4 distinct structural/sequence domains, two of which, namely coiled-coil and SPAZI domains, represent conserved motifs. The protein, its amino acid sequence and the four domains as seen in FIG. 6 .

AZU-1 gene, of which cDNA is shown in FIG. 4 , comprises a nucleotide sequence of 3813 nucleotides. The sequence is identified as SEQ ID NO: 1. The sequence of one of the AZU-1 gene variants, namely, AZU-2 gene is shown in FIG. 5 . AZU-2 cDNA sequence is identified as SEQ ID NO: 2. AZU-2 cDNA is longer and contains 4148 nucleotides. The divergent site is between nucleotide 764 and 765. The cDNA of AZU-1 gene variant TACC-2 is identified as SEQ ID NO: 5. Two genes which expressed homologous proteins to AZU-1 protein, namely, TACC-1 and TACC-3 gene, are identified as SEQ ID NO: 18 and SEQ ID NO: 20.

AZU-1 and AZU-2 genes are diverged at a location marked in FIGS. 4 and 5 as “T//T”, positioned at nucleotides 429 and 430 of AZU-1 gene and at nucleotides 764 and 765 of AZU-2 gene, respectively. AZU-1 and AZU-2 genes share identical sequence downstream of the divergent site indicated above.

1. AZU-1 Encoded Protein

AZU-1 sequence encodes a protein of 571 amino-acids. Sequence of AZU-1 encoded protein is identified as SEQ ID NO.:3. The AZU-1 variant AZU-2 encodes a protein of 1219 amino acids. Sequence of AZU-2 encoded protein is identified as SEQ ID NO: 4. Another AZU-1 variant TACC1 gene encodes protein identified as SEQ ID NO: 19.

FIG. 6 shows amino acid sequence of the protein encoded by AZU-1 gene (SEQ ID NO: 3).

FIG. 6A identifies amino acids in the sequence and separates them into four domains. The SPAZI domain contains amino acids 1-107 (SEQ ID NO: 7). REGION I (SEQ ID NO: 14) contains amino acids 109-248 (SEQ ID NO: 13). REGION II contains amino acids 250-360. The last and largest coiled-coil domain (CCD) contains amino acids 362-571 (SEQ ID NO: 8).

FIG. 6B is a schematic diagram comparing AZU-1 gene with TACC-1 gene. As seen, there are different degrees of homology in the SPAZI REGION I and coiled-coil domain of AZU-1 and TACC-1. There is a deletion in TACC-1 in the region corresponding to REGION II of AZU-1 gene. FIG. 6C shows point of homology between AZU-1 (SEQ ID NO: 7), TACC-1 (A) (SEQ ID NO: 9), TACC-1 (B) (SEQ ID NO: 15), TACC-3 (SEQ ID NO: 16) and BCK1 (SEQ ID NO: 10) in the SPAZI domain.

FIG. 6D shows points of homology between AZU-1 (SEQ ID NO: 8), TACC-1 (SEQ ID NO: 11), TACC-3 (SEQ ID NO: 17) and SB1.8 (SEQ ID NO: 12) in coiled-coil domain.

Sequence analysis of the full-length AZU-1 cDNA clone reveals an open reading frame that translates to a protein product of 571 amino acids (FIG. 6 A). At its N-terminus AZU-1 contains a novel serine and proline-rich domain, called a SPAZI domain, that is evolutionarily conserved and is predicted to exhibit an Ig-domain like fold. The C-terminal region of AZU-1 displays a series of heptad repeats consistent with the presence of an extensive, but discontinuous, coiled-coil domain.

Using this protein product sequence as the query for a PSI-BLAST database search ( Nucleic Acids Res ., 25:3389 (1997)), AZU-1 was found to share significant sequence similarity, particularly at its N- and C-termini, to TACC1 gene (Genbank locus AF049910) cDNA (SEQ ID NO: 18), the unpublished product of a gene cloned from the breast cancer amplicon 8p11. A second, unpublished gene product, TACC2 (AF095791) cDNA (SEQ ID NO: 5), seems to be a splice variant of AZU-1 gene since, apart from two insertions, it is identical to AZU-1 at both the nucleic acid and protein levels (FIGS. 19 and 20 ).

Inspection of the alignment of AZU-1 and TACC1 summarized in FIG. 6B suggests that AZU-1 can be partitioned into four domains. At its N-terminus, AZU-1 exhibits serine- and proline-rich “SPAZI” domain. SPAZI domain is shown in FIG. 6 C. The SPAZI domain of AZU-1 is compared to corresponding TACC1 (A), TACC2 (B), TACC3 and BCK1 domains. The serine and proline residues, which are distributed throughout this protein region, each comprise 18% of the AZU-1 sequence content for an overall proline/serine content of 36%. SPAZI domains are present once in AZU-1, twice in TACC1 and once in the Saccharomyces cerevisiae gene product BCK1, a member of the MAPKKK (mitigen activator protein kinase kinase kinase) family of serine/threonine kinases. In many instances, the abundant serine and proline residues are conserved in 2 or more of these sequences; 2 serine residues in the second half of the motif are invariant.

Fold recognition studies, using the GenTHREADER program ( J. Mol. Biol ., 287:797 (1999)), indicate that the SPAZI domain is likely to display an Ig-like beta-sandwich fold. For each SPAZI domain, at least one protein having a known, immunoglobulin-like (Ig-like) structure was reported. The most reliable prediction with estimated probability of correct match=0.59, was for the BCK1 SPAZI domain which matched an Ig-like domain in human Cd2, T lymphocyte adhesion glycoprotein, PDB (Protein Data Bank) identifier 1hnf. Based on these structural predictions, the SPAZI domain seems to be a new member of the hnf or C2-set superfamily.

The SPAZI domain of AZU-1 as seen in FIG. 6B , is followed by two domains, referred to as REGION I and REGION II, that are defined by virtue of their relationship to TACC1. REGION I of AZU-1 shows 20% identity with parallel amino acids of the TACC1 sequence. REGION II corresponds to those sequences of AZU-1 that are absent from TACC1 gene product. Fold recognition analyses of REGION I and REGION II predict that they too have Ig-like folds. AZU-1's REGION I matches known immunoglobulin structures, namely, PDB 1pfc, a fragment of an IgG1 with estimated probability of match=0.32, and PDB 1psk, an antibody Fab fragment, with estimated probability of match=0.51. Analysis of the third region present only in AZU-1 indicates a beta barrel fold, namely, PDB 1htp, H-protein with estimated probability of match=0.27.

The fourth and C-terminal region of AZU-1 displays a series of heptad repeats consistent with the presence of an extensive, but discontinuous, coiled-coil domain seen in FIG. 6 D. Structural studies have demonstrated that coiled-coil domains, like the one found in AZU-1, form amphipathic helices that associate with other like domains to form superhelical bundles comprised of anywhere from 2 to 4 helices. The seven structural positions of a heptad repeat are named a-g. Positions a and d, occupied by hydrophobic residues, form the helix interface whereas the remainder are hydrophilic and form the solvent-exposed part of the helix surface.

Apart from the closely related TACC1 coiled-coil domain, the highest scoring sequence from a PSI-BLAST search is the human SB1.8/DXS423E protein, a putative homologue of the Saccharomyces cerevisiae SMC-1 protein that is essential for proper chromosomal segregation during mitosis (PIR locus I54383). Alignments generated using the Multicoil program ( Protein Sci ., 6:1179 (1997)) indicate three major regions where the characteristic heptad repeats fall into register in all three proteins (FIG. 6 D). These regions in AZU-1, TACC1 and SB1.8 correspond to regions that the Multicoil program predicts to form dimers (probability >0.90). The two d positions towards the end of the coiled coil are occupied by notably charged residues E and K.

Cellular localization predictions generated using the PSORT program indicate that AZU-1 contains two putative nuclear localization sequences (NLSs). One NLS is positioned N-terminally in the SPAZI domain, while the second NLS starts at amino acid 122 in AZU-1's REGION I shown in FIG. 6A as underlined.

2. AZU-1 Gene Cloning

AZU-1 gene was identified, sequenced and cloned using methods known in the art.

Specifically, the nucleotide sequence of the 180 bp differential display cDNA fragment was determined and compared to existing Genbank sequences. The sequence of the 180 bp fragment was identical to three ESTs. All three sequences contained the 180 bp sequence plus additional 5′ and/or 3′ sequences; two of these clones exhibited polyadenylation sites. None displayed significant open reading frames, thereby indicating that the 180 bp fragment resided in the 3′ untranslated region of the gene product. This information indicated that the remainder of the gene product would be positioned 5′ to the isolated fragment and could be cloned by performing multiple rounds of 5′ RACE (rapid amplification of cDNA ends), according to protocols from Life Technologies, Inc. Grand Island, N.Y.).

Primers corresponding to the 180 bp differential display fragment were used to initiate the 5′ RACE cloning procedure according to the manufacturer's instructions. Ultimately the protocol was repeated 12 times to obtain 3.8 kb of AZU-1 sequence. In each cycle, 500-800 bp of additional overlapping 5′-end sequence was obtained. Sequencing of the 5′ RACE PCR products was conducted using cycle sequencing (Amersham Life Science, Cleveland, Ohio). Upon final analysis, the 3.8 kb of AZU-1 sequence contained a candidate translational start codon consistent with the Kozak consensus rules and an inframe stop codon ( Cell , 34:471 (1983); Nature , 308:241 (1984)).

To confirm the composition of the 3.8 kb AZU-1 sequence, and to generate a cDNA containing the entire AZU-1 open reading frame (ORF), primers corresponding to each end of the AZU-1 gene product were utilized in long template PCR (Boehringer Mannheim Corp. Indianapolis, Ind.). In two independent experiments, each using distinct pools of total S1 RNA, the RT-PCR resulted in the amplification 3.8 kb gene products whose sequences were identical to the AZU-1 sequence originally derived using 5′ RACE technology. The resulting cDNAs were subcloned into pCR 2.1 (Invitrogen, Carlsbad, Calif.) for further amplification and use.

Other AZU-1 constructs were also prepared. AZU-1 coding region sequences were amplified in polymerase-chain reactions using AZU-1-specific primers supplemented with EcoRI and XhoI restriction sites.

Forward primer: 5′ CTGAATTCATGGACCTGGACTCTGCCCTCCAG 3′ (SEQ ID NO: 22).

Reverse primer: 5′ GCCTCGAGTTAGGGCTGCTGGAACAGAAGGCC 3′ (SEQ ID NO: 23).

Amplified fragments, once digested with the appropriate enzymes, were ligated into the retroviral expression vector pLXSN (Clontech Inc., Palo Alto, Calif.). Cycle sequencing was performed to verify the sequence fidelity of each AZU-1 construct.

Probable sequence similarities between protein encoded by AZU-1 and other proteins were determined using BLAST computer-driven algorithm that calculates the sequence similarities. Based on the BLAST search results, the sequence of AZU-1 cDNA fragment did not match the cDNA sequences of any known proteins.

To determine whether any one of the GTGs or CTGs located upstream of the first ATG of the AZU-1 open reading frame can be used as an alternate initiator, long and short AZU-1 constructs, pCR-LAZU-1, (nucleotides 403-3325) and pCR-SAZU-1 (nucleotides 1610-3325) were generated. The major products of the in vitro synthesized proteins, using the rabbit reticulocyte lysates (Promega, Inc., Madison, Wis.) of these constructs exhibited a similar size predicated for the short construct (data not shown) suggesting that the first ATG in the deduced open reading frame was likely the translation start site. Based on these results, AZU-1 was predicted to encode a protein spanning 571 amino acids in length.

The BLAST search discovered that a splice variant of AZU-1, TACC2, has been cloned. TACC2 appeared to be isolated as a homolog of TACC1 which was located at 8p11, a breast cancer amplicon. The alignment differences between AZU-1 and TACC2 are shown in FIGS. 19 and 20 . TACC2 contains 31 additional upstream residues but no defined translation start site has been indicated. Two sequence insertions were located in the TACC2 protein (FIG. 20 ). The shorter insertion of DTFR residues was also noted in a small fraction of cDNAs transcribed from the premalignant S2 cell line RNA. The presence of these insertions seems to be characteristic of the premalignant cells and may serve as a marker for detection of premalignancy.

B. Antibody Characterization of Protein Encoded by AZU-1 Gene

This invention demonstrated that polyclonal antibodies directed against AZU-1 specific peptide specifically recognized the protein encoded by the cloned AZU-1 cDNA.

Polyclonal or monoclonal anti-AZU-1 antibodies were prepared using methods known in the art by raising the antibodies against either AZU-1 fusion proteins (full-length or N-terminus, as described below) or immunogenic AZU-1 peptides (amino acids 1-20 or amino acids 131-145). The studies performed during the development of this invention demonstrated the results obtained from using an affinity-purified polyclonal antibody directed against the AZU-1 peptide (amino acids 131-145), hereafter called the anti-AZU-1 antibody. This AZU-1 antibody specifically recognized the protein encoded by the cloned AZU-1 cDNA.

For these studies, different AZU-1 cDNA fragments, e.g., full length (pET-full length, nucleotides 1610-3325), N-terminus (pET-NT, nucleotides 1610-2692) and C-terminus (pET-CT, nucleotides 2693-3325) were subcloned into the pET28a+ bacterial expression vector (Novagen, Madison, Wis.) and were expressed in bacteria as a fusion protein containing an N-terminal T7 tag and a polyhistidine epitope. Bacterially-expressed AZU-1 fusion proteins isolated by His-Bind chromatography were analyzed by SDS-PAGE and Western blot hybridization. For each expressed fusion protein, antibody against T7 tag recognized a band corresponding to the protein size predicted for AZU-1 peptide and the N-terminally added T7 and his tag fragments (FIG. 7 ). On a parallel blot, the affinity-purified AZU-1 antibody (raised against AZU-1 amino acids 131-145) also recognized similar sizes of the full length and N-terminus AZU-1 bacterially expressed products (data not shown). Altogether, these experiments demonstrate that the anti-AZU-1 antibody recognizes the protein encoded by the AZU-1 cDNA.

Protein encoded by AZU-1 gene in the breast epithelial cells was also characterized by this AZU-1 antibody. FIG. 8 shows the Western blot analysis of AZU-1 protein in the S1 cell lysate with the AZU-1 antibody. Arrow indicates a 64-kd protein recognized by the AZU-1 antibody. To further test its specificity, the antibody was preincubaed with either 15 μg “+” or 30 μg “++” or AZU-1 immunogenic peptide before use in hybridization. In the presence of the antigenic peptide, the 64 kDa band was effectively competed away. On the other hand, the intensities of two minor bands (of higher molecular weight), were not diminished by the peptide competition. The results confirm that the 64 kDa band corresponds to the cellular AZU-1 protein.

III. Functionality of AZU-1 Gene

AZU-1 gene was found in abundance in the breast cells and its expression appears to be correlated with breast cell malignancy. The function of AZU-1 gene and its protein was, therefore, investigated.

A. Function of AZU-1 Gene in Breast Cells

Function of the AZU-1 gene was investigated in normal nonmalignant S1 breast cells, in premalignant S2 cells and in breast tumor cells T4-2 obtained as shown in FIG. 1 . Results show that a novel gene AZU-1 is expressed in nonmalignant breast cells and the expression is downregulated in malignant breast cells.

First, functionality studies were directed to identifying determinants of tumor progression by differential display.

The HMT-3522 breast culture model has the potential to provide significant insight into the molecular basis of tumor progression. By comparing the gene expression pattern of the model's tumorigenic cell population (HMT-3522-T4-2) with the nonmalignant S1 cell line and with that of its premalignant progenitor (HMT-3522-S2), genes that play a crucial role in the final stages of tumorigenic conversion were identified. Results of these studies are seen in FIG. 9 .

FIG. 9 illustrates differential expression of AZU-1 in premalignant and tumor breast cells where FIG. 9A shows differential display of gene messages in premalignant (S2) and tumor (T4-2) cells on a sequencing gel. The arrow indicates a band representing a more intense AZU-1 cDNA fragment in S2 cells than in T4-2 cells, in the absence (−), or in the presence (+) of a reverse transcription reaction (RT).

A PCR-based differential display seen in FIG. 9A strategy was used to screen for genes whose expression varies between S2 and T4-2 cell populations. Using this approach, a 180 bp partial cDNA that was reproducibly present at higher levels in the pre-malignant cell samples than in their tumorigenic counterparts was detected.

To confirm the expression pattern observed in the different display experiments, the cDNA fragment was isolated and used as a probe in Northern blot analyses of total RNA derived from S2 and T4-2 cell cultures seen in FIG. 9 B.

FIG. 9B shows Northern blot analysis where the top panel shows that the 4.4-kb AZU-1 message was highly abundant in S2 cells. In contrast, the similar mRNA was greatly reduced in the T4-2 cells. In the bottom panel, the GAPDH probe was used as a control for the amounts of RNA used. Two additional transcripts, 7-kb and 10-kb sizes, present in much less intensity, were also observed. The minor bands may correspond to RNA splice variants or unprocessed RNA species. Consistent with the differential display results, the tumor cell samples displayed a dramatic, more than 10-fold, reduction in the expression of the 4.4 kb message in comparison with the pre-malignant S2 cells.

Using the 180 bp cDNA fragment as starting material, 5′ RACE technology was used to recover a full-length cDNA clone. Additional probes derived from the complete cDNA sequence were used to establish the expression pattern of these gene product in the HMT-3522 human breast cell series and in other human breast cells. Results, shown in FIGS. 10A-C , indicate a consistent expression pattern using probes derived from different regions of AZU-1 cDNA sequence.

FIG. 10 shows expression of AZU-1 in nonmalignant breast epithelial cells and downregulation of AZU-1 in breast tumor cells and biopsies. AZU-1 expression patterns in nonmalignant and malignant breast cells were shown by Northern blot analysis.

FIG. 10A shows expression of AZU-1 gene in nonmalignant breast cells, namely, in S1 and MCF10A nonmalignant breast epithelial cell lines, and in primary luminal epithelial (luminal epi) and myoepithelial (myoepi) cells. As seen in FIG. 10A , the 4.4-kb message of AZU-1 gene was present in all nonmalignant breast epithelial cells examined.

Results seen in FIG. 10A show that an abundant and specific 4.4 kb message corresponding to AZU-1 expression was detected not only in the non-malignant human epithelial cell lines, HMT-3522-S1 and MCF10A, but also in primary cultures of human luminal epithelial and myoepithelial cells.

FIG. 10B shows the presence or absence of AZU-1 observed in premalignant S2 and in ten malignant breast epithelial cell lines. T4-1 (T4), HMT3909, MCF-7, CAMA-1, BT-20, MDA-MB-468, SKBR-3, T47D, MDA-MB-231, Hs578t, and BT549 cell lines. The 4.4-kb message of AZU-1 gene was absent in ten breast tumor cell lines examined. The low level of AZU-1 message in HMT3909 cells is probably due to the presence of some contaminating nonmalignant myoepithelial cells (personal communication, Ole Petersen, unpublished data). The RNA from premalignant cells (S2) was used as a positive control and, as expected, shows higher levels of AZU-1 expression.

Results seen in FIG. 10B show that the 4.4 kb message was significantly reduced in the 10 of the 11 breast carcinoma cell lines which were examined.

Gene message level was also examined in in situ ductal breast carcinoma cells. RNAs were isolated from in situ carcinoma in the breast and normal tissue from reduction mammaplasty. Three out of four samples taken from breast cancer patients exhibited a lower level of AZU-1 mRNA than the normal tissue. One sample exhibited a higher AZU-1 message level, presumably from a patient at the premalignant stage. Collectively, the gene expression profiles obtained above for the 4.4 kb transcript are consistent with a role for the identified gene product as a Class II tumor suppressor in breast epithelia.

In order to determine whether AZU-1 gene is tissue specific, that is if it is solely expressed in the breast cells or also in other tissues, expression of AZU-1 in normal human tissues was studied. Results are seen in FIG. 11 .

FIG. 11 shows Northern analysis of multiple human tissue RNA blot (Clontech, Inc., Palo alto, Calif.) probed with AZU-1 cDNA fragment. The 4.4-kb AZU-1 message was shown to be expressed in heart, brain, lung, kidney, and pancreas, whereas it was low or absent in placenta, liver, and skeletal muscle. The β-actin probe was used to indicate the amounts of RNA loaded. On a separate multiple human tissue RNA blot from the same source, similar AZU-1 message was shown to be expressed in prostate, testes, and colon. The message was absent or in very low abundance in spleen, thymus, ovary, small intestine, and peripheral blood leukocyte (data not shown).

B. Chromosomal Localization of AZU-1 Gene

Mapping of AZU-1 gene placed AZU-1 gene to chromosome 10q26. Localization of AZU-1 gene to chromosome 10q26 is seen in FIG. 12 .

FIG. 12 shows the chromosomal localization of AZU-1 gene by fluorescence in situ hybridization (FISH). The arrows indicate AZU-1 gene is located at chromosome 10q26. Deletions at 10q26, such as by loss of heterozygosity (LOH) correlated with the occurrence of a variety of human cancers, including brain tumors, endometrial carcinoma, and gliobastomas.

C. AZU-1 Association with Cytoskeletal Complexes

Association of AZU-1 with cytoskeletal complexes and its localization in nonmalignant HMT-3522 S1 cells were also studied. Results are shown in FIG. 13 .

FIG. 13 shows subcellular localization of AZU-1 protein. The immunostaining and fluorescent images were analyzed by confocal microscopy. FIG. 13A shows immunofluorescence images of AZU-1 in S1 cells grown on tissue culture plastic detected by the AZU-1 polyclonal antibody. AZU-1 was found to be present primarily in the cytoplasm and it existed as punctate, occasionally revealed in intense aggregates.

FIG. 13B shows that upon treatment with detergent such as Triton X-100, known to remove soluble components in the cytoplasm and nucleus, AZU-1 staining pattern remained, albeit having somewhat lower intensity. These results indicates that AZU-1 may be associated with cytoskeletal complexes.

In situ staining of AZU-1 in human breast tissues is shown in FIG. 14 . In these studies, localization of AZU-1 in normal human breast tissues was determined by AZU-1 antibody. FIG. 14 , top panel shows the control without the antibody. FIG. 14 , middle panel shows that AZU-1 is primarily present in the myoepithelial cells and in low abundance in the luminal epithelial cells of breast acini. Bottom panel shows AZU-1 to be present primarily in the myoepithelial cells of breast ductal tissues. Some AZU-1 protein could also be observed in the luminal epithelial cells.

D. AZU-1 Interaction with E-Cadherin and β-Catenin

AZU-1 interaction with the cell functional proteins, E-cadherin and β-catenin was also explored.

E-cadherin and β-catenin proteins function at cell-cell junctional complexes called adherens junctions. The adherens junctions are localized to sites of cell-cell contact along the lateral surface of epithelial cells. At this site, the adherens junctions interact with the active cytoskeleton and are believed to be crucial for maintaining the integrity of the cell structure. Loss of E-cadherin function correlates with increased cell invasion in many cell types, including the breast cells.

Interactions between adherens junction proteins E-cadherin (E-Cad), β-catenin (β-Cat) and AZU-1 were investigated by coimmunoprecipitation shown in FIG. 15 .

AZU-1 polyclonal antibody was used to immunoprecipitate AZU-1 protein in S1 and T4-2 cell lysates. The immunocomplexes were analyzed by SDS-PAGE and detected by Western analysis with AZU-1, β-catenin and E-cadherin antibodies. Left panel shows that the 64-kd AZU-1 protein was immunoprecipitated with AZU-1 antibody. The rabbit IgG (IgG) was used as a control. The same blot when probed with either β-catenin or E-cadherin antibody also detected the presence of both antigens. E-cadherin (lower panel) was used as a control for the amounts of cell lysates loaded. These results suggested the plausible interactions of AZU-1 with adherens junctional complexes, either through E-cadherin or β-catenin.

IV. Suppression and Reversion of Tumorigenicity of T4-2 Cells In Vitro and In Vivo

To determine the regulatory function of AZU-1 gene in suppression of tumorigenicity, T4-2 cancer cells were investigated in both in vitro and in vivo assays.

AZU-1 tumor suppression function in vivo and in vitro was assayed using a retroviral gene delivery system to introduce a full length AZU-1 transgene into the HMT-3522 T4-2 tumor cells.

The expression pattern of AZU-1 in non-malignant and tumorigenic cells suggests that AZU-1 may function as a Class II tumor suppressor and that, as such, AZU-1 may affect changes in cellular phenotype by virtue of its expression level.

A. Tumorigenicity Suppression In Vitro

Overexpression of AZU-1 gene in the HMT-3522 tumor cells (T4-2) was tested in order to confirm that such overexpression is sufficient to attenuate their tumorigenic phenotype. Results are seen in FIG. 16 .

FIG. 16 illustrates the findings that ectopically-expressed AZU-1 reduces invasiveness in vitro.

In. FIG. 16A , two pooled populations of cells containing stably incorporated DNA were screened for AZU-1 expression by performing Northern analysis of total cellular RNA. In both cases, exogenous expression of the AZU-1 gene in T4-2 cells resulted in the accumulation of AZU-1 message at levels comparable to those observed in the nonmalignant S1 cells. The levels observed in the AZU-1 overexpressed T4-2 cells were 2 to 3-fold higher than AZU-1 mRNA and protein expression in the mock-infected T4-2 cells.

Because the transcript derived from the AZU-1 transgene comigrates on gels with the endogenous AZU-1 gene product (at 4.4 kb), the expressed species were further characterized using transcript specific probes. Results obtained in these studies show that the increased expression observed in the T4-2+AZU-1 cells is entirely attributable to expression from the AZU-1 transgene (data not shown).

To test the potential tumor suppressor function of the AZU-1 gene product, assays of anchorage-independent growth, a generally accepted indicator of tumorigenicity in vitro, were performed on the AZU-1-overexpressing T4-2 cells and their unmodified counterparts. Results are seen in FIG. 16B where equal numbers of S1, T4-2, mock-infected T4-2 or T4-2+AZU-1 cells were embedded in semi-solid-agar and, after 4 weeks in culture, the number of viable colonies (>40 μm) was counted.

As expected, non-malignant S1 cells did not support growth in soft agar, whereas their tumorigenic T4-2 cell counterparts (both naive and mock-transfected) exhibited a markedly higher capacity for colony formation and anchorage-independent growth. Consistent with a role for the AZU-1 gene product in tumor suppression, the T4-2 cells overexpressing AZU-1 have shown an 80% diminished potential for growth capacity in soft agar assay, whereas the mock transfectants has around 10% reduction in its growth capacity. Ectopically expressed AZU-1 significantly suppressed tumorigenicity of T4-2 cells in vitro.

As an additional test of the tumorigenic cell phenotype, the capacity of the T4-2+AZU-1 cells to invade through basement membrane-coated filters in a modified Boyden chamber assay was performed. Using this approach, HMT-3522-S1 cells were found to be largely non-invasive ( Invasion Metastasis , 9:192 (1989)), whereas the tumorigenic T4-2 cells displayed the capacity to migrate effectively through the matrix-coated filters. AZU-1 overexpression diminished the tumor-like behavior of the T4-2 cells, in this case, by attenuating their invasive tendencies to 18% of that displayed by the mock-transfected T4-2 cells (FIG. 16 C).

B. Tumorigenicity Suppression In Vivo

In vivo tumorigenicity of T4-A2+AZU-1 cells was examined by injecting the cells subcutaneously into the rear flanks of nude mice. After 6-8 wks post-injection, the mice were inspected for palpable tumors. Results are seen in Table 1.

TABLE 1
		Number of
	Number of	Sites with	Mean Tumor Size
Cell Type	Injected 1 site	Tumors 2	(mm 3 ) ± S.E. (n)
S1	32	0	0 (0)
T4-2	32	28	250 ± 80 (28)
T4-2 + vector	32	28	265 ± 95 (28)
T4-2 + AZU-1	32	4	38 ± 19 (4)
1 Two injection sites per mouse
2 Lump > 10 mm 3

As seen in Table 1, the non-malignant S1 cells failed to give rise to tumors, while the T4-2 cells (naive or mock infected) gave rise to obvious tumor growth in 90% of the injected sites. Mice injected with T4-2 cells overexpressing AZU-1 gave a diminished tumorigenic response with only 4 of the 28 inoculated sites giving rise to detectable tumors. Moreover, the sizes of the T4-2+AZU-1 tumors were much smaller than those observed with the mock-transfected T4-2 cells.

These observations indicate that AZU-1 overexpression in human T4-2 cells is sufficient to reduce the tumorigenic behavior of these cells, both in vivo and in vitro.

C. AZU-1 Upregulation of Expression

Upregulation of AZU-1 expression or overexpression in phenotypically-reverted T4-2 cells was also investigated and AZU-1 overexpression was found to be sufficient to restore normal tissue architecture to tumorigenic MEC cells in culture.

According to PNAS , 89:9064 (1992), the behavioral phenotype of non-malignant and tumorigenic primary cultures or immortalized cell lines can be effectively reproduced in the context of a 3-dimensional reconstituted basement membrane assay.

Consequently, an additional test of AZU-1's tumor suppressor function was designed to investigate whether overexpression of AZU-1 in T4-2 cells would be sufficient to induce such phenotypic reversion of the HMEC tumor cells in the 3D basement membrane assay. These studies are illustrated in FIG. 17 .

S1 cells, the mock-infected cells and AZU-1 expressing-T4-2 cells were embedded in 3-dimensional basement membrane gels. After 10 days in culture, the cell colonies were measured for size and tested with immunofluorescence microscopy. Results show that the S1 cells formed polarized, growth-arrested acinar structures with organized endogenous basement membranes. The mock infected T4-2 cells continued to grow and formed large irregular colonies, as seen in FIG. 17B , that failed to deposit an organized (polarized) endogenous basement membrane. Tumorigenic T4-2 cells, grown under the same conditions, formed large disorganized cell colonies that continued to grow.

When the overexpression of AZU-1 gene was induced in T4-2 cells, the T4-2+AZU-1 colonies underwent phenotypic reversion. They adopted sizes comparable to the S1 cell colonies and were capable of depositing an organized basement membrane at the basal perimeter of the acinar structures. These results, seen in FIG. 17 , indicate that AZU-1 overexpression was sufficient not only to reduce the size of the tumor colonies, but also to facilitate the structural reorganization that is required to give rise to polarized, organotypic acinar structures of malignant cells.

When the colony size was measured in the three above groups, as seen in FIG. 17C , the size of T4-2+AZU-1 colonies reverted to the size of the normal nonmalignant cells.

The acinus-like structure of T4-2+AZU-1 cells in the 3D culture were reminiscent of the morphologically-reverted T4-2 cells in the presence of an inhibitor anti-β1 integrin (A2BII) or an EGFR specific inhibitor (tyrphostin AG1478).

Accordingly, studies were performed to examine whether the AZU-1 message level was modulated in the reverted T4-2 cells.

Phenotypic reversion of T4-2 cells in the 3D rBM assay is dependent on the establishment of bi-direction reciprocal cross-talk between at least three intracellular signal mediators, including β1 integrin, EGFR and MAP kinase (ibid) ( PNAS , 95:14821 (1998)). Functional inhibition of any one of these elements abrogates the signaling activity of the other two and results in the reduction of total β1 integrin and EGFR protein levels. The AZU-1 gene product might also play a role in the observed cross-talk phenomenon, and its expression might be modulated in the presence of the previously described reverting agents ( J. Of Cell. Biol ., 137:231 (1997), PNAS , 95:14821 (1998)).

To test this, total RNA was extracted from 3D rBM cultures of S1 cells and T4-2 cells treated with or without inhibitors of either β1 integrin (mAb AIIB2) or EGFR (Tyrphostin). Results are seen in FIG. 18 . Northern blot analysis seen in FIG. 18 revealed that AZU-1 levels, while significantly down-modulated in untreated T4-2 cells (FIG. 18 C), were restored to S1-like levels in cultures treated with either inhibitors of β1 integrin (T4β1) or EGFR (T4tyr) ( FIG. 18C , two right panels). When T4-2 cells were cultured in 3D rBM in the presence of functional inhibitors of β1 integrin or EGFR, the T4-2 cells become “phenotypically reverted”, that is they became reorganized to form S1-like organotypic spheres. Collectively, these findings suggest that AZU-1 expression is coupled to β1 integrin and EGFR activity. By virtue of its connections with β1 integrin and EGFR, AZU-1 may provide essential cellular information that dictates cellular structure and phenotype.

In this regard, the 3D rBM assay served as another assay in testing tumor suppression, not only with respect to inhibition of cell growth, but also with respect to restoration of the appropriate tissue polarity and architecture.

D. Detection of AZU-1 Protein in Breast Tumor Biopsies

In order to determine the degree of malignancy in relation to the presence or absence of AZU-1 expressed protein and to determine whether the protein may be useful for detection of malignancy and tumorigenic progression, biopsies were obtained from 19 patients with confirmed stages of breast cancer progression. Results are seen in Table 2.

	TABLE 2
			No. of	No. of
		Malignancy	Samples	AZU-1
	Carcinomas	Stage	Tested	Positive
	Infiltrating ductal	1	6	3
	carcinoma
	Mucinous carcinoma	1	1	1
	Infiltrating ductal	2	6	1
	carcinoma
	Infiltrating ductal	3	3	0
	carcinoma
	Medullary carcinoma	3	1	0
	Metaplastic carcinoma	3	2	0

Table 2 shows in situ staining of AZU-1 protein in breast tumor biopsies. Nineteen in situ breast carcinoma biopsies with diagnosed mucinous carcinoma (stage 1), infiltrating ductal carcinoma (stage 1, stage 2 and stage 3), medullary carcinoma (stage 3) and metaplastic carcinoma (stage 3), were investigated to determine relative abundance of AZU-1 protein by immunostaining with AZU-1 antibody. The malignancy state of the carcinomas was-graded by a scale of 1 to 3. Number 1 indicates more differentiated and an early stage of malignancy, whereas number 3 indicates samples at a more advanced stage.

The results show that AZU-1 protein was present in 60% (4/7) of breast tumors at early stage 1 of malignancy whereas it was nearly absent (1/12) in the biopsies taken at more advanced state. Specifically, 1 out of 6 samples in the malignancy stage 2 showed the presence of AZU-1 protein. None of the six samples of the advanced stage 3 showed the presence of AZU-1 protein.

V. Methods for Detection, Diagnosis, Treatment, Prophylaxis and a Kit for Diagnosis

The current invention further concerns methods for detection of breast cancer, for its treatment and prophylaxis and kits suitable for diagnostic detection of breast cancer growth.

A. A Method For Treatment of Breast Cancer

The proteins encoded by AZU-1 gene or its variants AZU-2 or TACC2 genes were found to be present in large amounts in the normal nonmalignant epithelial breast cells. Its level was found to be significantly decreased or nonexistent in the breast malignant cells.

AZU-1 gene or its variants, which express the protein, are thus actively expressing the protein in nonmalignant cells. Such expression, however, is decreased or absent in malignant breast cells.

The presence of protein expressed by AZU-1 gene, therefore, affects tumorigenicity of the breast tissue and is believed to act, and the findings described herein support its function, as the tumor suppressor.

The method for treatment of breast tumor, thus, comprises providing the subject patient with either the tumor suppressing protein directly targeted to the breast tissue or with genetic material able to express such protein. The protein may be delivered to a patient encapsulated within liposomes or formulated for target delivery using any other targeting means known in the art and used for targeted delivery of drugs to specific organs and tissue.

The second mode for treatment provides the subject patient with genetic material able to express AZU-1 protein. This is typically achieved through gene therapy.

A method for treatment comprises in vivo and ex vivo therapeutic approaches as well as in vivo gene therapy and ex vivo methods.

The gene therapy according to the invention utilizes two approaches. One approach comprises genetic modifications of the tumorigenic cells of a subject to be treated. Such modifications may be induced in the cells in vivo by, for example, developing and transferring a genetic material for expression of the specific protein, or the genetic manipulation may be performed in the subject's own cells or other mammalian cells outside of the body, under ex vivo conditions. The resulting protein then may be imported and delivered to the cells or tissue of the treated subject.

In vivo gene therapy consists of transferring the genetic material directly into the subject's cells.

Ex vivo gene therapy consists of removing cells from the subject and inserting the genetic material into these cells in vitro, prior to replacing the cells in the treated subject.

In the in vivo treatment, cDNA and the expression vectors are prepared. The plasmid encoding the AZU-1 protein is prepared and used to transfect subject's cells. In the cells, the plasmid is transcribed into mRNA and the protein is expressed.

In vivo, thus, the genetic material encoding the protein is transferred directly into the subject's cells or tissue. To ensure the efficiency of the method and expression of the AZU-1 protein at suitably high levels, the coding DNA sequence is engineered to be flanked by an appropriate regulatory sequence such as a viral promoter for ensuring high level expression or tissue specific promoter for ensuring specific organ target.

The transfer of genetic material according to the invention is designed to incorporate AZU-1 gene into breast cells by integrating it into chromosome 10q26. The genetic treatment is intended to permanently alter patient's genetic apparatus ensuring continuous long-term expression of AZU-1 gene.

The method for transfer of genetic material in vivo utilizes, for example, adenovirus vectors, herpes simplex vectors, receptor mediated endocytosis and liposomes, among others, as well as nonviral systems or replication incompetent viruses. Genetic material transfer is achieved, for example, by direct injection, electroporation, particle bombardment, receptor-mediated endocytosis or using liposomes targeted for specific cells or tissues.

In the ex vivo approach, the genetic material, that is AZU-1 cDNA, are built into the expression vector, for example plasmid, cloned and transferred into cells, preferably subject's tumorigenic breast cells, grown in culture, that is grown in vitro and extracorporally. These cells are then transformed and expanded by cell culture in vitro and only then introduced into the subject. To avoid immune system rejection the autologous cells are normally used. For this purpose, the cells are collected initially from the subject to be treated, grown in culture, transformed and reintroduced by implantation into the same subject.

The ex vivo transfer of genetic material is achieved by and utilizes, for example, retrovirus vectors, adeno-associated virus vectors, and to a lesser degree, adenovirus vectors, herpes simplex vectors and liposomes.

The ex vivo transfer of genetic material involves essentially four steps:

(a) cloning a dual-function genetic material into a vector, such as retroviral vector;

(b) transfecting the subject's cells where the targeted AZU-1 protein synthesis is to occur with the genetic material encoding recombinant AZU-1 protein;

(c) verifying the expression of the AZU-1 protein in the cells; and

(d) reimplanting these cells in the patient.

The methods used for in vivo gene therapy applicable to the breast cancer therapy according to the invention are known in the art and are described, for example, in Molecular Biotechnology: Therapeutic Applications and Strategies , Sunil Maulik, Solil D. Patel, WILEY-LISS, A JOHN WILEY & SONS, Inc., New York, (1997); Medical Genetics , pp. 252-257, George H. Sack, McGraw-Hill, New York (1999); Human Molecular Genetics , 551-588, T. Strachan, A. P. Read, Bios Scientific Publishers, WILEY-LISS, A JOHN WILEY & SONS, Inc., New York (1998); and Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors , Ed. Enc Wickstrom, Marcell Decker, Inc., New York (1998), all the above hereby incorporated by reference.

The administration of the genetic material, such as DNA, to the subject is done by means of a composition comprising the cDNA expressing the AZU-1 protein and a pharmaceutically-acceptable carrier and/or other agents such as recombinase enzymes, a lipid agent, a lipid and protein agent, and the like.

Typically, the carrier may comprise solid, liquid or gaseous carriers. Examples of carriers are aqueous solutions, including water, buffered, aqueous solutions and the like.

While it is possible for the cDNA to be administered alone it is preferable to administer it as a pharmaceutical formulation.

In the preferred embodiment of the invention, the DNA of the above composition comprises AZU-1 gene cDNA sequence (SEQ ID NO: 1) encoding the AZU-1 protein.

The delivery of the cDNA into the cell may be conducted by a variety of techniques discussed above. These encompass providing the DNA enveloped by a lipid layer (liposomes), further complexed with a protein and a lipid or a dendrimer.

The complexing the cDNA encoding the protein with lipid, lipid-protein, or dendrimer is especially applicable to in vivo transfection since less cell lethality is encountered, the DNA is protected from DNase degradation and the method is compatible with intracorporeal injection or administration.

One concern about the direct intravenous delivery of genetic material in vivo is the ability of the polynucleotide to survive in circulation long enough to arrive at the desired cellular destination.

In this respect, the coating or masking of the DNA is of extreme utility. The utilization of liposomes, a lipoproteic, or a dendrimer coating is extremely useful. In addition, a successful liposome system uses the cationic lipid reagent dioleyloxytrimethalammonium (DOTMA). DOTMA may be mixed with phosphatidyl ethanolamine (PE) to form the reagent LIPOFECTIN®. When this reagent is utilized to carry the polynucleotides the liposomes are mixed with the DNA and readied for administration.

The DNA may be conveniently enveloped by a lipid layer (liposomes), encapsulated by a lipid and a protein layer, or is complexed to dendrimer. The choice of the foregoing preparations will vary depending on the cell type used, the in vitro, ex vivo or in vivo conditions and the inherent limitations of each transfection method. Preferred conditions for enveloping the cDNA with a lipid layer are as follows. The cDNA is admixed with a lipid such as dioleophosphatidyl ethanolamine, dipalmitoylphosphatidylethanolamine (dipalmitoyl PtdEtn), palmitoyloleoylphosphatidylethanolamine (palmitoyloleoyl PtdEtn), dioleoylphosphatidylcholine (PtdCho), dimyristoylphospatidylethanolamine (dimyristoyl PtdEtn), diphytanoylgeycero-phosphatidylethanolamine (diphytanoyl PtdEtn), N-monomethyl PtdEtn, and N-dimethyl PtdEtn in a proportion of about 1 μg: 1 nmole to 1 μg: 500 nmoles, in an aqueous solution. Other components and proportions are permissible when this technology is applied to the in vivo method. The pH of the solution may be adjusted to about 8 to 10, and more preferably about 9. In addition to the above, ingredients such as a buffer and other known components may also be added to this composition. The amounts in which these components may be added are standard in the art and need not be further described herein.

In addition to the above, efficient transfer of genetic material requires the targeting of the genetic material encoding the AZU-1 protein to the breast cells. This can be attained by procedures based upon receptor mediated endocytosis according to J. Biol. Chem ., 262:4429(1987) or J. Biol. Chem ., 263:14, 621 (1988)). This technology utilizes a cell-specific ligand-polylysine complex bound to the DNA polynucleotide sequence through charge interactions. This complex is taken up by the target cells. The successful transfection of a similar hepatoma cell line resulting in stable expression of enzymatic activity following directed targeting was reported in Biochem. Pharmacol ., 40:253 (1985)). PNAS , (USA), 87:3410 (1990) and PNAS , (USA) 88:4255 (1991) utilized a transferrin-polycation to attain the delivery of a plasmid into a human leukemic cell line and observed expression of the encoded luciferase gene. These proteins require attachment to the polynucleotide via, for example, a polylysine linker.

Moreover, in many receptor-mediated systems as chloroquine or other disrupters of intracellular trafficking may be required for high levels of transfection. Adenovirus, for instance, has been used to enhance the delivery of polynucleotides in receptor-mediated systems ( PNAS (USA), 88:8850 (1991)).

Alternatively, the genetic material may be masked through association with lipids. In one embodiment, the DNA is encased in standard liposomes as described, for example, in U.S. Pat. No. 4,394,448, the relevant portion of the specification of which is hereby incorporated by reference. In another embodiment, the DNA is incubated with a synthetic cationic lipid similar to those described in U.S. Pat. No. 4,897,355. The above-described synthetic cationic lipid effectively mask the DNA when associated therewith. The methods described in the above and below references are hereby incorporated by reference.

The cell recognition element is a molecule capable of recognizing a component on the surface of a targeted cell, covalently linked with a DNA-associating moiety by conventional methods. Cell recognition components include antibodies to cell surface antigens, ligands for cell surface receptors including those involved in receptor-mediated endocytosis, peptide hormones, etc.

Specific ligands contemplated by this invention include carbohydrate ligands such as galactose, mannose, mannosyl 5-phosphate, fucose, sialic groups, N-acetylglucosamine or combinations of these groups as complex carbohydrates such as those found on glycolipids of the blood groups or on various secreted proteins. Other ligands include folate, biotin, various peptides that can interact with cell surface or intracellular receptors such as the chemoattractants peptide containing N-formyl peptides that contain a cystine residue or that interact with cell surface protein such as the human immunodeficiency virus GP-120, and peptides that interact with CD-4.

Other ligands include antibodies or antibody fragments. The specificity of the antibodies can be directed against a variety of epitopes that can be expressed on cell surfaces including histocompatibility, macromolecules, autoimmune antigens, viral, parasitic or bacterial proteins. Other protein ligands include hormones such as growth hormone and insulin or protein growth factors such as GM-CSF, G-CSF, erythropoietin, epidermal growth factor, basic and acidic fibroblast growth factor and the like. Other protein ligands would include various cytokines that work through cell surface receptors such as interleukin 2, interleukin 1, tumor necrosis factor and suitable peptide fragments from such macromolecules.

The membrane-permeabilizing element of this system is a molecule that aids in the passage of a polynucleotide across a membrane. The liposomes, synthetic cationic lipids, lipid-proteins, and dendrimer described above as DNA-masking components also may function as membrane-permeabilization components.

Additional membrane-permeabilizing components that will facilitate delivery of the genetic material of this invention also include polycations that neutralize the large negative charge on polynucleotides. Polycations of this invention include polylysine, polyarginine, poly (lysine-arginine) and similar polypeptides, and the polyamines and the polycationic dendrimers.

The membrane-permeabilizing component that facilitates transfer of the protein or DNA of this invention may be an amphiphathic cationic peptide. Amphipathic cationic peptides are peptides whose native configuration is such that the peptide is considered to have a cationic face and a neutral, hydrophobic face. In a preferred embodiment, the peptide is a cyclic peptide. Examples of the amphipathic cationic cyclic peptizes of this invention are gramicidin S, and tyrocidines. The peptide may also contain some or all of the amino acids in the D configuration as opposed to the naturally occurring L configuration.

The membrane permeabilizing elements, i.e., the cyclic peptide and optional phospholipid and polyamine, may be added to the composition simultaneously or consecutively. Preferably, the cyclic peptide is added first, and the phospholipid or polyamine is added later. The molar ratio of added cyclic peptide to added polyamine is preferably from about 1:1 to about 1:3. The molar ratio of added cyclic peptide to added phospholipid is preferably from about 1:1 to about 1:20.

The subcellular-localization element of this system is a molecule capable of recognizing a subcellular component in a targeted cell, covalently linked with a DNA-associating moiety by conventional methods. Particular subcellular components include the nucleus, ribosomes, mitochondria, and chloroplasts. In a preferred embodiment of this invention, the subcellular-localization component is a nuclear-localization component.

The nuclear-localization components include known peptides of defined amino acid sequences, and longer sequences containing these peptides.

For the conventional therapy, the patient is provided with the recombinant AZU-1 protein.

Using either in vitro or ex vivo methods described above and in examples, the AZU-1 recombinant protein is prepared and delivered in the conventional way using pharmaceutically acceptable delivery vehicles and routes. This type of delivery needs to assure that the protein is properly protected from the destruction by digestive proteases when administered orally, or destroyed in the body before it reaches the target breast cells or tissue.

In this mode, the AZU-1 protein may be delivered orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, as aerosol, or using any other mode of delivery known in the art.

In order to avoid the major drawbacks of delivery of proteins, such as instability in the proteolytic environment of the GI tract and poor absorbability through the mucosa, AZU-1 protein containing compositions are preferably prepared as sterile solutions and administered to patients by daily injection. In this particular instance, the protein is prepared ex vivo, isolated, purified and administered to a subject. However, this form of drug delivery could cause pain and inconvenience to patients and thus could be poorly accepted. Many novel delivery systems have been developed to address these problems ( Trends Biotech ;, 16(8): 343-9, (1998), J. Pharmaceut. Sci , 87(11): 1331:1334, (1998)). Examples of such deliveries of proteins include, but are not limited to combinations of:

1. Targeted delivery of the protein to cells or tissues to be treated or administered orally, nasally, as injectable, etc., as alternate sites of delivery to a site where the target protein should be delivered.

2. Formulations containing the AZU-1 protein for sustained release.

3. Formulations for administration of concentrated AZU-1 protein into cells at the mucosal surface.

4. Formulations modified for enhanced absorption of the protein into breast cells.

5. Formulation for inhibition of proteolysis including protease inhibitors, chemical modification of the peptide molecule to produce prodrugs and analogs ( Nippon Rinsho. Jap. J. Clin. Med ., 56(3): 601-7 (1998) and genetic engineering of proteolytically resistant forms.

A number of novel delivery systems have been approved by the FDA ( J. Pharmaceut. Sci ., 87:1331-4 (1998)). In many cases, a given formulation incorporates elements of several of the above mentioned approaches. Development and optimization of an oral drug delivery systems tends to be specific for each protein or peptide drug. ( J. Pharmaceut. Sci ., 85:1282-5 (1996)).

Examples of formulations which prevents proteolytic degradation of the AZU-1 protein, sustained release and enhanced absorption follow.

The AZU-1 protein may be delivered via microparticles or microsphere. Gelatin capsules coated with various concentrations of sodium alginate, for example, 20% w/v, and cross-linked with appropriate concentrations of calcium chloride, are resistant to the harsh environment of the stomach and deliver the drug to the distal gastrointestinal tract where drug absorption occurs. ( J. Biomaterials Sci., Polymer Ed ., 7(1): 39-48 (1995).

The protein can also be incorporated into biodegradable microparticles to reduce the effect of gut secretions and to enable the absorption of the protein in an unaltered form. The uptake of micropartidulates through the gut wall is accepted as a true biological phenomenon but the mechanism and route of uptake have not been established.

Lipid delivery vehicles enhance microparticle uptake and the selective transport of microspheres across M cells ( J. Anatomy , 189 (Pt3): 487-90 (1996)). Microparticles and microspheres also allow sustained release. Gelatin nanoparticle-poly(lactic-co-glycolic acid) (PLGA) microsphere composites can be prepared by encapsulating protein-loaded gelatin nanoparticles in PLGA microspheres. This encapsulation is conducted by using a phase separation method and a solvent extraction method.

Protein release experiments described in J. Pharmaceut. Sci ., 86(8): 891-5 (1997), indicate that this composite system possesses sustained release characteristics. This system also demonstrates the capability of preventing the denaturation of the AZU-1 protein.

Poly(vinyl alcohol) (PVA) hydrogel nanoparticles may be prepared by using a water-in-oil emulsion technology plus cyclic freezing-thawing process. The PVA hydrogel nanoparticles prepared by this method are suitable for the AZU-1 protein drug delivery since formation of the hydrogel does not require crosslinking agents or other adjuvants and does not involve any residual monomer. The PVA hydrogel nanoparticles swell in an aqueous solution and the swelling degree increases with the increase of temperature.

Another route of delivery for AZU-1 protein is through mucoadhesives. Mucoadhesives are polymeric delivery systems for use to concentrate protein and peptide pharmaceuticals at the mucosal surface. Some of the mucoadhesive polymers were found to display other important biological activities, i.e., inhibition of proteolytic enzymes and or modulation of the permeability of usually tight epithelial tissue barriers. Rather than being just adhesives, mucoadhesive polymers may therefore be considered as a novel class of multifunctional macromolecules with a number of desirable properties for their use as biologically active drug delivery adjuvants which are particularly useful in the context of peptide and protein drug delivery.

Carbopol (polyacrylic) polymers with strong bioadhesive properties also can inhibit lumenal degradation of peptide or proteins, offering multiple advantages for their uses in oral drug delivery ( J. Pharm. Pharmacol ., 48(1): 17-21, (1996), Pharmaceut. Res , 12(9): 1293-8 (1995). The mucoadhesive polymers carbomer 934P and chitosan hydrochloride are able to enhance the intestinal absorption of some agents such as buserelin in vivo, and are therefore suitable excipients in peroral delivery system for AZU-1 protein.

Mucoadhesives include monolithic type devices in which the drug is dispersed throughout the polymer and protein-polymer conjugates where the drug is covalently bound to the polymer. Advanced delivery systems include systems containing mucoadhesive polymers providing an intimate contact to the mucosa, thereby reducing the drug degradation between delivery system and absorbing membrane. They also may contain controlled release systems which provide a simultaneous release of protein and inhibitor or inhibitor prodrug or the immobilization of enzyme inhibitors on delivery systems ( J. Controlled Release , 52(1-2):1-16 (1998); J. Med. Chem , 41(13): 2339-44 (1998).

For treatment of the breast cancer, the patient will be provided either with the gene therapy or with the AZU-1 protein formulated as described above.

Treatment regimen would be 1-3 times a day, daily, 1-2 times a week or as needed and will be continued until the normal levels of AZU-1 protein are detectable, until the AZU-1 gene expression is restored and until the tumorigenicity reversion is achieved and confirmed by immunostaining morphological micrography or any other means.

B. A Method For Prophylaxis of Breast Cancer

The method for prophylaxis of cancer growth in the breast cells is achieved in the same way as described above for the treatment.

A patient to be prophylactically treated would have either a family history of the breast cancer or the low level of expression of AZU-1 gene, or the low level of AZU-1 protein will be detected in the biopsy, although there will not yet be any visible, palpable observable or detectable tumor growth.

For prophylaxis, the dosages of the protein will typically be lower than for treatment, treatment will be administered 1-2 times weekly and the patient will be monitored weekly or monthly for AZU-1 gene expression or for the levels of AZU-1 protein in the breast tissue biopsies.

C. A Method For Detection of Breast Cancer

Detection and diagnosis of the breast cancer comprises determining a level of expression of AZU-1 message or detecting a level of protein expressed in breast biopsies.

The level of AZU-1 expression is determined by, for example, in situ hybridization of AZU-1 RNA using a complimentary DNA probe or by RT-PCR assay using gene specific primers according to Example 5.

The level of AZU-1 expressed protein is determined, for example, by detecting with polyclonal or monoclonal AZU-1 antibodies specifically prepared (Example 8) against AZU-1 protein or peptide using indirect immunofluorescence assay as described in Example 18.

D. Kits for Detection and Diagnosis of Breast Cancer

Kits for detection and diagnosis of breast cancer are based on two approaches outlined in Section C, namely, on detecting the AZU-1 protein or on detecting AZU-1 gene message.

1. The Kit for Detection of AZU-1 Protein

The kit for detection of AZU-1 protein in breast biopsies (either cryosections or parafilm embedded blocks) for diagnosis of breast cancer-comprises:

(a) polyclonal or monoclonal anti AZU-1 antibodies;

(b) secondary antibody, e.g. FITC or Texas Red conjugated;

(c) permeabilizing/fixing reagent (optional);

(d) blocking/hybridization solution;

(e) means of tabulating detected AZU-1 protein in the breast tissue samples and correlating the level with the presence, absence, or the stage of breast tumorigenicity.

Examples of results using this kit are shown in FIG. 14 and Table II following the method described in Example 18.

2. The Kit for Detection of AZU-1 Message

The kit for detection of AZU-1 message in breast biopsies for diagnosis of breast cancer comprises components needed for in situ hybridization or for detection of the message by RT reverse transcription PCR.

(A) In situ hybridization kit comprises:

(a) means for preparing intact breast biopsies, e.g., cryosection and mounting condition;

(b) AZU-1 gene specific cDNA probe;

(c) P-actin cDNA as a positive control;

(d) means for labeling of cDNA probes;

(e) means for hybridization/blocking; and

(f) means for tabulating and correlating the detected AZU-1 message levels with the malignant stages of breast cancer patients.

(B) RT-PCR kit comprises:

(a) means for preparing intact breast biopsies, e.g., cryosection and preservation of fresh tissues;

(b) means for RNA extraction from breast biopsies;

(c) AZU-1 gene specific cDNA primers;

(d) means for RT-PCR amplification of RNA extracts;

(e) means for detection of amplified cDNA product, e.g., by ethidium bromide staining/agarose gels;

(f) means for quantification of detected cDNA products; and

(g) means for correlation of obtained values to a degree of tumorigenicity.

UTILITY

The current invention is useful for diagnostic and therapeutic purposes for detection and treatment of human breast cancer. For diagnostic purposes, the level of AZU-1 gene encoded protein is determined in the breast biopsy from the patient. When the amount of AZU-1 protein is high, then there are no tumor cells present in the biopsy. When the AZU-1 encoded protein is absent or at a low level, then there are breast tumor cells present. The protein is useful also as a marker for the malignancy progression. The protein is also useful as a diagnostic marker for tumorigenic reversion in cancer patients undergoing conventional cancer therapy.

The invention is also useful for therapy, particularly gene therapy of breast tumors wherein the specifically targeted AZU-1 gene is introduced into the breast cells, using, for example, retrovial delivery system for gene therapy or the AZU-1 protein is administered in tissue targeted formulation.

EXAMPLE 1

Cell Separation

This example describes the procedure used for cell separation.

Human breast luminal epithelial and myoepithelial cells were purified from organoids after these had spread out to form monolayers in primary culture or had been passaged once. Cells were trypsinized and resuspended in N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer with 0.5% (W/V) bovine serum albumin (BSA, fraction V, A4919, Sigma) and filtered through a 100 mM nylon mesh (Millipore, Hedehusene, Denmark) to remove residual cell clumps. The cell suspension was incubated 30 minutes at 4° C. with the primary mAb, 115D8, directed against sialomucin (provided by Jo Hilgers, Amsterdam, The Netherlands) or J5 directed against common acute lymphoblastic leukemia antigen (CALLA) or CD10 antigen (Coulter Clone, Struers Kebo Lab., Albertslund, Denmark) diluted 1:100 and 1:10, respectively in HEPES/BSA. The cells were then washed twice in HEPES/BSA and incubated 15 minutes at 4° C. with goat anti-mouse IgG microbeads (AH Diagnostics, {dot over (A)}rhus, Denmark) diluted 1:5 in HEPES/BSA and washed twice in HEPES/BSA. Cell separation was carried out by use of the MiniMACS magnetic cell separation system obtained from AH Diagnostics according to the kit instructions.

EXAMPLE 2

Cell Culture and Human Luminal Epithelial Cells

This example describes cell culture conditions used for culturing human luminal epithelial cells.

The HMT-3522 human mammary epithelial cells were grown in H14 medium consisting of DMEM/F12 medium (GIBCO/BRL, St. Louis, Mo.) and additives including 250 ng/ml insulin, 10 μg/ml transferrin, 2.6 ng/ml sodium selenite, 10 −10 M estradiol, 1.4×10 −6 M hydrocortisone, and 5 μg/ml prolactin. The S-1 and MCF10A cells were propagated in H14 medium as monolayers on plastic in the presence of 10 ng/ml epidermal growth factor (EGF) whereas the S2 and T4-2 cells were cultured as monolayers on flasks coated with collagen Type I (Vitrogen 100, Celtrix Laboratories, Palo Alto, Calif.) in the absence of EGF. HMT3909 and MCF-7 cells were cultured as monolayers on collagen type I in DMEM/F12 medium supplemented with 1.4×10 −6 M hydrocortisone and 2 μM glutamine, respectively. Breast tumor cell lines, e.g., CAMA-1, BT-20, MDA-MB468, SKBR3, T47D, MDA-MB231, Hs578T and BT549, were cultured as monolayers in DMEM/F12 with 5% bovine serum.

Human breast luminal epithelial cells were purified organoids grown as monolayers in primary culture. Three dimensional (3D) cultures were prepared by growing S1, T4-2 cells, and T4-2 transfectants to confluence as monolayers, followed by trypsinization and embedding (8.5×10 5 cells/ml) as single cells into a commercially prepared reconstituted basement membrane (Matrigel, Collaborative Research, Waltham, Mass.) from Englebreth-Holm-Swarm mouse tumors.

EXAMPLE 3

Probe Mapping on Metaphase Chromosome

This example describes probe mapping on metaphore chromosome and primers and procedure used for chromosome localization of AZU-1 gene.

U4 (CGTATGCACTACTGTATTTCCTTTC) (SEQ ID NO: 24) and L3 (GGGCAAGGGCCAAGGTCCAGCAATG) (SEQ ID NO: 25) primers were used to generate 199-bp genomic DNA to screen for P1/BAC/PAC clones to determine location of AZU-1 on human chromosome by fluorescence in situ hybridization (FISH).

P1/BAC/PAC clone was used to determine the location of AZU-1 on human chromosomes. DNA was extracted from an overnight culture using alkaline lysis technique. Probe DNA was labeled with digoxigenin-11-dUTP by nick translation. Hybridization was carried out in the presence of human Cot 1 DNA to suppress the background signal and hybridized to metaphase chromosomes overnight. The hybridized signal was detected by anti-digoxigenin conjugated with FITC. The location of the probes was determined by digital image microscopy following FISH and localized by the fractional length from the p-terminus (FLpter) described previously in Human Genet . 83: 335 ( 1989).

EXAMPLE 4

RNA Extraction, Quantification and Northern Blot Analysis

This example describes conditions and procedures used for RNA isolation and Northern blot analysis.

Total RNA was extracted from cells cultured as monolayers or in 3D rBM or cells from normal tissues or in situ carcinoma with TRIzol reagent (Life Technologies, Inc. Grand Island, N.Y.). For Northern blots, total RNA (20 μg/lane) was resolved on denaturing agarose gels and transferred to Hybond-N + membranes (Amersham). Resulting blots were hybridized with 32 P-labeled cDNA probes and analyzed by autoradiography. A GAPDH probe was used to normalize variations in loading.

EXAMPLE 5

Differential Display

This example describes conditions used for differential display and RACE cDNA amplification.

Differential display was performed using the RNA image protocol (GenHunter Corp., Nashville, Tenn.) following manufacturer's instructions.

The total RNA (DNA-free) from S2 and T4-2 cell lines was reverse transcribed and the cDNA products were amplified by polymerase chain reaction using the anchored (H-T 11 M, M=A,C,G) and arbitrary primers (H-AP and H-T 11 A) provided in the kit. Amplified products were resolved on 6% acrylamide gels and differential expression of the amplified species was evaluated by autoradiography.

The expression patterns of these two cell lines were observed on the sequencing gel. The differences in the intensity of bands representing differential gene expression were further confirmed by agarose gel electrophoresis analysis of the reamplified cDNA fragments that had been eluted from the gel. To confirm observed differential expression patterns, cDNA fragments of interest were excised from the gel, subject to a second PCR amplification and analyzed on agarose gels. Gene identification of the cDNA products and differences in the message levels were then verified by northern blot analysis.

One of the DNA fragments from the PCR products of H-T 11 A (5-AAGCTTTTTTTTTTTA) SEQ ID NO: 28 and H-AP1 (5′-AAGCTTGATTGCC) SEQ ID NO: 29 primers showed a significantly higher intense band in S2 than in T4-2 cells on both sequencing and agarose gel analyses. Northern blot analysis using this cDNA fragment as a probe confirmed its gene product was greatly more abundant in the S2 cells than in T4-2 cells. Sequence analysis revealed it was novel. This gene has been named AZU-1.

EXAMPLE 6

Rapid Amplification of 5′ cDNA Ends (RACE)

This example describes methods used for rapid amplification of 5′ cDNA end.

5′ RACE system (Life Technologies, Inc. Grand Island, N.Y.) was performed according to the manufacturer's protocol to extend the 5′ end of the cDNA length. The procedure was repeated approximately twelve times to map a total length of 3.8 kb of AZU-1 sequence. In each run, 500-800 bp of additional 5′-end sequence was obtained. To further determine the contiquousness and accuracy of AZU-1 sequence, 3.8 kb AZU-1 cDNA fragments were prepared from two separate reverse transcription products using Expand Long Template PCR System (Boehringer Mannheim Corp., Indianapolis, Ind.). A complete match of the sequence of these two cDNA clones confirmed AZU-1 cDNA sequence.

EXAMPLE 7

Sequencing of 5′ RACE PCR Products

This example describes sequencing of 5′ RACE PCR product.

Sequencing of the 5′ RACE PCR products was conducted by thermo sequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science, Cleveland, Ohio). AZU-1 cDNA clones described in Example 6 were sequenced by the sequencing facility in the University of California at Berkeley.

EXAMPLE 8

Preparation of Anti AZU-1 Antibodies

This example describes procedures used for preparation of antibodies against AZU-1 peptides, AZU-1 N-terminal (fragment 1-368), and full length AZU-1 protein.

Polyclonal Antibodies:

AZU-1 polyclonal antibody was raised against an immunogenic peptide AZU-1-A (residues 121-135, KPAKKKKTPLKTVKK) (SEQ ID NO: 26) in rabbits by Animal Pharm Services, Inc. (Healdsburg, Calif.). The immunoglobulin G fraction of the antiserum was further purified by AZU-1 peptide-linked affinity chromatography.

AZU-1 polyclonal antibodies are raised against immunogenic peptides AZU-1-B (residues 1-20, MPLRPPKMKKTPEKLDNTPA) (SEQ ID NO: 27), and purified His-tagged fusion protein containing residues 1-368 of AZU-1 protein fragment in rabbits by ImmunoVision Inc., (Daly City, Calif.). The immunoglobulin G fractions of the antisera are further purified by AZU-1 peptide or purified protein-linked affinity chromatography. The antibody is further purified by affinity chromatography and analyzed by enzyme-linked immunosorbent assay (ELISA).

Monoclonal antibody:

AZU-1 monoclonal antibody is raised against purified His-tagged full length AZU-1 fusion protein in mice by ImmunoVision Inc. The antibody is further purified by affinity chromatography and analyzed by enzyme-linked immunosorbent assay (ELISA).

EXAMPLE 9

Reversion Assays

This example describes reversion assays.

The β1-integrin function-blocking mAb AIIB2 (C. Damsky, UCSF) was introduced into the cell-embedded substratum at a concentration of 100 μg/ml ascites protein (which corresponds to 4-10 μg/ml purified rat IgG1) at the time of Matrigel gelation. Tyrphostin AG 1478 (Calbiochem) dissolved in dimethyl sulfoxide was added to the medium at a concentration of 100 nM on alternate days. Control cultures were treated with mouse IgG and vehicle only for AIIB2 antibody and inhibitor experiments, respectively.

EXAMPLE 10

Immunoblotting and Immunoprecipitation

This example describes immunoblotting and immunoprecipitation methods.

Cells grown as monolayers were lysed in situ in RIPA buffer [1% Nonidet P-40, 0.5% deoxycholate, 0.2% SDS, 150 mM sodium chloride, 50 mM Tris-HCl (pH 7.4) containing 2 mM sodium fluoride, 1 mM sodium orthovanadate, 10 μg/ml E64, and 1 mM Pefabloc]. Cells grown in 3D rBM cultures for 10 days were isolated as colonies with ice-cold PBS/EDTA [0.01 M sodium phosphate (pH 7.2) containing 138 mM sodium chloride and 5 mM EDTA] and thereafter were lysed in RIPA buffer. Protein lysates were resolved upon 7.5% SDS-PAGE gels, electrotransferred to immobilon-P blots (Millipore Corp.) And the blots were then subjected to Western analyses and enhanced chemiluminescence (ECL) (Amersham Corp., Arlington Heights, Ill.) detection. For reprobing, the blots were stripped by incubating in 2% SDS, 62.5 mM Tris-HCl, (pH 6.7), 2-mercaptoethanol, at 50° C. for 30 minutes.

For immunoprecipitation of AZU-1, the RIPA lysates were first precleared by incubating with rabbit IgG and protein A coupled to Sepharose 4B beads (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) before immunoprecipitation. The precleared lysates were incubated with affinity-purified polyclonal anti AZU-1 antibodies, monoclonal antibody, or normal rabbit IgG antibodies (negative control) together with protein A coupled to Sepharose-4B beads. Immunoprecipitates were washed five times in RIPA buffer and dissolved in equal volume of 2×SDS sample buffer (0.125M Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 4% β-mercaptoethanol) for SDS-PAGE analysis and subsequent Western analyses.

EXAMPLE 11

AZU-1 Plasmid Construct

This example describes preparation of plasmid AZU-1 constructs.

pCR-LAZU-1, pCR-SAZU-1, pET- full length AZU-1 pET-NT, pET-CT, and pLXSN-AZU-1 constructs were used. Full length AZU-1 open reading frame (nucleotides 1610-3325) was inserted into SacI-SalI sites of pET28 a(+) vector (Noragen), and EcoRI-XhoI sites of pLXSN vector (Clontech Inc.) to generate pET- full length AZU-1 and pLXSN-AZU-1 constructs.

N-terminus (nucleotides 1610-2692) and C-terminus (nucleotides 2693-3325) of AZU-1 protein sequences were inserted into SacI-SalI sites of pET28 vector to generate pET-NT and pET-CT constructs. Two AZU-1 cDNA sequences, positioned at nucleotides 403-3325 and nucleotides 1610-3325 were inserted into pCR 2.1 vector (Invitrogen) to generate pCR-LAZU-1 and pCR-SAZU-1 constructs.

EXAMPLE 12

Transfection Assay

This example describes methods used for transfection.

PLXSN vector and pLXSN-AZU-1 were transfected into PT60 cells provided in the Retro-X system (Clontech Laboratories, Inc., Palo Alto, Calif.) and the stable virus-packaging PT60 cells were generated by selection with 500 μg/ml G418 (Genetisen; Gibco Inc.). The retrovirus particles collected from the growth media selection of the stably transfected PT67 cells were then used to infect T4-2 cells and stable transfectants were selected in 50 μg/ml G418.

EXAMPLE 13

In Vitro Transcription and Translation

This example describes transcription and translation methods.

The CR-LAZU-1 and pCR-SAZU-1 constructs were used to generate in vitro translated product by a TNT coupled reticulocyte lysate system (Promega, Madison, Wis.).

EXAMPLE 14

Soft Agar Assay

This example describes anchorage-independent growth assay.

S1, T4-2, T4-2 (mock) and T4-2 +AZU-1 cells were seeded at 1×10 5 cells/well in 0.35% soft agar on 12-well plate for 4 weeks and the size of the colony was measured by eyepiece. Colonies greater than 40 μm was scored as positive and counted. Four repeats were performed on each cell and the experiments performed in triplicate.

EXAMPLE 15

In Vivo Tumorigenicity

This example describes assay used for testing in vivo tumorigenicity.

S1, T4-2, T4-2 (mock) and T4-2+AZU-1 cells propagated as monolayers were trypsinized and dispersed in DMEM:F12 medium at a concentration of 2.5×10 7 /ml. An aliquot of 100 μl (2.5×10 6 cells) was subcutaneously injected into each flank of 4-6 week old BalbC nu/nu mice. The size of nodule on the flank was measured by a caliper and recorded at 6-8 weeks after injection.

EXAMPLE 16

In Vitro Invasion Assay

This example describes assay used for testing in vitro invasion.

8 μM Falcon cell culture PET inserts (Becton Dickinson Labware, Franklin Lakes, N.J.) were coated with 10 μl of 1:2 dilution (50 μg/filter) of matrigel in DMEM/E12. 1×10 5 of S1, T4-2, T4-2 (mock), T4-2-AZU-1 cells resuspended in 200 μl H14 medium were grown on top of coated insert in 24 well plate. After 18-24 hours, the cells migrated through the matrigel was fixed in glutaldehyde, stained with toluidine blue and counted. Four repeats were performed for each cell line and the experiment was repeated three times.

EXAMPLE 17

Morphogenesis Assessment and Criteria

This example describes assays used for assessment of morphogenesis.

Morphology was assessed in situ by examining the degree of colony organization visually by phase contrast microscopy, and by measuring colony diameter using an eyepiece equipped with a micrometer spindle. Polarity was indicated by the presence of a basally organized basement membrane (BM) as determined by collagen IV and β4-integrin immunostaining.

EXAMPLE 18

Indirect Immunofluorescence

This example describes the procedure used for measurement of indirect immunofluorescence.

Cells or breast tissues were permeabilized in situ (0.5% Triton X-100 in 100 mM NaCl/300 mM sucrose/10 mM PIPES, pH 6.8/5 mM MgCl 2 containing 1 mM Pefabloc Sc (AEBSF) (Boehringer Mannheim)/10 g/ml leupeptin/10 μg/ml aprotinin/10 μg trypsin inhibitor type II/250 μM NaF), fixed in 2% paraformaldehyde, and immunostained in the presence or absence of AZU-1 antibody using essentially the assay as described in J. of Cell Biology , 137:231 (1997).

EXAMPLE 19

Homology Search and Secondary Structure Prediction Programs

This example describes methods and programs used for homology search and secondary structure prediction.

The gapped BLAST (National Center for Biotechnology Information, NCBI), BEAUTY+BLAST (Baylor College of Medicine), and TFASTA (University of Wisconsin) were the used sequence homology search programs.

Website was used to predict coiled coil structure and to calculate the probability that AZU-1 sequence adopts a coiled-coil conformation.

EXAMPLE 20

AZU-1 Recombinant Proteins

This example describes production of AZU-1 recombinant proteins.

Different AZU-1 cDNA fragments, e.g., full length (pET-full length, nucleotides 1610-3325), N-terminus (pET-NT, nucleotides 1610-2692) and C-terminus (pET-CT, nucleotides 2693-3325) were subcloned into pET 28 bacterial expression vector (Novagen, Madison, Wis.) and were expressed in bacteria as a fusion protein containing an N-terminus T7 tag and a polyhistidine epitope. The expressed proteins in the solubilized bacterial cell lysates were purified by His Bind column chromatography and following the manufacturer's procedures. The AZU-1 recombinant proteins were eluted with 1×elute buffer (150 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9).

29 1 3813 DNA Homo sapiens 1ggaaagccct ttgcacacat cggcctattg aagcactttg cttgattcag ctattctcct 60ctcaggacct gcctggatca tcccagcctg tagaatcctg ggtttctgtg gcagtttgtt 120cttctggtat cacctgctat gctctgaatg tttgtatccc tctcagcccc gaaattcctg 180tgttgaaatc ctaaccccta aggtgatggt atgaggaggt ggggccttcg ggaggtgatt 240aggccataag ggcggagcct ttgtgaatgg gattagtgcc cttataaaaa gaggccccac 300agcactgcct tgcctcttct gccacgtgaa gatgcagtga gaaggagctg tctgtgaact 360tggaaggggt cctcatgaga cactgaacct gctggtgcct tgatcttgga cttcccagcc 420tccagaactt tcacctgtgg cagatgatat catccagccc gctgcccctg cagacctgga 480aagcccaacc ttagctgcct cttcctacca cagtgatgtt gttggccagg tctctacgga 540tctgatagcc cagaggagtt ccgattctga agaggcattt gagaccccgg agtcaacgac 600ccctgtcaaa gctccgccag ctccaccccc accacccccc gaagtcatcc cagaacccga 660ggtcagcaca cagccacccc cggaagaacc aggatgtggt tctgagacag tccctgtccc 720tgatggccca cggagcgact cggtggaagg aagtcccttc cgtcccccgt cacacccctt 780ctctgccgtc ttcgatgaag accagccgat agccagcagt gggacttaca acttggactt 840tgacaacatt gagcttgtgg atacctttca gaccttggag cctcgtgcct cagacgctaa 900gaatcaggag ggcaaagtga acacacggag gaagtccacg gattccgtcc ccatctctaa 960gtctacactg tcccggtcgc tcagcctgca agccagtgac tttgatggtg cttcttcctc 1020aggcaatccc gaggccgtgg cccttgcccc agatgcatat agcacgggtt ccagcagtgc 1080ttctagtacc cttaagcgaa ctaaaaaacc gaggccgcct tccttaaaaa agaaacagac 1140caccaagaaa cccacagaga cccccccagt gaaggagacg caacaggagc cagatgaaga 1200gagccttgtc cccagtgggg agaatctagc atctgagacg aaaacggaat ctgccaagac 1260ggaaggtcct agcccagcct tattggagga gacgcccctt gagcccgctg tggggcccaa 1320agctgcctgc cctctggact cagagagtgc agaaggggtt gtccccccgg cttctggagg 1380tggcagagtg cagaactcac cccctgtcgg gaggaaaacg ctgcctctta ccacggcccc 1440ggaggcaggg gaggtaaccc catcggatag cggggggcaa gaggactctc cagccaaagg 1500gctctccgta aggctggagt ttgactattc tgaggacaag agtagttggg acaaccagca 1560ggaaaacccc cctcctacca aaaagatagg caaaaagcca gttgccaaaa tgcccctgag 1620gaggccaaag atgaaaaaga cacccgagaa acttgacaac actcctgcct cacctcccag 1680atcccctgct gaacccaatg acatccccat tgctaaaggt acttacacct ttgatattga 1740caagtgggat gaccccaatt ttaacccttt ttcttccacc tcaaaaatgc aggagtctcc 1800caaactgccc caacaatcat acaactttga cccagacacc tgtgatgagt ccgttgaccc 1860ctttaagaca tcctctaaga cccccagctc accttctaaa tccccagcct cctttgagat 1920cccggccagt gctatggaag ccaatggagt ggacggggat gggctaaaca agcccgccaa 1980gaagaagaag acgcccctaa agacggtgaa aaagtcgcca aaacggtctc ctctctctga 2040tccaccttcc caggacccca ccccagctgc tacaccagaa acaccaccag tgatctctgc 2100ggtggtccac gccacagatg aggaaaagct ggcggtcacc aaccagaagt ggacgtgcat 2160gacagtggac ctagaggctg acaaacagga ctacccgcag ccctcggacc tgtccacctt 2220tgtaaacgag accaaattca gttcacccac tgaggagttg gattacagaa actcctatga 2280aattgaatat atggagaaaa ttggctcctc cttacctcag gacgacgatg ccccgaagaa 2340gcaggccttg taccttatgt ttgacacttc tcaggagagc cctgtcaagt catctcccgt 2400ccgcatgtca gagtccccga cgccgtgttc agggtcaagt tttgaagaga ctgaagccct 2460tgtgaacact gctgcgaaaa accagcatcc tgtcccacga ggactggccc ctaaccaaga 2520gtcacacttg caggtgccag agaaatcctc ccagaaggag ctggaggcca tgggcttggg 2580caccccttca gaagcgattg aaattagaga ggctgctcac ccaacagacg tctccatctc 2640caaaacagcc ttgtactccc gcatcgggac cgctgaggtg gagaaacctg caggccttct 2700gttccagcag cccgacctgg actctgccct ccagatcgcc agagcagaga tcataaccaa 2760ggagagagag gtctcagaat ggaaagataa atatgaagaa agcaggcggg aagtgatgga 2820aatgaggaaa atagtggccg agtatgagaa gaccatcgct cagatgatag aggacgaaca 2880gagagagaag tcagtctccc accagacggt gcagcagctg gttctggaga aggagcaagc 2940cctggccgac ctgaactccg tggagaagtc tctggccgac ctcttcagaa gatatgagaa 3000gatgaaggag gtcctagaag gcttccgcaa gaatgaagag gtgttgaaga gatgtgcgca 3060ggagtacctg tcccgggtga agaaggagga gcagaggtac caggccctga aggtgcacgc 3120ggaggagaaa ctggacaggg ccaatgctga gattgctcag gttcgaggca aggcccagca 3180ggagcaagcc gcccaccagg ccagcctgcg gaaggagcag ctgcgagtgg acgccctgga 3240aaggacgctg gagcagaaga ataaagaaat agaagaactc accaagattt gtgacgaact 3300gattgccaaa atggggaaaa gctaactctg aaccgaatgt tttggactta actgttgcgt 3360gcaatatgac cgtcggcaca ctgctgttcc tccagttcca tggacaggtt ctgttttcac 3420tttttcgtat gcactactgt atttcctttc taaataaaat tgatttgatt gtatgcagta 3480ctaaggagac tatcagaatt tcttgctatt ggtttgcatt ttcctagtat aattcatagc 3540aagttgacct cagagttcct gtatcaggga gattgtctga ttctctaata aaagacacat 3600tgctgacctt ggccttgccc tttgtacaca agttcccagg gtgagcagct tttggattta 3660atatgaacat gtacagcgtg catagggact cttgccttaa ggagtgtaaa cttgatctgc 3720atttgctgat ttgtttttaa aaaaacaaga aatgcatgtt tcaaataaaa ttctctattg 3780taaataaaat tttttctttg gatcttggca ata 3813 2 4148 DNA Homo sapiens 2ggcacgagcg acagtccaca tggtagaaga tggtcctggg actttgctca cacaggggtt 60ccaggacatg tgccaaggtc cacgtgtgcc ccttctcctc agagggaggt tttgactgtg 120cctgaggcca acagtgagcc ctggaccctt gacacgcttg ggggtgaaag gagacccgga 180gtcactgctg gcatcttgga aatgcgaaat gccctgggca accagagcac ccctgcacca 240ccaactggag aagtggcaga cactcccctg gagcctggca aggtggcagg cgctgctggg 300gaagcagagg gtgacatcac cctgagcaca gctgagacac aggcatgtgc gtccggtgat 360ctgcctgaag caggtactac gaggacattc tccgttgtgg caggtgactt ggtgctgcca 420ggaagctgtc aggacccagc ctgctctgac aaggctccgg ggatggaggg tacagctgcc 480cttcatgggg acagcccagc caggccccag caggataagg agcagccagg acctgagcgc 540cccattccag ctggggatgg gaaggtgtgc gtctcctcac ctccagagcc tgacgaaact 600cacgacccga agctgcaaca tttggctcca gaagagctcc acactgacag agagagcccc 660aggcctggcc catccatgtt accttcggtt cctaagaagg atgctccaag agtcatggat 720aaagtcactt cagatgagac cagaggtgcg gaaggaacag aaagttcacc tgtggcagat 780gatatcatcc agcccgctgc ccctgcagac ctggaaagcc caaccttagc tgcctcttcc 840taccacagtg atgttgttgg ccaggtctct acggatctga tagcccagag gagttccgat 900tctgaagagg catttgagac cccggagtca acgacccctg tcaaagctcc gccagctcca 960cccccaccac cccccgaagt catcccagaa cccgaggtca gcacacagcc acccccggaa 1020gaaccaggat gtggttctga gacagtccct gtccctgatg gcccacggag cgactcggtg 1080gaaggaagtc ccttccgtcc cccgtcacac cccttctctg ccgtcttcga tgaagaccag 1140ccgatagcca gcagtgggac ttacaacttg gactttgaca acattgagct tgtggatacc 1200tttcagacct tggagcctcg tgcctcagac gctaagaatc aggagggcaa agtgaacaca 1260cggaggaagt ccacggattc cgtccccatc tctaagtcta cactgtcccg gtcgctcagc 1320ctgcaagcca gtgactttga tggtgcttct tcctcaggca atcccgaggc cgtggccctt 1380gccccagatg catatagcac gggttccagc agtgcttcta gtacccttaa gcgaactaaa 1440aaaccgaggc cgccttcctt aaaaaagaaa cagaccacca agaaacccac agagaccccc 1500ccagtgaagg agacgcaaca ggagccagat gaagagagcc ttgtccccag tggggagaat 1560ctagcatctg agacgaaaac ggaatctgcc aagacggaag gtcctagccc agccttattg 1620gaggagacgc cccttgagcc cgctgtgggg cccaaagctg cctgccctct ggactcagag 1680agtgcagaag gggttgtccc cccggcttct ggaggtggca gagtgcagaa ctcaccccct 1740gtcgggagga aaacgctgcc tcttaccacg gccccggagg caggggaggt aaccccatcg 1800gatagcgggg ggcaagagga ctctccagcc aaagggctct ccgtaaggct ggagtttgac 1860tattctgagg acaagagtag ttgggacaac cagcaggaaa acccccctcc taccaaaaag 1920ataggcaaaa agccagttgc caaaatgccc ctgaggaggc caaagatgaa aaagacaccc 1980gagaaacttg acaacactcc tgcctcacct cccagatccc ctgctgaacc caatgacatc 2040cccattgcta aaggtactta cacctttgat attgacaagt gggatgaccc caattttaac 2100cctttttctt ccacctcaaa aatgcaggag tctcccaaac tgccccaaca atcatacaac 2160tttgacccag acacctgtga tgagtccgtt gaccccttta agacatcctc taagaccccc 2220agctcacctt ctaaatcccc agcctccttt gagatcccgg ccagtgctat ggaagccaat 2280ggagtggacg gggatgggct aaacaagccc gccaagaaga agaagacgcc cctaaagacg 2340gtgaaaaagt cgccaaaacg gtctcctctc tctgatccac cttcccagga ccccacccca 2400gctgctacac cagaaacacc accagtgatc tctgcggtgg tccacgccac agatgaggaa 2460aagctggcgg tcaccaacca gaagtggacg tgcatgacag tggacctaga ggctgacaaa 2520caggactacc cgcagccctc ggacctgtcc acctttgtaa acgagaccaa attcagttca 2580cccactgagg agttggatta cagaaactcc tatgaaattg aatatatgga gaaaattggc 2640tcctccttac ctcaggacga cgatgccccg aagaagcagg ccttgtacct tatgtttgac 2700acttctcagg agagccctgt caagtcatct cccgtccgca tgtcagagtc cccgacgccg 2760tgttcagggt caagttttga agagactgaa gcccttgtga acactgctgc gaaaaaccag 2820catcctgtcc cacgaggact ggcccctaac caagagtcac acttgcaggt gccagagaaa 2880tcctcccaga aggagctgga ggccatgggc ttgggcaccc cttcagaagc gattgaaatt 2940agagaggctg ctcacccaac agacgtctcc atctccaaaa cagccttgta ctcccgcatc 3000gggaccgctg aggtggagaa acctgcaggc cttctgttcc agcagcccga cctggactct 3060gccctccaga tcgccagagc agagatcata accaaggaga gagaggtctc agaatggaaa 3120gataaatatg aagaaagcag gcgggaagtg atggaaatga ggaaaatagt ggccgagtat 3180gagaagacca tcgctcagat gatagaggac gaacagagag agaagtcagt ctcccaccag 3240acggtgcagc agctggttct ggagaaggag caagccctgg ccgacctgaa ctccgtggag 3300aagtctctgg ccgacctctt cagaagatat gagaagatga aggaggtcct agaaggcttc 3360cgcaagaatg aagaggtgtt gaagagatgt gcgcaggagt acctgtcccg ggtgaagaag 3420gaggagcaga ggtaccaggc cctgaaggtg cacgcggagg agaaactgga cagggccaat 3480gctgagattg ctcaggttcg aggcaaggcc cagcaggagc aagccgccca ccaggccagc 3540ctgcggaagg agcagctgcg agtggacgcc ctggaaagga cgctggagca gaagaataaa 3600gaaatagaag aactcaccaa gatttgtgac gaactgattg ccaaaatggg gaaaagctaa 3660ctctgaaccg aatgttttgg acttaactgt tgcgtgcaat atgaccgtcg gcacactgct 3720gttcctccag ttccatggac aggttctgtt ttcacttttt cgtatgcact actgtatttc 3780ctttctaaat aaaattgatt tgattgtatg cagtactaag gagactatca gaatttcttg 3840ctattggttt gcattttcct agtataattc atagcaagtt gacctcagag ttcctgtatc 3900agggagattg tctgattctc taataaaaga cacattgctg accttggcct tgccctttgt 3960acacaagttc ccagggtgag cagcttttgg atttaatatg aacatgtaca gcgtgcatag 4020ggactcttgc cttaaggagt gtaaacttga tctgcatttg ctgatttgtt tttaaaaaaa 4080caagaaatgc atgtttcaaa taaaattctc tattgtaaat aaaatttttt ctttggatct 4140tggcaata 4148 3 571 PRT Homo sapiens 3Met Pro Leu Arg Arg Pro Lys Met Lys Lys Thr Pro Glu Lys Leu Asp 1 5 10 15Asn Thr Pro Ala Ser Pro Pro Arg Ser Pro Ala Glu Pro Asn Asp Ile 20 25 30Pro Ile Ala Lys Gly Thr Tyr Thr Phe Asp Ile Asp Lys Trp Asp Asp 35 40 45Pro Asn Phe Asn Pro Phe Ser Ser Thr Ser Lys Met Gln Glu Ser Pro 50 55 60Lys Leu Pro Gln Gln Ser Tyr Asn Phe Asp Pro Asp Thr Cys Asp Glu 65 70 75 80Ser Val Asp Pro Phe Lys Thr Ser Ser Lys Thr Pro Ser Ser Pro Ser 85 90 95Lys Ser Pro Ala Ser Phe Glu Ile Pro Ala Ser Ala Met Glu Ala Asn 100 105 110Gly Val Asp Gly Asp Gly Leu Asn Lys Pro Ala Lys Lys Lys Lys Thr 115 120 125Pro Leu Lys Thr Val Lys Lys Ser Pro Lys Arg Ser Pro Leu Ser Asp 130 135 140Pro Pro Ser Gln Asp Pro Thr Pro Ala Ala Thr Pro Glu Thr Pro Pro145 150 155 160Val Ile Ser Ala Val Val His Ala Thr Asp Glu Glu Lys Leu Ala Val 165 170 175Thr Asn Gln Lys Trp Thr Cys Met Thr Val Asp Leu Glu Ala Asp Lys 180 185 190Gln Asp Tyr Pro Gln Pro Ser Asp Leu Ser Thr Phe Val Asn Glu Thr 195 200 205Lys Phe Ser Ser Pro Thr Glu Glu Leu Asp Tyr Arg Asn Ser Tyr Glu 210 215 220Ile Glu Tyr Met Glu Lys Ile Gly Ser Ser Leu Pro Gln Asp Asp Asp225 230 235 240Ala Pro Lys Lys Gln Ala Leu Tyr Leu Met Phe Asp Thr Ser Gln Glu 245 250 255Ser Pro Val Lys Ser Ser Pro Val Arg Met Ser Glu Ser Pro Thr Pro 260 265 270Cys Ser Gly Ser Ser Phe Glu Glu Thr Glu Ala Leu Val Asn Thr Ala 275 280 285Ala Lys Asn Gln His Pro Val Pro Arg Gly Leu Ala Pro Asn Gln Glu 290 295 300Ser His Leu Gln Val Pro Glu Lys Ser Ser Gln Lys Glu Leu Glu Ala305 310 315 320Met Gly Leu Gly Thr Pro Ser Glu Ala Ile Glu Ile Arg Glu Ala Ala 325 330 335His Pro Thr Asp Val Ser Ile Ser Lys Thr Ala Leu Tyr Ser Arg Ile 340 345 350Gly Thr Ala Glu Val Glu Lys Pro Ala Gly Leu Leu Phe Gln Gln Pro 355 360 365Asp Leu Asp Ser Ala Leu Gln Ile Ala Arg Ala Glu Ile Ile Thr Lys 370 375 380Glu Arg Glu Val Ser Glu Trp Lys Asp Lys Tyr Glu Glu Ser Arg Arg385 390 395 400Glu Val Met Glu Met Arg Lys Ile Val Ala Glu Tyr Glu Lys Thr Ile 405 410 415Ala Gln Met Ile Glu Asp Glu Gln Arg Glu Lys Ser Val Ser His Gln 420 425 430Thr Val Gln Gln Leu Val Leu Glu Lys Glu Gln Ala Leu Ala Asp Leu 435 440 445Asn Ser Val Glu Lys Ser Leu Ala Asp Leu Phe Arg Arg Tyr Glu Lys 450 455 460Met Lys Glu Val Leu Glu Gly Phe Arg Lys Asn Glu Glu Val Leu Lys465 470 475 480Arg Cys Ala Gln Glu Tyr Leu Ser Arg Val Lys Lys Glu Glu Gln Arg 485 490 495Tyr Gln Ala Leu Lys Val His Ala Glu Glu Lys Leu Asp Arg Ala Asn 500 505 510Ala Glu Ile Ala Gln Val Arg Gly Lys Ala Gln Gln Glu Gln Ala Ala 515 520 525His Gln Ala Ser Leu Arg Lys Glu Gln Leu Arg Val Asp Ala Leu Glu 530 535 540Arg Thr Leu Glu Gln Lys Asn Lys Glu Ile Glu Glu Leu Thr Lys Ile545 550 555 560Cys Asp Glu Leu Ile Ala Lys Met Gly Lys Ser 565 570 4 1219 PRT Homo sapiens 4Gly Thr Ser Asp Ser Pro His Gly Arg Arg Trp Ser Trp Asp Phe Ala 1 5 10 15His Thr Gly Val Pro Gly His Val Pro Arg Ser Thr Cys Ala Pro Ser 20 25 30Pro Gln Arg Glu Val Leu Thr Val Pro Glu Ala Asn Ser Glu Pro Trp 35 40 45Thr Leu Asp Thr Leu Gly Gly Glu Arg Arg Pro Gly Val Thr Ala Gly 50 55 60Ile Leu Glu Met Arg Asn Ala Leu Gly Asn Gln Ser Thr Pro Ala Pro 65 70 75 80Pro Thr Gly Glu Val Ala Asp Thr Pro Leu Glu Pro Gly Lys Val Ala 85 90 95Gly Ala Ala Gly Glu Ala Glu Gly Asp Ile Thr Leu Ser Thr Ala Glu 100 105 110Thr Gln Ala Cys Ala Ser Gly Asp Leu Pro Glu Ala Gly Thr Thr Arg 115 120 125Thr Phe Ser Val Val Ala Gly Asp Leu Val Leu Pro Gly Ser Cys Gln 130 135 140Asp Pro Ala Cys Ser Asp Lys Ala Pro Gly Met Glu Gly Thr Ala Ala145 150 155 160Leu His Gly Asp Ser Pro Ala Arg Pro Gln Gln Asp Lys Glu Gln Pro 165 170 175Gly Pro Glu Arg Pro Ile Pro Ala Gly Asp Gly Lys Val Cys Val Ser 180 185 190Ser Pro Pro Glu Pro Asp Glu Thr His Asp Pro Lys Leu Gln His Leu 195 200 205Ala Pro Glu Glu Leu His Thr Asp Arg Glu Ser Pro Arg Pro Gly Pro 210 215 220Ser Met Leu Pro Ser Val Pro Lys Lys Asp Ala Pro Arg Val Met Asp225 230 235 240Lys Val Thr Ser Asp Glu Thr Arg Gly Ala Glu Gly Thr Glu Ser Ser 245 250 255Pro Val Ala Asp Asp Ile Ile Gln Pro Ala Ala Pro Ala Asp Leu Glu 260 265 270Ser Pro Thr Leu Ala Ala Ser Ser Tyr His Ser Asp Val Val Gly Gln 275 280 285Val Ser Thr Asp Leu Ile Ala Gln Arg Ser Ser Asp Ser Glu Glu Ala 290 295 300Phe Glu Thr Pro Glu Ser Thr Thr Pro Val Lys Ala Pro Pro Ala Pro305 310 315 320Pro Pro Pro Pro Pro Glu Val Ile Pro Glu Pro Glu Val Ser Thr Gln 325 330 335Pro Pro Pro Glu Glu Pro Gly Cys Gly Ser Glu Thr Val Pro Val Pro 340 345 350Asp Gly Pro Arg Ser Asp Ser Val Glu Gly Ser Pro Phe Arg Pro Pro 355 360 365Ser His Pro Phe Ser Ala Val Phe Asp Glu Asp Gln Pro Ile Ala Ser 370 375 380Ser Gly Thr Tyr Asn Leu Asp Phe Asp Asn Ile Glu Leu Val Asp Thr385 390 395 400Phe Gln Thr Leu Glu Pro Arg Ala Ser Asp Ala Lys Asn Gln Glu Gly 405 410 415Lys Val Asn Thr Arg Arg Lys Ser Thr Asp Ser Val Pro Ile Ser Lys 420 425 430Ser Thr Leu Ser Arg Ser Leu Ser Leu Gln Ala Ser Asp Phe Asp Gly 435 440 445Ala Ser Ser Ser Gly Asn Pro Glu Ala Val Ala Leu Ala Pro Asp Ala 450 455 460Tyr Ser Thr Gly Ser Ser Ser Ala Ser Ser Thr Leu Lys Arg Thr Lys465 470 475 480Lys Pro Arg Pro Pro Ser Leu Lys Lys Lys Gln Thr Thr Lys Lys Pro 485 490 495Thr Glu Thr Pro Pro Val Lys Glu Thr Gln Gln Glu Pro Asp Glu Glu 500 505 510Ser Leu Val Pro Ser Gly Glu Asn Leu Ala Ser Glu Thr Lys Thr Glu 515 520 525Ser Ala Lys Thr Glu Gly Pro Ser Pro Ala Leu Leu Glu Glu Thr Pro 530 535 540Leu Glu Pro Ala Val Gly Pro Lys Ala Ala Cys Pro Leu Asp Ser Glu545 550 555 560Ser Ala Glu Gly Val Val Pro Pro Ala Ser Gly Gly Gly Arg Val Gln 565 570 575Asn Ser Pro Pro Val Gly Arg Lys Thr Leu Pro Leu Thr Thr Ala Pro 580 585 590Glu Ala Gly Glu Val Thr Pro Ser Asp Ser Gly Gly Gln Glu Asp Ser 595 600 605Pro Ala Lys Gly Leu Ser Val Arg Leu Glu Phe Asp Tyr Ser Glu Asp 610 615 620Lys Ser Ser Trp Asp Asn Gln Gln Glu Asn Pro Pro Pro Thr Lys Lys625 630 635 640Ile Gly Lys Lys Pro Val Ala Lys Met Pro Leu Arg Arg Pro Lys Met 645 650 655Lys Lys Thr Pro Glu Lys Leu Asp Asn Thr Pro Ala Ser Pro Pro Arg 660 665 670Ser Pro Ala Glu Pro Asn Asp Ile Pro Ile Ala Lys Gly Thr Tyr Thr 675 680 685Phe Asp Ile Asp Lys Trp Asp Asp Pro Asn Phe Asn Pro Phe Ser Ser 690 695 700Thr Ser Lys Met Gln Glu Ser Pro Lys Leu Pro Gln Gln Ser Tyr Asn705 710 715 720Phe Asp Pro Asp Thr Cys Asp Glu Ser Val Asp Pro Phe Lys Thr Ser 725 730 735Ser Lys Thr Pro Ser Ser Pro Ser Lys Ser Pro Ala Ser Phe Glu Ile 740 745 750Pro Ala Ser Ala Met Glu Ala Asn Gly Val Asp Gly Asp Gly Leu Asn 755 760 765Lys Pro Ala Lys Lys Lys Lys Thr Pro Leu Lys Thr Val Lys Lys Ser 770 775 780Pro Lys Arg Ser Pro Leu Ser Asp Pro Pro Ser Gln Asp Pro Thr Pro785 790 795 800Ala Ala Thr Pro Glu Thr Pro Pro Val Ile Ser Ala Val Val His Ala 805 810 815Thr Asp Glu Glu Lys Leu Ala Val Thr Asn Gln Lys Trp Thr Cys Met 820 825 830Thr Val Asp Leu Glu Ala Asp Lys Gln Asp Tyr Pro Gln Pro Ser Asp 835 840 845Leu Ser Thr Phe Val Asn Glu Thr Lys Phe Ser Ser Pro Thr Glu Glu 850 855 860Leu Asp Tyr Arg Asn Ser Tyr Glu Ile Glu Tyr Met Glu Lys Ile Gly865 870 875 880Ser Ser Leu Pro Gln Asp Asp Asp Ala Pro Lys Lys Gln Ala Leu Tyr 885 890 895Leu Met Phe Asp Thr Ser Gln Glu Ser Pro Val Lys Ser Ser Pro Val 900 905 910Arg Met Ser Glu Ser Pro Thr Pro Cys Ser Gly Ser Ser Phe Glu Glu 915 920 925Thr Glu Ala Leu Val Asn Thr Ala Ala Lys Asn Gln His Pro Val Pro 930 935 940Arg Gly Leu Ala Pro Asn Gln Glu Ser His Leu Gln Val Pro Glu Lys945 950 955 960Ser Ser Gln Lys Glu Leu Glu Ala Met Gly Leu Gly Thr Pro Ser Glu 965 970 975Ala Ile Glu Ile Arg Glu Ala Ala His Pro Thr Asp Val Ser Ile Ser 980 985 990Lys Thr Ala Leu Tyr Ser Arg Ile Gly Thr Ala Glu Val Glu Lys Pro 995 1000 1005Ala Gly Leu Leu Phe Gln Gln Pro Asp Leu Asp Ser Ala Leu Gln Ile 1010 1015 1020Ala Arg Ala Glu Ile Ile Thr Lys Glu Arg Glu Val Ser Glu Trp Lys1025 1030 1035 1040Asp Lys Tyr Glu Glu Ser Arg Arg Glu Val Met Glu Met Arg Lys Ile 1045 1050 1055Val Ala Glu Tyr Glu Lys Thr Ile Ala Gln Met Ile Glu Asp Glu Gln 1060 1065 1070Arg Glu Lys Ser Val Ser His Gln Thr Val Gln Gln Leu Val Leu Glu 1075 1080 1085Lys Glu Gln Ala Leu Ala Asp Leu Asn Ser Val Glu Lys Ser Leu Ala 1090 1095 1100Asp Leu Phe Arg Arg Tyr Glu Lys Met Lys Glu Val Leu Glu Gly Phe1105 1110 1115 1120Arg Lys Asn Glu Glu Val Leu Lys Arg Cys Ala Gln Glu Tyr Leu Ser 1125 1130 1135Arg Val Lys Lys Glu Glu Gln Arg Tyr Gln Ala Leu Lys Val His Ala 1140 1145 1150Glu Glu Lys Leu Asp Arg Ala Asn Ala Glu Ile Ala Gln Val Arg Gly 1155 1160 1165Lys Ala Gln Gln Glu Gln Ala Ala His Gln Ala Ser Leu Arg Lys Glu 1170 1175 1180Gln Leu Arg Val Asp Ala Leu Glu Arg Thr Leu Glu Gln Lys Asn Lys1185 1190 1195 1200Glu Ile Glu Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala Lys Met 1205 1210 1215Gly Lys Ser 5 2446 DNA Homo sapiens 5tggagtttga ctattctgag gacaagagta gttgggacaa ccagcaggaa aacccccctc 60ctaccaaaaa gataggcaaa aagccagttg ccaaaatgcc cctgaggagg ccaaagatga 120aaaagacacc cgagaaactt gacaacactc ctgcctcacc tcccagatcc cctgctgaac 180ccaatgacat ccccattgct aaaggtactt acacctttga tattgacaag tgggatgacc 240ccaattttaa ccctttttct tccacctcaa aaatgcagga gtctcccaaa ctgccccaac 300aatcatacaa ctttgaccca gacacctgtg atgagtccgt tgaccccttt aagacatcct 360ctaagacccc cagctcacct tctaaatccc cagcctcctt tgagatccca gccagtgcta 420tggaagccaa tggagtggac ggggatgggc taaacaagcc cgccaagaag aagaagacgc 480ccctaaagac tgacacattt agggtgaaaa agtcgccaaa acggtctcct ctctctgatc 540caccttccca ggaccccacc ccagctgcta caccagaaac accaccagtg atctctgcgg 600tggtccacgc cacagatgag gaaaagctgg cggtcaccaa ccagaagtgg acgtgcatga 660cagtggacct agaggctgac aaacaggact acccgcagcc ctcggacctg tccacctttg 720taaacgagac caaattcagt tcacccactg aggagttgga ttacagaaac tcctatgaaa 780ttgaatatat ggagaaaatt ggctcctcct tacctcagga cgacgatgcc ccgaagaagc 840aggccttgta ccttatgttt gacacttctc aggagagccc tgtcaagtca tctcccgtcc 900gcatgtcaga gtccccgacg ccgtgttcag ggtcaagttt tgaagagact gaagcccttg 960tgaacactgc tgcgaaaaac cagcatcctg tcccacgagg actggcccct aaccaagagt 1020cacacttgca ggtgccagag aaatcctccc agaaggagct ggaggccatg ggtttgggca 1080ccccttcaga agcgattgaa attacagctc ccgagggctc ctttgcctct gctgacgccc 1140tcctcagcag gctagctcac cccgtctctc tctgtggtgc acttgactat ctggagcccg 1200acttagcaga aaagaacccc ccactattcg ctcagaaact ccagagagag gctgttcacc 1260caacagacgt ctccatctcc aaaacagcct tgtactcccg catcgggacc gctgaggtgg 1320agaaacctgc aggccttctg ttccagcagc ccgacctgga ctctgccctc cagatcgcca 1380gagcagagat cataaccaag gagagagagg tctcagaatg gaaagataaa tatgaagaaa 1440gcaggcggga agtgatggaa atgaggaaaa tagtggccga gtatgagaag accatcgctc 1500agatgataga ggacgaacag agagagaagt cagtctccca ccagacggtg cagcagctgg 1560ttctggagaa ggagcaagcc ctggccgacc tgaactccgt ggagaagtct ctggccgacc 1620tcttcagaag atatgagaag atgaaggagg tcctagaagg cttccgcaag aatgaagagg 1680tgttgaagag atgtgcgcag gagtacctgt cccgggtgaa gaaggaggag cagaggtacc 1740aggccctgaa ggtgcacgcg gaggagaaac tggacagggc caatgctgag attgctcagg 1800ttcgaggcaa ggcccagcag gagcaagccg cccaccaggc cagcctgcgg aaggagcagc 1860tgcgagtgga cgccctggaa aggacgctgg agcagaagaa taaagaaata gaagaactca 1920ccaagatttg tgacgaactg attgccaaaa tggggaaaag ctaactctga accgaatgtt 1980ttggacttaa ctgttgcggc aatatgaccg tcggcacact gctgttcctc cagttccatg 2040gacaggttct gttttcactt tttcgtatgc actactgtat ttcctttcta aataaaattg 2100atttgattgt atgcagtact aaggagacta tcagaatttc ttgctattgg tttgcatttt 2160cctagtataa ttcatagcaa gttgacctca gagttcctgt atcagggaga ttgtctgatt 2220ctctaataaa agacacattg ctgaccttgg ccttgccctt tgtacacaag ttcccagggt 2280gagcagcttt tggatttaat atgaacatgt acagcgtgca tagggactct tgccttaagg 2340agtgtaaact tgatctgcat ttgctgattt gtttttaaaa aaacaagaaa tgcatgtttc 2400aaataaaatt ctctattgta aataaaattt tttctttgga tcttga 2446 6 653 PRT Homo sapiens 6Glu Phe Asp Tyr Ser Glu Asp Lys Ser Ser Trp Asp Asn Gln Gln Glu 1 5 10 15Asn Pro Pro Pro Thr Lys Lys Ile Gly Lys Lys Pro Val Ala Lys Met 20 25 30Pro Leu Arg Arg Pro Lys Met Lys Lys Thr Pro Glu Lys Leu Asp Asn 35 40 45Thr Pro Ala Ser Pro Pro Arg Ser Pro Ala Glu Pro Asn Asp Ile Pro 50 55 60Ile Ala Lys Gly Thr Tyr Thr Phe Asp Ile Asp Lys Trp Asp Asp Pro 65 70 75 80Asn Phe Asn Pro Phe Ser Ser Thr Ser Lys Met Gln Glu Ser Pro Lys 85 90 95Leu Pro Gln Gln Ser Tyr Asn Phe Asp Pro Asp Thr Cys Asp Glu Ser 100 105 110Val Asp Pro Phe Lys Thr Ser Ser Lys Thr Pro Ser Ser Pro Ser Lys 115 120 125Ser Pro Ala Ser Phe Glu Ile Pro Ala Ser Ala Met Glu Ala Asn Gly 130 135 140Val Asp Gly Asp Gly Leu Asn Lys Pro Ala Lys Lys Lys Lys Thr Pro145 150 155 160Leu Lys Thr Asp Thr Phe Arg Val Lys Lys Ser Pro Lys Arg Ser Pro 165 170 175Leu Ser Asp Pro Pro Ser Gln Asp Pro Thr Pro Ala Ala Thr Pro Glu 180 185 190Thr Pro Pro Val Ile Ser Ala Val Val His Ala Thr Asp Glu Glu Lys 195 200 205Leu Ala Val Thr Asn Gln Lys Trp Thr Cys Met Thr Val Asp Leu Glu 210 215 220Ala Asp Lys Gln Asp Tyr Pro Gln Pro Ser Asp Leu Ser Thr Phe Val225 230 235 240Asn Glu Thr Lys Phe Ser Ser Pro Thr Glu Glu Leu Asp Tyr Arg Asn 245 250 255Ser Tyr Glu Ile Glu Tyr Met Glu Lys Ile Gly Ser Ser Leu Pro Gln 260 265 270Asp Asp Asp Ala Pro Lys Lys Gln Ala Leu Tyr Leu Met Phe Asp Thr 275 280 285Ser Gln Glu Ser Pro Val Lys Ser Ser Pro Val Arg Met Ser Glu Ser 290 295 300Pro Thr Pro Cys Ser Gly Ser Ser Phe Glu Glu Thr Glu Ala Leu Val305 310 315 320Asn Thr Ala Ala Lys Asn Gln His Pro Val Pro Arg Gly Leu Ala Pro 325 330 335Asn Gln Glu Ser His Leu Gln Val Pro Glu Lys Ser Ser Gln Lys Glu 340 345 350Leu Glu Ala Met Gly Leu Gly Thr Pro Ser Glu Ala Ile Glu Ile Thr 355 360 365Ala Pro Glu Gly Ser Phe Ala Ser Ala Asp Ala Leu Leu Ser Arg Leu 370 375 380Ala His Pro Val Ser Leu Cys Gly Ala Leu Asp Tyr Leu Glu Pro Asp385 390 395 400Leu Ala Glu Lys Asn Pro Pro Leu Phe Ala Gln Lys Leu Gln Arg Glu 405 410 415Ala Val His Pro Thr Asp Val Ser Ile Ser Lys Thr Ala Leu Tyr Ser 420 425 430Arg Ile Gly Thr Ala Glu Val Glu Lys Pro Ala Gly Leu Leu Phe Gln 435 440 445Gln Pro Asp Leu Asp Ser Ala Leu Gln Ile Ala Arg Ala Glu Ile Ile 450 455 460Thr Lys Glu Arg Glu Val Ser Glu Trp Lys Asp Lys Tyr Glu Glu Ser465 470 475 480Arg Arg Glu Val Met Glu Met Arg Lys Ile Val Ala Glu Tyr Glu Lys 485 490 495Thr Ile Ala Gln Met Ile Glu Asp Glu Gln Arg Glu Lys Ser Val Ser 500 505 510His Gln Thr Val Gln Gln Leu Val Leu Glu Lys Glu Gln Ala Leu Ala 515 520 525Asp Leu Asn Ser Val Glu Lys Ser Leu Ala Asp Leu Phe Arg Arg Tyr 530 535 540Glu Lys Met Lys Glu Val Leu Glu Gly Phe Arg Lys Asn Glu Glu Val545 550 555 560Leu Lys Arg Cys Ala Gln Glu Tyr Leu Ser Arg Val Lys Lys Glu Glu 565 570 575Gln Arg Tyr Gln Ala Leu Lys Val His Ala Glu Glu Lys Leu Asp Arg 580 585 590Ala Asn Ala Glu Ile Ala Gln Val Arg Gly Lys Ala Gln Gln Glu Gln 595 600 605Ala Ala His Gln Ala Ser Leu Arg Lys Glu Gln Leu Arg Val Asp Ala 610 615 620Leu Glu Arg Thr Leu Glu Gln Lys Asn Lys Glu Ile Glu Glu Leu Thr625 630 635 640Lys Ile Cys Asp Glu Leu Ile Ala Lys Met Gly Lys Ser 645 650 7 107 PRT Homo sapiens 7Met Pro Leu Arg Arg Pro Lys Met Lys Lys Thr Pro Glu Lys Leu Asp 1 5 10 15Asn Thr Pro Ala Ser Pro Pro Arg Ser Pro Ala Glu Pro Asn Asp Ile 20 25 30Pro Ile Ala Lys Gly Thr Tyr Thr Phe Asp Ile Asp Lys Trp Asp Asp 35 40 45Pro Asn Phe Asn Pro Phe Ser Ser Thr Ser Lys Met Gln Glu Ser Pro 50 55 60Lys Leu Pro Gln Gln Ser Tyr Asn Phe Asp Pro Asp Thr Cys Asp Glu 65 70 75 80Ser Val Asp Pro Phe Lys Thr Ser Ser Lys Thr Pro Ser Ser Pro Ser 85 90 95Lys Ser Pro Ala Ser Phe Glu Ile Pro Ala Ser 100 105 8 210 PRT Homo sapiens 8Gly Leu Leu Phe Gln Gln Pro Asp Leu Asp Ser Ala Leu Gln Ile Ala 1 5 10 15Arg Ala Glu Ile Ile Thr Lys Glu Arg Glu Val Ser Glu Trp Lys Asp 20 25 30Lys Tyr Glu Glu Ser Arg Arg Glu Val Met Glu Met Arg Lys Ile Val 35 40 45Ala Glu Tyr Glu Lys Thr Ile Ala Gln Met Ile Glu Asp Glu Gln Arg 50 55 60Glu Lys Ser Val Ser His Gln Thr Val Gln Gln Leu Val Leu Glu Lys 65 70 75 80Glu Gln Ala Leu Ala Asp Leu Asn Ser Val Glu Lys Ser Leu Ala Asp 85 90 95Leu Phe Arg Arg Tyr Glu Lys Met Lys Glu Val Leu Glu Gly Phe Arg 100 105 110Lys Asn Glu Glu Val Leu Lys Arg Cys Ala Gln Glu Tyr Leu Ser Arg 115 120 125Val Lys Lys Glu Glu Gln Arg Tyr Gln Ala Leu Lys Val His Ala Glu 130 135 140Glu Lys Leu Asp Arg Ala Asn Ala Glu Ile Ala Gln Val Arg Gly Lys145 150 155 160Ala Gln Gln Glu Gln Ala Ala His Gln Ala Ser Leu Arg Lys Glu Gln 165 170 175Leu Arg Val Asp Ala Leu Glu Arg Thr Leu Glu Gln Lys Asn Lys Glu 180 185 190Ile Glu Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala Lys Met Gly 195 200 205Lys Ser 210 9 80 PRT Homo sapiens 9Gly Asn Ser Cys Pro Glu Leu Val Pro Ser Arg Arg Ser Lys Leu Arg 1 5 10 15Lys Pro Lys Pro Val Pro Leu Arg Lys Lys Ala Ile Gly Gly Glu Phe 20 25 30Ser Asp Thr Asn Ala Ala Val Glu Gly Thr Pro Leu Pro Lys Ala Ser 35 40 45Tyr His Phe Ser Pro Glu Glu Leu Asp Glu Asn Thr Ser Pro Leu Leu 50 55 60Gly Asp Ala Arg Phe Gln Lys Ser Pro Pro Asp Ile Lys Glu Thr Pro 65 70 75 80 10 81 PRT Homo sapiens 10Ser Val Ala Ser Thr Lys Ser Ser Ser Lys Ser Pro Arg Ala Thr Ser 1 5 10 15Arg Lys Ser Ile Tyr Asp Asp Ile Arg Ser Gln Phe Pro Asn Leu Thr 20 25 30Pro Asn Ser Thr His Ser Gln Phe Tyr Glu Ser Thr Pro Val Ile Glu 35 40 45Gln Ser Phe Asn Trp Thr Thr Asp Asp His Ile Ser Ala Gly Thr Leu 50 55 60Glu Asn Pro Thr Ser Phe Thr Asn Ser Ser Tyr Lys Asn Asp Asn Gly 65 70 75 80Pro 11 210 PRT Homo sapiens 11Gly Ile Cys Leu Ser Glu Ser Asp Lys Thr Ala Val Leu Thr Leu Ile 1 5 10 15Arg Glu Glu Ile Ile Thr Lys Glu Ile Glu Ala Asn Glu Trp Lys Lys 20 25 30Lys Tyr Glu Glu Thr Arg Gln Glu Val Leu Glu Met Arg Lys Ile Val 35 40 45Ala Glu Tyr Glu Lys Thr Ile Ala Gln Met Ile Glu Asp Glu Gln Arg 50 55 60Thr Ser Met Thr Ser Gln Lys Ser Phe Gln Gln Leu Thr Met Glu Lys 65 70 75 80Glu Gln Ala Leu Ala Asp Leu Asn Ser Val Glu Arg Ser Leu Ser Asp 85 90 95Leu Phe Arg Arg Tyr Glu Asn Leu Lys Gly Val Leu Glu Gly Phe Lys 100 105 110Lys Asn Glu Glu Ala Leu Lys Lys Cys Ala Gln Asp Tyr Leu Ala Arg 115 120 125Val Lys Gln Glu Glu Gln Arg Tyr Gln Ala Leu Lys Ile His Ala Glu 130 135 140Glu Lys Leu Asp Lys Ala Asn Glu Glu Ile Ala Gln Val Arg Thr Lys145 150 155 160Ala Lys Ala Glu Ser Ala Ala Leu His Ala Gly Leu Arg Lys Glu Gln 165 170 175Met Lys Val Glu Ser Leu Glu Arg Ala Leu Gln Gln Lys Asn Gln Glu 180 185 190Ile Glu Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala Lys Leu Gly 195 200 205Lys Thr 210 12 223 PRT Homo sapiens 12Leu Gln Leu Phe Lys Leu Tyr His Asn Glu Val Glu Ile Glu Lys Leu 1 5 10 15Asn Lys Glu Leu Ala Ser Lys Asn Lys Glu Ile Glu Lys Asp Lys Lys 20 25 30Arg Met Asp Lys Val Glu Asp Glu Leu Lys Glu Lys Lys Lys Glu Leu 35 40 45Gly Lys Met Met Arg Glu Gln Gln Gln Ile Glu Lys Glu Ile Lys Glu 50 55 60Lys Asp Ser Glu Leu Asn Gln Lys Arg Pro Gln Tyr Ile Lys Ala Lys 65 70 75 80Glu Asn Thr Ser His Lys Ile Lys Lys Leu Glu Ala Ala Lys Lys Ser 85 90 95Leu Gln Asn Ala Gln Lys His Tyr Lys Lys Arg Lys Gly Asp Met Asp 100 105 110Glu Leu Glu Lys Glu Met Leu Ser Val Glu Lys Ala Arg Gln Glu Phe 115 120 125Glu Glu Arg Met Glu Glu Glu Ser Gln Ser Gln Gly Arg Asp Leu Thr 130 135 140Leu Glu Glu Asn Gln Val Lys Lys Tyr His Arg Leu Lys Glu Glu Ala145 150 155 160Ser Lys Arg Ala Ala Thr Leu Ala Gln Glu Leu Glu Lys Phe Asn Arg 165 170 175Asp Gln Lys Ala Asp Gln Asp Arg Leu Asp Leu Glu Glu Arg Lys Lys 180 185 190Val Glu Thr Glu Ala Lys Ile Lys Gln Lys Leu Arg Glu Ile Glu Glu 195 200 205Asn Gln Lys Arg Ile Glu Lys Leu Glu Glu Tyr Ile Thr Thr Ser 210 215 220 13 140 PRT Homo sapiens 13Met Glu Ala Asn Gly Val Asp Gly Asp Gly Leu Asn Lys Pro Ala Lys 1 5 10 15Lys Lys Lys Thr Pro Leu Lys Thr Val Lys Lys Ser Pro Lys Arg Ser 20 25 30Pro Leu Ser Asp Pro Pro Ser Gln Asp Pro Thr Pro Ala Ala Thr Pro 35 40 45Glu Thr Pro Pro Val Ile Ser Ala Val Val His Ala Thr Asp Glu Glu 50 55 60Lys Leu Ala Val Thr Asn Gln Lys Trp Thr Cys Met Thr Val Asp Leu 65 70 75 80Glu Ala Asp Lys Gln Asp Tyr Pro Gln Pro Ser Asp Leu Ser Thr Phe 85 90 95Val Asn Glu Thr Lys Phe Ser Ser Pro Thr Glu Glu Leu Asp Tyr Arg 100 105 110Asn Ser Tyr Glu Ile Glu Tyr Met Glu Lys Ile Gly Ser Ser Leu Pro 115 120 125Gln Asp Asp Asp Ala Pro Lys Lys Gln Ala Leu Tyr 130 135 140 14 111 PRT Homo sapiens 14Met Phe Asp Thr Ser Gln Glu Ser Pro Val Lys Ser Ser Pro Val Arg 1 5 10 15Met Ser Glu Ser Pro Thr Pro Cys Ser Gly Ser Ser Phe Glu Glu Thr 20 25 30Glu Ala Leu Val Asn Thr Ala Ala Lys Asn Gln His Pro Val Pro Arg 35 40 45Gly Leu Ala Pro Asn Gln Glu Ser His Leu Gln Val Pro Glu Lys Ser 50 55 60Ser Gln Lys Glu Leu Glu Ala Met Gly Leu Gly Thr Pro Ser Glu Ala 65 70 75 80Ile Glu Ile Arg Glu Ala Ala His Pro Thr Asp Val Ser Ile Ser Lys 85 90 95Thr Ala Leu Tyr Ser Arg Ile Gly Thr Ala Glu Val Glu Lys Pro 100 105 110 15 87 PRT Homo sapiens 15Lys Ser Ala Gly Leu Glu Gln Pro Thr Asp Pro Val Ala Arg Asp Gly 1 5 10 15Pro Leu Ser Gln Thr Ser Ser Lys Pro Asp Pro Ser Gln Trp Glu Ser 20 25 30Pro Ser Phe Asn Pro Phe Gly Ser His Ser Val Leu Gln Asn Ser Pro 35 40 45Pro Leu Ser Ser Glu Gly Ser Tyr His Phe Asp Pro Asp Asn Phe Asp 50 55 60Glu Ser Met Asp Pro Phe Lys Pro Thr Thr Thr Leu Thr Ser Ser Asp 65 70 75 80Phe Cys Ser Pro Thr Gly Asn 85 16 91 PRT Homo sapiens 16Gly Ala Thr Ser Lys Arg Ala Pro Pro Pro Arg Arg Leu Gly Glu Arg 1 5 10 15Ser Gly Leu Lys Pro Pro Leu Arg Lys Ala Ala Val Arg Gln Gln Lys 20 25 30Ala Pro Gln Glu Val Glu Glu Asp Asp Gly Arg Ser Gly Ala Gly Glu 35 40 45Asp Pro Pro Met Pro Ala Ser Arg Gly Ser Tyr His Leu Asp Trp Asp 50 55 60Lys Met Asp Asp Pro Asn Phe Ile Pro Phe Gly Gly Asp Thr Lys Ser 65 70 75 80Gly Cys Ser Glu Ala Gln Pro Pro Glu Ser Pro 85 90 17 203 PRT Homo sapiens 17Leu Leu Gln Tyr Ser Gln Lys Asp Leu Asp Ala Val Val Lys Ala Thr 1 5 10 15Gln Glu Glu Asn Arg Glu Leu Arg Ser Arg Cys Glu Glu Leu His Gly 20 25 30Lys Asn Leu Glu Leu Gly Lys Ile Met Asp Arg Phe Glu Glu Val Val 35 40 45Tyr Gln Ala Met Glu Glu Val Gln Lys Gln Lys Glu Leu Ser Lys Ala 50 55 60Glu Ile Gln Lys Val Leu Lys Glu Lys Asp Gln Leu Thr Thr Asp Leu 65 70 75 80Asn Ser Met Glu Lys Ser Phe Ser Asp Leu Phe Lys Arg Phe Glu Lys 85 90 95Gln Lys Glu Val Ile Glu Gly Tyr Arg Lys Asn Glu Glu Ser Leu Lys 100 105 110Lys Cys Val Glu Asp Tyr Leu Ala Arg Ile Thr Gln Glu Gly Gln Arg 115 120 125Tyr Gln Ala Leu Lys Ala His Ala Glu Glu Lys Leu Gln Leu Ala Asn 130 135 140Glu Glu Ile Ala Gln Val Arg Ser Lys Ala Gln Ala Glu Ala Leu Ala145 150 155 160Leu Gln Ala Ser Leu Arg Lys Glu Gln Met Arg Ile Gln Ser Leu Glu 165 170 175Lys Thr Val Glu Gln Lys Thr Lys Glu Asn Glu Glu Leu Thr Arg Ile 180 185 190Cys Asp Asp Leu Ile Ser Lys Met Glu Lys Ile 195 200 18 7736 DNA Homo sapiens 18cagaggtcta gcagccgggc gccgcgggcc gggggcctga ggaggccaca ggacgggcgt 60cttcccggct agtggagccc ggcgcggggc ccgctgcggc cgcaccgtga ggggaggagg 120ccgaggagga cgcggcgccg gctgccggcg ggaggaagcg ctccaccagg gcccccgacg 180gcactcgttt aaccacatcc gcgcctctgc tggaaacgct tgctggcgcc tgtcaccggt 240tccctccatt ttgaaaggga aaaaggctct ccccacccat tcccctgccc ctaggagctg 300gagccggagg agccgcgctc atggcgttca gcccgtggca gatcctgtcc cccgtgcagt 360gggcgaaatg gacgtggtct gcggtacgcg gcggggccgc cggcgaggac gaggctggcg 420ggcccgaggg cgaccccgag gaggaggatt cgcaagccga gaccaaatcc ttgagtttca 480gctcggattc tgaaggtaat tttgagactc ctgaagctga aaccccgatc cgatcacctt 540tcaaggagtc ctgtgatcca tcactcggat tggcaggacc tggggccaaa agccaagaat 600cacaagaagc tgatgaacag cttgtagcag aagtggttga aaaatgttca tctaagactt 660gttctaaacc ttcagaaaat gaagtgccac agcaggccat tgactctcac tcagtcaaga 720atttcagaga agaacctgaa catgatttta gcaaaatttc catcgtgagg ccattttcaa 780tagaaacgaa ggattccacg gatatctcgg cagtcctcgg aacaaaagca gctcatggct 840gtgtaactgc agtctcaggc aaggctctgc cttccagccc gccagacgcc ctccaggacg 900aggcgatgac agaaggcagc atgggggtca ccctcgaggc ctccgcagaa gctgatctaa 960aagctggcaa ctcctgtcca gagcttgtgc ccagcagaag aagcaagctg agaaagccca 1020agcctgtccc cctgaggaag aaagcaattg gaggagagtt ctcagacacc aacgctgctg 1080tggagggcac acctctcccc aaggcatcct atcacttcag tcctgaagag ttggatgaga 1140acacaagtcc tttgctagga gatgccaggt tccagaagtc tccccctgac attaaagaaa 1200ctcccggcac tctcagtagt gacaccaacg actcaggggt tgagctgggg gaggagtcga 1260ggagctcacc tctcaagctt gagtttgatt tcacagaaga tacaggaaac atagaggcca 1320ggaaagccct tccaaggaag cttggcagga aactgggtag cacactgact cccaagatac 1380aaaaagatgg catcagtaag tcagcaggtt tagaacagcc tacagaccca gtggcacgag 1440acgggcctct ctcccaaaca tcttccaagc cagatcctag tcagtgggag agccccagct 1500tcaacccctt tgggagccac tctgttctgc agaactcccc acccctctct tctgagggct 1560cctaccactt tgacccagat aactttgacg aatccatgga tccctttaaa ccaactacga 1620ccttaacaag cagtgacttt tgttctccca ctggtaatca cgttaatgaa atcttagaat 1680cacccaagaa ggcaaagtcg cgtttaataa cgagtggctg taaggtgaag aagcatgaaa 1740ctcagtctct cgccctggat gcatgttctc gggatgaagg ggcagtgatc tcccagattt 1800cagacatttc taatagggat ggccatgcta ctgatgagga gaaactggca tccacgtcat 1860gtggtcagaa atcagctggt gccgaggtga aaggtgagcc agaggaagac ctggagtact 1920ttgaatgttc caatgttcct gtgtctacca taaatcatgc gttttcatcc tcagaagcag 1980gcatagagaa ggagacgtgc cagaagatgg aagaagacgg gtccactgtg cttgggctgc 2040tggagtcctc tgcagagaag gcccctgtgt cggtgtcctg tggaggtgag agccccctgg 2100atgggatctg cctcagcgaa tcagacaaga cagccgtgct caccttaata agagaagaga 2160taattactaa agagattgaa gcaaatgaat ggaagaagaa atacgaagag acccggcaag 2220aagttttgga gatgaggaaa attgtagctg aatatgaaaa gactattgct caaatgattg 2280aagatgaaca aaggacaagt atgacctctc agaagagctt ccagcaactg accatggaga 2340aggaacaggc cctggctgac cttaactctg tggaaaggtc cctttctgat ctcttcagga 2400gatatgagaa cctgaaaggt gttctggaag ggttcaagaa gaatgaagaa gccttgaaga 2460aatgtgctca ggattactta gccagagtta aacaagagga gcagcgatac caggccctga 2520aaatccacgc agaagagaaa ctggacaaag ccaatgaaga gattgctcag gttcgaacaa 2580aagcaaaggc tgagagtgca gctctccatg ctggactccg caaagagcag atgaaggtgg 2640agtccctgga aagggccctg cagcagaaga accaagaaat tgaagaactg acaaaaatct 2700gtgatgagct gattgcaaag ctgggaaaga ctgactgaga cactccccct gttagctcaa 2760cagatctgca tttggctgct tctcttgtga ccacaattat cttgccttat ccaggaataa 2820ttgccccttt gcagagaaaa aaaaaaaact taaaaaaagc acatgcctac tgctgcctgt 2880cccgctttgc tgccaatgca acagccctgg aagaaaccct agagggttgc atagtctaga 2940aaggagtgtg acctgacagt gctggagcct cctagtttcc ccctatgaag gttcccttag 3000gctgctgagt ttgggtttgt gatttatctt tagtttgttt taaagtcatc tttactttcc 3060caaatgtgtt aaatttgtaa ctcctctttg gggtcttctc caccacctgt ctgatttttt 3120tgtgatctgt ttaatctttt aattttttag tatcagtggt tttatttaag gagacagttt 3180ggcctattgt tacttccaat ttataatcaa gaaggggctc tggatcccct tttaaattac 3240acacactctc acacacatac atgtatgttt atagatgctg ctgctctttt ccctgaagca 3300tagtcaagta agaactgctc tacagaagga catatttcct tggatgtgag accctatttt 3360gaaatagagt cctgactcag aacaccaact taagaatttg ggggattaaa gatgtgaaga 3420ccacagtctt gggttttcat atctggagaa gactatttgc catgacgttt tgttgccctg 3480gtatttggac actcctcagc tttaatgggt gtggcccctt tagggttagt cctcagacta 3540atgatagtgt ctgctttctg catgaacggc aatatgggac tccctccaag ctagggtttg 3600gcaagtctgc cctagagtca tttactctcc tctgcctcca tttgttaata cagaatcaac 3660atttagtctt cattatcttt tttttttttt ttgagacaga gtttcgatct attttaagta 3720tgtgaagaaa atctacttgt aaaaggctca gatcttaatt aaaaggtaat tgtagcacat 3780taccaattat aaggtgaaga aatgtttttt cccaagtgtg atgcattgtt cttcagatgt 3840tgaaaagaaa gcaaaaaata ccttctaact taagacagaa tttttaacaa aatgagcagt 3900aaaagtcaca tgaaccactc caaaaatcag tgcattttgc atatttttaa acaaagacag 3960cttgttgaat actgagaaga ggagtgcaag gagaaggtct gtactaacaa agccaaattc 4020ctcaagctct tactggactc agttcagagt ggtgggccat taaccccaac atggaatttt 4080tccatataaa tctcaatgaa ttccctttca tttgaatagg caaacccaaa tccatgcaag 4140tgttttaaag cactgtcctg tcttaatctt acatgctgaa agtcttcatg gtgatatgca 4200ctatattcag tatacgtatg ttttcctact tctcttgtaa aactgttgca tgatccaact 4260tcagcaatga attgtgccta gtggagaacc tctatagatc ttaaaaaatg aattattctt 4320tagcagtgta ttactcacat gggtgcaatc tttagcccca gggaggtcaa taatgtcttt 4380taaagccaga agtcacattt taccaatatg catttatcat aattggtgct taggctgtat 4440attcaagcct gttgtcttaa cattttgtat aaaaaagaac aacagaaatt atctgtcatt 4500tgagaagtgg cttgacaatc atttgagctt tgaaagcagt cactgtggtg taatatgaat 4560gctgtcctag tggtcatagt accaagggca cgtgtctccc cttggtataa ctgatttcct 4620ttttagtcct ctactgctaa ataagttaat tttgcatttt gcagaaagaa acattgattg 4680ctaaatcttt ttgctgctgt gttttggtgt tttcatgttt acttgtttta tattgatctg 4740ttttaagtat gagaggctta tagtgccctc cattgtaaat ccatagtcat ctttttaagc 4800ttattgtgtt taagaaagta gctatgtgtt aaacagaggt gatggcagcc cttccctagc 4860acactggtgg aagagacccc ttaagaacct gaccccagtg aatgaagctg atgcacaggg 4920acgaccaaag gaccttcgtt aagtgataat tgtcctggcc tctcagccat gaccgttatg 4980aggaaatatc ccccattcga acttaacaga tgcctcctct ccaaagagaa ttaaaatcgt 5040agcttgtaca gatcaagaga atatactggg cagaatgaag tatgtttgtt tatttttctt 5100taaaaataaa ggattttgga actctggaga gtaagaatat agtatagagt ttgcctcaac 5160acatgtgagg gccaaataac ctgctagcta ggcagtaata aactctgtta cagaagagaa 5220aaagggccgg gcacagtggc ttattcctgt aatcccaaca ctgtggaagg ccgaggcagg 5280aggatcactt gagtccagga gtttgaaacc tacctaggca acatggtgaa accttgtctc 5340taccaaaata aaaattagct gggcatggtg gcacgtgcct gtggtcccag ctacttggga 5400ggctgaggtg ggagcctggg aggtcaaggc tgcagtgagc catgatcatg ccactgcact 5460ccatcctggg tgacagcaag atcttgtctc aaaaaaaaaa aaaaaaacca ggagtgaaaa 5520aggaaagtag aaggcagctg ctggcctaga tgttggtttg ggaatattag gtgatcctgt 5580tgagattctg gatccagagc aatttcttta gcttttgact ttgccaaagt gtagatagcc 5640tttatccagc agtattttaa gtggggaatg caacgtgagg ccaactgaac aattcccccc 5700gtggctgccc agatagtcac agtcaaggtt ggagagtctc cttccagcca gtgacctacc 5760caaacctttt gttctgtaaa actgctctgg aaataccggg aagcccagtt ttctcacgtg 5820gtttctagct tcttcagact cagcccaaat taggaagtgc agaagcacat gatggtgaaa 5880aacctaggat ttggcagcct tccagaatgg tatggaatct gagggaagat ttatgtttcg 5940ttttggagga tagctcaagt tgaattttct ttccagccag ttaccctttc aacctaccca 6000tactttgtac aactcttaca caaatactta gatatttatt agatagccct gaattcactc 6060taattataaa cagggagtgt aaactgcccc cagatgttcc tgggctgggt aaaagcagct 6120ggagtgaagc actcattttc cataaaggta acaaagggca gctcagtggt tactcaagct 6180caaaagggtt tttttaagag caagcattgg ttaagtctgt gtatactgag ttggaagtga 6240tttcagcaca ttctttttta gtgggagtga aagttctgaa gccccctttt aacttcctct 6300tggtttttca ttataattgg tagccatctc atgaactgtc tctgactgtt gtctctttgt 6360ggtcatgtga ttgtgagctt gctttctgac ttgcatttct gactttatcc tgttgttagg 6420aagatagaaa ctaggttttg aaagattaca tgattcaagc gagggatttt aaagtaaaga 6480tgtatttatt ctgaagaatc taaaagataa cagattattt gcttatgaaa gaacaatata 6540gtctgggaat cccagaatgt caagccaaag gtctaagaag tcatctcctt caaatacttt 6600aataaagaag tatttcgagg agatatctgt ccaaaaaggt ttgactggcc tccagattcc 6660agttattttt aaaaagcaac ttaccactaa atccttgagt ctccatagag taacagtaaa 6720gaaactgatg taacagactc tcctctcaaa ggatctcctc tggaagagac tatcagcggc 6780agatctctcc agggaagacc catcccctag tgccagagct tgcatcctgg agactaaaga 6840ttgcactttt ttgtagtttt ttgtccaaat gcaatcccat ttctgtgcct cttagcatgc 6900agttagattt ggacaaacaa gattcctaag gaatgacttt attaactata atatggttac 6960agctattata taaatatata ttctggttat agttctaata tggagatgtt gtgtgcaatg 7020ctggcctgtg gtggtctgtg taatgcttta acttgtatgg aggaggccag gctcagagct 7080gagatgtggc ctgaaccttc cctgtatcga tcctttaatt tagaactgtc aagatgtcac 7140tttctccccc tctgcctttt agtggtatct gacatatact caaaacagta atttcctggt 7200cacatcatta actgctaatt ctgtatttat aaagaatttt cagatggaca tgtacaaatt 7260tgaactcaaa ccatccccag tccagataca gggcagcgtg taggtgacca caccagagcc 7320tcagcctcgg tccttctcag ccgtcgggat aggatccagg catttctttt aaatctcaga 7380ggtagcagta aacttttcag tattgctgtt agcaagtgtg tgtttgccaa tagataccca 7440ttatactaat gtgccaagta aatgttcatt gcacatctgc ttccactgtg ttcccacggg 7500tgccatgaag tgtgtgagga gcccctcatc tggagggatg agtgctgcgt tgactactgc 7560tatcaggatt gtgttgtgtg gaatattcat ctacataaat tttatatgca cagtaatttc 7620cctttttata tgtcaagtaa ctatttgtaa aagttatact cacaaattat tataatgatt 7680actaatatat tttttccatg tttcattgcc tgaataaaaa ctgtttacca ctgtta 7736 19 805 PRT Homo sapiens 19Met Ala Phe Ser Pro Trp Gln Ile Leu Ser Pro Val Gln Trp Ala Lys 1 5 10 15Trp Thr Trp Ser Ala Val Arg Gly Gly Ala Ala Gly Glu Asp Glu Ala 20 25 30Gly Gly Pro Glu Gly Asp Pro Glu Glu Glu Asp Ser Gln Ala Glu Thr 35 40 45Lys Ser Leu Ser Phe Ser Ser Asp Ser Glu Gly Asn Phe Glu Thr Pro 50 55 60Glu Ala Glu Thr Pro Ile Arg Ser Pro Phe Lys Glu Ser Cys Asp Pro 65 70 75 80Ser Leu Gly Leu Ala Gly Pro Gly Ala Lys Ser Gln Glu Ser Gln Glu 85 90 95Ala Asp Glu Gln Leu Val Ala Glu Val Val Glu Lys Cys Ser Ser Lys 100 105 110Thr Cys Ser Lys Pro Ser Glu Asn Glu Val Pro Gln Gln Ala Ile Asp 115 120 125Ser His Ser Val Lys Asn Phe Arg Glu Glu Pro Glu His Asp Phe Ser 130 135 140Lys Ile Ser Ile Val Arg Pro Phe Ser Ile Glu Thr Lys Asp Ser Thr145 150 155 160Asp Ile Ser Ala Val Leu Gly Thr Lys Ala Ala His Gly Cys Val Thr 165 170 175Ala Val Ser Gly Lys Ala Leu Pro Ser Ser Pro Pro Asp Ala Leu Gln 180 185 190Asp Glu Ala Met Thr Glu Gly Ser Met Gly Val Thr Leu Glu Ala Ser 195 200 205Ala Glu Ala Asp Leu Lys Ala Gly Asn Ser Cys Pro Glu Leu Val Pro 210 215 220Ser Arg Arg Ser Lys Leu Arg Lys Pro Lys Pro Val Pro Leu Arg Lys225 230 235 240Lys Ala Ile Gly Gly Glu Phe Ser Asp Thr Asn Ala Ala Val Glu Gly 245 250 255Thr Pro Leu Pro Lys Ala Ser Tyr His Phe Ser Pro Glu Glu Leu Asp 260 265 270Glu Asn Thr Ser Pro Leu Leu Gly Asp Ala Arg Phe Gln Lys Ser Pro 275 280 285Pro Asp Ile Lys Glu Thr Pro Gly Thr Leu Ser Ser Asp Thr Asn Asp 290 295 300Ser Gly Val Glu Leu Gly Glu Glu Ser Arg Ser Ser Pro Leu Lys Leu305 310 315 320Glu Phe Asp Phe Thr Glu Asp Thr Gly Asn Ile Glu Ala Arg Lys Ala 325 330 335Leu Pro Arg Lys Leu Gly Arg Lys Leu Gly Ser Thr Leu Thr Pro Lys 340 345 350Ile Gln Lys Asp Gly Ile Ser Lys Ser Ala Gly Leu Glu Gln Pro Thr 355 360 365Asp Pro Val Ala Arg Asp Gly Pro Leu Ser Gln Thr Ser Ser Lys Pro 370 375 380Asp Pro Ser Gln Trp Glu Ser Pro Ser Phe Asn Pro Phe Gly Ser His385 390 395 400Ser Val Leu Gln Asn Ser Pro Pro Leu Ser Ser Glu Gly Ser Tyr His 405 410 415Phe Asp Pro Asp Asn Phe Asp Glu Ser Met Asp Pro Phe Lys Pro Thr 420 425 430Thr Thr Leu Thr Ser Ser Asp Phe Cys Ser Pro Thr Gly Asn His Val 435 440 445Asn Glu Ile Leu Glu Ser Pro Lys Lys Ala Lys Ser Arg Leu Ile Thr 450 455 460Ser Gly Cys Lys Val Lys Lys His Glu Thr Gln Ser Leu Ala Leu Asp465 470 475 480Ala Cys Ser Arg Asp Glu Gly Ala Val Ile Ser Gln Ile Ser Asp Ile 485 490 495Ser Asn Arg Asp Gly His Ala Thr Asp Glu Glu Lys Leu Ala Ser Thr 500 505 510Ser Cys Gly Gln Lys Ser Ala Gly Ala Glu Val Lys Gly Glu Pro Glu 515 520 525Glu Asp Leu Glu Tyr Phe Glu Cys Ser Asn Val Pro Val Ser Thr Ile 530 535 540Asn His Ala Phe Ser Ser Ser Glu Ala Gly Ile Glu Lys Glu Thr Cys545 550 555 560Gln Lys Met Glu Glu Asp Gly Ser Thr Val Leu Gly Leu Leu Glu Ser 565 570 575Ser Ala Glu Lys Ala Pro Val Ser Val Ser Cys Gly Gly Glu Ser Pro 580 585 590Leu Asp Gly Ile Cys Leu Ser Glu Ser Asp Lys Thr Ala Val Leu Thr 595 600 605Leu Ile Arg Glu Glu Ile Ile Thr Lys Glu Ile Glu Ala Asn Glu Trp 610 615 620Lys Lys Lys Tyr Glu Glu Thr Arg Gln Glu Val Leu Glu Met Arg Lys625 630 635 640Ile Val Ala Glu Tyr Glu Lys Thr Ile Ala Gln Met Ile Glu Asp Glu 645 650 655Gln Arg Thr Ser Met Thr Ser Gln Lys Ser Phe Gln Gln Leu Thr Met 660 665 670Glu Lys Glu Gln Ala Leu Ala Asp Leu Asn Ser Val Glu Arg Ser Leu 675 680 685Ser Asp Leu Phe Arg Arg Tyr Glu Asn Leu Lys Gly Val Leu Glu Gly 690 695 700Phe Lys Lys Asn Glu Glu Ala Leu Lys Lys Cys Ala Gln Asp Tyr Leu705 710 715 720Ala Arg Val Lys Gln Glu Glu Gln Arg Tyr Gln Ala Leu Lys Ile His 725 730 735Ala Glu Glu Lys Leu Asp Lys Ala Asn Glu Glu Ile Ala Gln Val Arg 740 745 750Thr Lys Ala Lys Ala Glu Ser Ala Ala Leu His Ala Gly Leu Arg Lys 755 760 765Glu Gln Met Lys Val Glu Ser Leu Glu Arg Ala Leu Gln Gln Lys Asn 770 775 780Gln Glu Ile Glu Glu Leu Thr Lys Ile Cys Asp Glu Leu Ile Ala Lys785 790 795 800Leu Gly Lys Thr Asp 805 20 2781 DNA Homo sapiens 20ggcggcggta gcagccaggc ttggcccccg gcgtggagca gacgcggacc cctccttcct 60ggcggcggcg gcgcgggctc agagcccggc aacgggcggg cgggcagaat gagtctgcag 120gtcttaaacg acaaaaatgt cagcaatgaa aaaaatacag aaaattgcga cttcctgttt 180tcgccaccag aagttaccgg aagatcgtct gttcttcgtg tgtcacagaa agaaaatgtg 240ccacccaaga acctggccaa agctatgaag gtgacttttc agacacctct gcgggatcca 300cagacgcaca ggattctaag tcctagcatg gccagcaaac ttgaggctcc tttcactcag 360gatgacaccc ttggactgga aaactcacac ccggtctgga cacagaaaga gaaccaacag 420ctcatcaagg aagtggatgc caaaactact catggaattc tacagaaacc agtggaggct 480gacaccgacc tcctggggga tgcaagccca gcctttggga gtggcagctc cagcgagtct 540ggcccaggtg ccctggctga cctggactgc tcaagctctt cccagagccc aggaagttct 600gagaaccaaa tggtgtctcc aggaaaagtg tctggcagcc ctgagcaagc cgtggaggaa 660aaccttagtt cctattcctt agacagaaga gtgacacccg cctctgagac cctagaagac 720ccttgcagga cagagtccca gcacaaagcg gagactccgc acggagccga ggaagaatgc 780aaagcggaga ctccgcacgg agccgaggag gaatgccggc acggtggggt ctgtgctccc 840gcagcagtgg ccacttcgcc tcctggtgca atccctaagg aagcctgcgg aggagcaccc 900ctgcagggtc tgcctggcga agccctgggc tgccctgcgg gtgtgggcac ccccgtgcca 960gcagatggca ctcagaccct tacctgtgca cacacctctg ctcctgagag cacagcccca 1020accaaccacc tggtggctgg cagggccatg accctgagtc ctcaggaaga agtggctgca 1080ggccaaatgg ccagctcctc gaggagcgga cctgtaaaac tagaatttga tgtatctgat 1140ggcgccacca gcaaaagggc acccccacca aggagactgg gagagaggtc cggcctcaag 1200cctcccttga ggaaagcagc agtgaggcag caaaaggccc cgcaggaggt ggaggaggac 1260gacggtagga gcggagcagg agaggacccc cccatgccag cttctcgggg ctcttaccac 1320ctcgactggg acaaaatgga tgacccaaac ttcatcccgt tcggaggtga caccaagtct 1380ggttgcagtg aggcccagcc cccagaaagc cctgagacca ggctgggcca gccagcggct 1440gaacagttgc atgctgggcc tgccacggag gagccaggtc cctgtctgag ccagcagctg 1500cattcagcct cagcggagga cacgcctgtg gtgcagttgg cagccgagac cccaacagca 1560gagagcaagg agagagcctt gaactctgcc agcacctcgc ttcccacaag ctgtccaggc 1620agtgagccag tgcccaccca tcagcagggg cagcctgcct tggagctgaa agaggagagc 1680ttcagagacc ccgctgaggt tctaggcacg ggcgcggagg tggattacct ggagcagttt 1740ggaacttcct cgtttaagga gtcggccttg aggaagcagt ccttatacct caagttcgac 1800cccctcctga gggacagtcc tggtagacca gtgcccgtgg ccaccgagac cagcagcatg 1860cacggtgcaa atgagactcc ctcaggacgt ccgcgggaag ccaagcttgt ggagttcgat 1920ttcttgggag cactggacat tcctgtgcca ggcccacccc caggtgttcc cgcgcctggg 1980ggcccacccc tgtccaccgg acctatagtg gacctgctcc agtacagcca gaaggacctg 2040gatgcagtgg taaaggcgac acaggaggag aaccgggagc tgaggagcag gtgtgaggag 2100ctccacggga agaacctgga actggggaag atcatggaca ggttcgaaga ggttgtgtac 2160caggccatgg aggaagttca gaagcagaag gaactttcca aagctgaaat ccagaaagtt 2220ctaaaagaaa aagaccaact taccacagat ctgaactcca tggagaagtc cttctccgac 2280ctcttcaagc gttttgagaa acagaaagag gtgatcgagg gctaccgcaa gaacgaagag 2340tcactgaaga agtgcgtgga ggattacctg gcaaggatca cccaggaggg ccagaggtac 2400caagccctga aggcccacgc ggaggagaag ctgcagctgg caaacgagga gatcgcccag 2460gtccggagca aggcccaggc ggaagcgttg gccctccagg ccagcctgag gaaggagcag 2520atgcgcatcc agtcgctgga gaagacagtg gagcagaaga ctaaagagaa cgaggagctg 2580accaggatct gcgacgacct catctccaag atggagaaga tctgacctcc acggagccgc 2640tgtccccgcc cccctgctcc cgtctgtctg tcctgtctga ttctcttagg tgtcatgttc 2700ttttttctgt cttgtcttca acttttttta aaactagatt gctttgaaaa catgactcaa 2760taaaagtttc ctttcaattt a 2781 21 838 PRT Homo sapiens 21Met Ser Leu Gln Val Leu Asn Asp Lys Asn Val Ser Asn Glu Lys Asn 1 5 10 15Thr Glu Asn Cys Asp Phe Leu Phe Ser Pro Pro Glu Val Thr Gly Arg 20 25 30Ser Ser Val Leu Arg Val Ser Gln Lys Glu Asn Val Pro Pro Lys Asn 35 40 45Leu Ala Lys Ala Met Lys Val Thr Phe Gln Thr Pro Leu Arg Asp Pro 50 55 60Gln Thr His Arg Ile Leu Ser Pro Ser Met Ala Ser Lys Leu Glu Ala 65 70 75 80Pro Phe Thr Gln Asp Asp Thr Leu Gly Leu Glu Asn Ser His Pro Val 85 90 95Trp Thr Gln Lys Glu Asn Gln Gln Leu Ile Lys Glu Val Asp Ala Lys 100 105 110Thr Thr His Gly Ile Leu Gln Lys Pro Val Glu Ala Asp Thr Asp Leu 115 120 125Leu Gly Asp Ala Ser Pro Ala Phe Gly Ser Gly Ser Ser Ser Glu Ser 130 135 140Gly Pro Gly Ala Leu Ala Asp Leu Asp Cys Ser Ser Ser Ser Gln Ser145 150 155 160Pro Gly Ser Ser Glu Asn Gln Met Val Ser Pro Gly Lys Val Ser Gly 165 170 175Ser Pro Glu Gln Ala Val Glu Glu Asn Leu Ser Ser Tyr Ser Leu Asp 180 185 190Arg Arg Val Thr Pro Ala Ser Glu Thr Leu Glu Asp Pro Cys Arg Thr 195 200 205Glu Ser Gln His Lys Ala Glu Thr Pro His Gly Ala Glu Glu Glu Cys 210 215 220Lys Ala Glu Thr Pro His Gly Ala Glu Glu Glu Cys Arg His Gly Gly225 230 235 240Val Cys Ala Pro Ala Ala Val Ala Thr Ser Pro Pro Gly Ala Ile Pro 245 250 255Lys Glu Ala Cys Gly Gly Ala Pro Leu Gln Gly Leu Pro Gly Glu Ala 260 265 270Leu Gly Cys Pro Ala Gly Val Gly Thr Pro Val Pro Ala Asp Gly Thr 275 280 285Gln Thr Leu Thr Cys Ala His Thr Ser Ala Pro Glu Ser Thr Ala Pro 290 295 300Thr Asn His Leu Val Ala Gly Arg Ala Met Thr Leu Ser Pro Gln Glu305 310 315 320Glu Val Ala Ala Gly Gln Met Ala Ser Ser Ser Arg Ser Gly Pro Val 325 330 335Lys Leu Glu Phe Asp Val Ser Asp Gly Ala Thr Ser Lys Arg Ala Pro 340 345 350Pro Pro Arg Arg Leu Gly Glu Arg Ser Gly Leu Lys Pro Pro Leu Arg 355 360 365Lys Ala Ala Val Arg Gln Gln Lys Ala Pro Gln Glu Val Glu Glu Asp 370 375 380Asp Gly Arg Ser Gly Ala Gly Glu Asp Pro Pro Met Pro Ala Ser Arg385 390 395 400Gly Ser Tyr His Leu Asp Trp Asp Lys Met Asp Asp Pro Asn Phe Ile 405 410 415Pro Phe Gly Gly Asp Thr Lys Ser Gly Cys Ser Glu Ala Gln Pro Pro 420 425 430Glu Ser Pro Glu Thr Arg Leu Gly Gln Pro Ala Ala Glu Gln Leu His 435 440 445Ala Gly Pro Ala Thr Glu Glu Pro Gly Pro Cys Leu Ser Gln Gln Leu 450 455 460His Ser Ala Ser Ala Glu Asp Thr Pro Val Val Gln Leu Ala Ala Glu465 470 475 480Thr Pro Thr Ala Glu Ser Lys Glu Arg Ala Leu Asn Ser Ala Ser Thr 485 490 495Ser Leu Pro Thr Ser Cys Pro Gly Ser Glu Pro Val Pro Thr His Gln 500 505 510Gln Gly Gln Pro Ala Leu Glu Leu Lys Glu Glu Ser Phe Arg Asp Pro 515 520 525Ala Glu Val Leu Gly Thr Gly Ala Glu Val Asp Tyr Leu Glu Gln Phe 530 535 540Gly Thr Ser Ser Phe Lys Glu Ser Ala Leu Arg Lys Gln Ser Leu Tyr545 550 555 560Leu Lys Phe Asp Pro Leu Leu Arg Asp Ser Pro Gly Arg Pro Val Pro 565 570 575Val Ala Thr Glu Thr Ser Ser Met His Gly Ala Asn Glu Thr Pro Ser 580 585 590Gly Arg Pro Arg Glu Ala Lys Leu Val Glu Phe Asp Phe Leu Gly Ala 595 600 605Leu Asp Ile Pro Val Pro Gly Pro Pro Pro Gly Val Pro Ala Pro Gly 610 615 620Gly Pro Pro Leu Ser Thr Gly Pro Ile Val Asp Leu Leu Gln Tyr Ser625 630 635 640Gln Lys Asp Leu Asp Ala Val Val Lys Ala Thr Gln Glu Glu Asn Arg 645 650 655Glu Leu Arg Ser Arg Cys Glu Glu Leu His Gly Lys Asn Leu Glu Leu 660 665 670Gly Lys Ile Met Asp Arg Phe Glu Glu Val Val Tyr Gln Ala Met Glu 675 680 685Glu Val Gln Lys Gln Lys Glu Leu Ser Lys Ala Glu Ile Gln Lys Val 690 695 700Leu Lys Glu Lys Asp Gln Leu Thr Thr Asp Leu Asn Ser Met Glu Lys705 710 715 720Ser Phe Ser Asp Leu Phe Lys Arg Phe Glu Lys Gln Lys Glu Val Ile 725 730 735Glu Gly Tyr Arg Lys Asn Glu Glu Ser Leu Lys Lys Cys Val Glu Asp 740 745 750Tyr Leu Ala Arg Ile Thr Gln Glu Gly Gln Arg Tyr Gln Ala Leu Lys 755 760 765Ala His Ala Glu Glu Lys Leu Gln Leu Ala Asn Glu Glu Ile Ala Gln 770 775 780Val Arg Ser Lys Ala Gln Ala Glu Ala Leu Ala Leu Gln Ala Ser Leu785 790 795 800Arg Lys Glu Gln Met Arg Ile Gln Ser Leu Glu Lys Thr Val Glu Gln 805 810 815Lys Thr Lys Glu Asn Glu Glu Leu Thr Arg Ile Cys Asp Asp Leu Ile 820 825 830Ser Lys Met Glu Lys Ile 835 22 32 DNA Artificial Sequence Description of Artificial Sequence primer 22ctgaattcat ggacctggac tctgccctcc ag 32 23 32 DNA Artificial Sequence Description of Artificial Sequence primer 23gcctcgagtt agggctgctg gaacagaagg cc 32 24 25 DNA Artificial Sequence Description of Artificial Sequence primer 24cgtatgcact actgtatttc ctttc 25 25 25 DNA Artificial Sequence Description of Artificial Sequence primer 25gggcaagggc caaggtccag caatg 25 26 15 PRT Homo sapiens 26Lys Pro Ala Lys Lys Lys Lys Thr Pro Leu Lys Thr Val Lys Lys 1 5 10 15 27 20 PRT Homo sapiens 27Met Pro Leu Arg Arg Pro Lys Met Lys Lys Thr Pro Glu Lys Leu Asp 1 5 10 15Asn Thr Pro Ala 20 28 15 DNA Artificial Sequence Description of Artificial Sequence primer 28aagttttttt tttta 15 29 13 DNA Artificial Sequence Description of Artificial Sequence primer 29aagcttgatt gcc 13

Claims

1. A method for diagnosis of breast cell malignancy comprising steps of:(a) detecting, in a patient's tissue, a degree of expression of AZU-1 gene or a level of a protein encoded therein; and (b) correlating the degree of expression of AZU-1 gene with breast cells malignancy wherein the high expression of AZU-1 gene and high level of the protein encoded therein is correlated with nonmalignancy and the low or nonexistent AZU-1 gene expression and a low or nonexistent level of the protein encoded by AZU-1 gene is correlated with malignancy; (c) wherein the detection of the degree of expression of AZU-1 gene comprises determination of the presence and quantity of a protein depicted by SEQ ID NO: 3.

2. The method of claim 1 wherein a diagnostic detection according to claim 1 is repeated every month to determine malignancy progression.

3. The method of claim 1 wherein the degree of expression of AZU-1 gene is detected by the determination of a presence of AZU-1 DNA sequence depicted by SEQ ID NO: 1.

4. The method of claim 3 wherein the presence of DNA sequence depicted by SEQ ID NO: 1 is determined by in situ hybridization or by reverse transcription polymerase chain reaction (RT-PCR).

5. The method of claim 4 wherein the presence of DNA sequence depicted by SEQ ID NO: 1 is determined by RT-PCR using gene specific primers depicted by the SEQ ID NO: 28 and SEQ ID NO: 29.

6. The method of claim 4 wherein the presence of DNA sequence depicted by SEQ ID NO: 1 is determined by in situ hybridization of AZU-1 RNA using a complimentary DNA probe.

7. The method of claim 2 wherein the detected protein is the protein depicted by SEQ ID NO: 3 encoded by the nucleotide sequence depicted by the sequence SEQ ID NO: 1.

8. The method of claim 2 wherein the presence and quantity of the protein depicted by SEQ ID NO: 3 is detected with a polyclonal or monoclonal anti-AZU-1 antibodies.

9. The method of claim 8 wherein the antibodies are polyclonal.

10. The method of claim 9 wherein the polyclonal anti-AZU-1 antibodies are raised against a protein depicted by SEQ ID NO: 26 or SEQ ID NO: 27.

11. The method of claim 8 wherein the antibodies are monoclonal.

12. The method of claim 11 wherein the monoclonal anti-AZU-1 antibodies are raised against a purified His-tagged full length AZ-1 fusion protein.

13. The method of claim 8 wherein said protein is detected in breast biopsies by contacting a breast tissue with the anti AZU-1 antibody and detecting the presence or absence of the protein by immunostaining.