Patent Grant
7205109
This invention relates to the human tumor related gene HC56 in the region of sub-band 3 of band 3 of zone 1 of human chromosome 17 (17p13.3) and its uses. In particular, it relates to the method and kit for detecting tumor susceptibility based on the single nucleotide polymorphism or SNP of HC56.
The mortality rate of malignant tumor is just lower than that of cardio-and cerebro-vascular disease in China. The mortality rate of hepatocarcinoma is the third in common tumors, just below lung cancer and gastric cancer. The diagnostic and therapeutic means are lacking for hepatocarcinoma, except for microhepatocarcinoma. The development of carcinoma is a complex process involving multiple genes and steps and effected by the activation of many oncogenes and inactivation of anti-oncogenes. The anti-oncogenes are much important. Therefore, to find anti-oncogenes is one of the focuses of the current studies. The loss of heterozygosity (LOH) of a gene may result in the loss of normal characters. Anti-oncogenes may exist in the site having high frequency of LOH.
The key state laboratory in Shanghai Cancer Institute has studied human hepatocarcinoma gene for long time. In 1991, it was found that the development of hepatocarcinoma was related to the inactivation of p53, which had codon mutations not only on position 249 but also on other positions. (Li D. Z; Gu JR et al. Carcinogenesis 14 (2): 169, 1993). P53 is located in chromosome 17p13.1.
The researches on 17p13.3 have been reported overseas. Nishida et al. selected 10 polymorphism probes on 17p and studied the relationship between the LOH on 17p of human hepatocarcinoma and p53 mutation (Noashi Nishida et al., Cancer Research 53: 368–372, 1993). He deemed that the development of hepatocarcinoma might be related to unknown anti-oncogenes on 17p13.3. Schultz et al. (David C. Schultz, et al., Cancer Research 56: 1997–2002, 1996) proved that two new genes, OVCA1 and OVCA2, were located on 17p13.3 by allele deletion mapping and site-directed cloning. These two genes were expressed in normal epithelial cells of ovary, but were low-expressed or not expressed in oophoroma or ovary cancer cell lines. Wales et al. (Michele M. Wales et al., Nature Medicine: 570–577, 1995) discovered a candidate gene Hic-1 on 17p13.3 by analyzing the DNA methylation degree in tumor tissues, which was a transcription factor having a zinc-finger structure and was expressed widely in normal tissues but low-expressed in tumor cells.
However, the previous reports did not indicate the minimum range of LOH in hepatocarcinoma tissue and the corresponding sites. Further, the relationship between the above candidate genes and hepatocarcinoma was not reported.
Since cancer is one of the main diseases harmful to human health, people are concerned about the early diagnosis and gene therapy of cancer so as to effectively cure and prevent tumors, such as hepatocarcinoma. Therefore, there is a keen need in the art to develop new tumor-related and/or tumor-inhibiting human proteins and the corresponding detection kits.
One purpose of the invention is to provide a novel hepatoma related protein, named HC56 protein, and its fragments, analogs and derivatives.
Another purpose of the invention is to provide a polynucleotide encoding the polypeptides.
Still another purpose of the invention is to provide a method for HC56-based method and detection kit for detecting tumor susceptibility.
In the first aspect, the invention provides an isolated normal human HC56 polypeptide, which comprises the amino acid sequence of SEQ ID NO: 4. Further, it provides the amino acid mutation caused by SNPs when compared with the normal human HC56. One example of the mutated HC56 amino acid sequence is shown in SEQ ID NO: 2.
In the second aspect, the invention provides an isolated polynucleotide encoding the normal HC56 polypeptide, e.g., SEQ ID NO: 3. It further provides the SNPs in human HC56 gene. One HC56 sequence having SNPs is shown in SEQ ID NO: 1.
In the third aspect, the invention provides a vector comprising the above polynucleotide and a host cell transformed with the vector or a host cell transformed with the polynucleotide.
In the fourth aspect, the invention provides a method for producing a polypeptide having the activity of HC56 protein, which comprises (a) culturing the above transformed host cell under the conditions suitable for the expression of protein; (b) isolating the polypeptides having the activity of HC56 protein from the culture.
In the fifth aspect, the invention provides nucleic acid molecules for detection, which consist of 20–4750 consecutive nucleotides of SEQ ID NO: 3 and having mutations selected from the group consisting of:
Alternatively, the nucleic acid molecule has sequence of SEQ ID NO: 1. These nucleic acid molecules can be used as probes, primers or antisense fragments.
In the sixth aspect, the invention provides a method for detecting the carcinomatous change or cancer susceptibility of hepatocytes, comprising the steps of: detecting whether there is any change of HC56 gene, transcript and/or protein in the hepatocyte of the subject when compared with the normal HC56 gene, transcript and/or protein, and the change indicating that the possibility of suffering cancer in the subject is higher than that in the normal population. Preferably, the change is nucleotide deletion, insertion or substitution.
In one preferred embodiment, the HC56 gene or transcript are detected and compared with the normal HC56 nucleotide sequence and the change is selected from the group consisting of:
More preferably, the change is selected from the group consisting of:
In one preferred embodiment, the HC56 protein is detected and compared with the normal HC56 amino acid sequence and the change is selected from the group consisting of:
More preferably, the change is selected from the group consisting of:
In the seventh aspect, the invention provides a kit for detecting tumor (e.g., hepatocarcinoma) susceptibility, which comprises primers which specifically amplify human HC56 gene or transcript to produce an amplification product having a SNP selected from the group consisting of:
Preferably, the kit further comprises the probes specifically binding to the SNP site and/or a restriction enzyme specifically recognizing the SNP site wherein the nucleotide change in the SNP site results in the appearance or disappearance of the cleavage site of restriction enzyme. More preferably, the restriction enzyme is selected from SecI and EarI.
Preferably, the change is selected from the group consisting of:
The other aspects of the invention will be apparent to the skilled in the art in light of the technical disclosure of the invention.
In the hepatocarcinoma study, the inventors first found that there was high frequency of LOH (60–100%) of 17p13.3 in hepatocarcinoma (HC) tissue. Recently, the full genomic scanning of HC also proved that 17p13.3 was the region having the highest frequency. The inventors isolated and cloned the tumor related ESTs or expressed sequence tags in region 17p13.3 by screening the normal liver cDNA library using PAC P579 clone corresponding to site 926 in region 17p13.3. The cDNA clone was obtained. The full-length nucleotide sequence, named HC56, and the encoded amino acid sequence were obtained by RACE. The Northern blotting, RT-PCR and Southern blotting results proved that HC56 was related to tumor. The in vitro experiment proved that HC56 inhibited the growth of liver cancer cell line 7721. The study also indicated that there were several SNPs in the exon regions of HC56. Therefore, HC56 gene can be used in the diagnosis, treatment and prevention of tumor.
HC56 protein has various uses including, but not to be limited to: curing disorders (e.g., tumor) caused by low or no activity of HC56 protein, and screening out antibodies, polypeptides or ligands as agonists of HC56. The expressed recombinant HC56 protein can be used to screen polypeptide library to find out therapeutically valuable polypeptide molecules that activate HC56 protein.
In another aspect, the invention also provides the polyclonal and monoclonal antibodies, especially the monoclonal antibody, which are specifically against the polypeptide encoded by human HC56 DNA or fragments thereof. By “specificity”, it is meant an antibody that binds to the HC56 gene products or fragments thereof. Preferably, the antibody specifically binds to the protein having an amino acid sequence of SEQ ID NO: 2 and does not bind to the protein having an amino acid sequence of SEQ ID NO: 4.
The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically-active antibody fragments, e.g., a Fab′ or (Fab)2 fragment, an antibody light chain, an antibody heavy chain, a genetically engineered single chain Fv molecule, or a chimeric antibody.
The antibodies in the present invention can be prepared by various techniques known to those skilled in the art. For example, purified HC56 gene products, or its antigenic fragments can be administrated to animals to induce the production of polyclonal antibodies. Similarly, cells expressing HC56 or its antigenic fragments can be used to immunize animals to produce antibodies. Antibodies of the invention can be monoclonal antibodies that can be prepared by using hybridoma technique. The polyclonal antibodies can be prepared by immunizing animals, such as rabbit, mouse, and rat, with HC56 protein. Various adjuvants, e.g., Freund's adjuvant, can be used to enhance the immunization. The antibody against HC56 protein can be used in immunohistochemical method to detect the presence of HC56 protein in the biopsy specimen.
The invention further provides diagnostic assays for quantitative and in situ measurement of HC56 protein level. These assays are well known in the art and include FISH assay and radioimmunoassay. The level of HC56 protein detected in the assay can be used to illustrate the importance of HC56 protein in diseases and to determine the diseases associated with HC56 protein.
A method of detecting the presence of HC56 protein in a sample by utilizing the antibody specifically against HC56 protein comprises the steps of: contacting the sample with the antibody specifically against HC56 protein; observing the formation of antibody complex, which indicates the presence of HC56 protein in a sample.
The polynucleotide encoding HC56 protein can be used in the diagnosis of HC56 protein related diseases. The polynucleotide encoding HC56 can be used to detect whether HC56 is expressed or not, and whether the expression of HC56 is normal or abnormal, e.g., in the case of diseases. HC56 DNA sequences can be used in the hybridization with biopsy samples to determine the expression of HC56. The hybridization methods include Southern blotting, Northern blotting and in situ blotting, etc., which are public and sophisticated techniques. The corresponding kits are commercially available. A part of or all of the polynucleotides of the invention can be used as probe and fixed on a microarray or DNA chip for analysis the differential expression of genes in tissues and for the diagnosis of genes. The HC56 specific primers can be used in RNA-polymerase chain reaction and in vitro amplification to detect the transcripts of HC56.
Further, detection of the mutation of HC56 gene is useful for the diagnosis of HC56 protein related diseases. The mutation forms of HC56 include site mutation, translocation, deletion, rearrangement and any other mutations compared with the wild-type HC56 DNA sequence. The conventional methods, such as Southern blotting, DNA sequencing, PCR and in situ blotting, can be used to detect mutation. A preferred method for detecting the mutation of HC56 nucleic acid is to detect the presence of SNPs. Moreover, mutation sometimes affects the expression of protein. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether the gene is mutated or not.
The invention also provides a kit for detecting the presence of SNPs of HC56 in the analyte, which comprises a suitable container, primers located in the container which specifically amplify HC56 gene or transcripts to produce an amplification products having the SNP sites identified in the invention, and the specification.
The invention has the following characterizations: it has been proved that the LOH or DNA rearrangement of HC56 exists in HC tissues by Southern blotting; it has been proved that the expression of HC56 is inhibited in certain HC tissues by Northern blotting. Therefore, it is suggested HC56 is related to the development of HC and is a candidate for HC anti-oncogene. The in vitro DNA transfection has shown that HC56 remarkably inhibits the growth of HC cells. The SNP detection has shown that at least two SNPs have sufficient statistical difference (p<0.01) between the HC patient peripheral blood DNA and normal peripheral blood DNA. Therefore, the SNP detection kit is useful for detecting HC susceptibility.
The invention is further illustrated by the following examples. It is appreciated that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples, they are performed under routine conditions, e.g., those described by Sambrook. et al., in Molecule Clone: A Laboratory Manual, N.Y.: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturers, unless otherwise specified.
DNA was extracted from P579 (Genome System Co.) and cut with NotI. The DNA fragments about 100 bp were recovered and used as probes. After labeling with 32P-dATP, the probes were used to screen the human liver cDNA library conventionally constructed. After pre-hybridization and hybridization, the positive clones were obtained.
The sequencing of one terminal sequence (about 500 bp) of cDNA clones was carried out on ABI 337 DNA auto-sequencer using dideoxy chain termination method. After analysis, the sequencing of the other terminal was carried out for some clones, which were determined to be new genes, until the full-length sequences were obtained. One clone was named HC56, whose nucleotide and amino acid sequences were shown in SEQ ID NOs: 3 and 4. The full-length sequence was 4750 bp and the ORF was from position 1341 to 4484, encoding a protein having 1047 amino acids.
The homology comparison did not find any polypeptide or protein homologous to HC56.
The normal human liver tissue was taken, and the total RNA was extracted using Trizol™ agents (GIBCO. BRL) according to the specification, and mRNA was extracted using mRNA Purification Kit (Pharmacia). The reverse transcription was carried out at 42 degree using MMLV-RT-Superscript II (GIBCO BRL) to obtain liver cDNA. Based on SEQ ID NO:3, the HC56 specific primers were synthesized. The protocol was 97° C., 3 min, 1 cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 35 cycles; 72° C. 10 min, 1 cycles. The amplification product containing complete ORF sequence was obtained. The sequencing confirmed the sequence of the products was in accord with sequence of Example 1.
DNA was extracted from HC56 clone prepared in Example 1. According to the scheme shown in
1 μl of total tissue RNA was taken. In a 20 μl reaction system, the Superscript II RT kit (GIBCO. BRL) was used to synthesize the first strand of cDNA. Then, the following primers were used in PCR:
HC56 upstream primer: 5′-ctgcccacca ccatctgcca-3′ (SEQ ID NO: 5)
HC56 downstream primer: 5′-agcagcatgg ccaacagggg-3′ (SEQ ID NO: 6).
β2-M was used as internal control DNA. After reaction, 3 μl PCR product was taken and run on 6% PAGE. The results were shown in
The multi-tissue Northern hybridization membrane (MTN) commercially available from Clontech was pre-hybridized at 42° C. for 3–4 hrs (Pre-hybridization solution contained 50% formamide, 0.05M Tris, 1% SDS, 5× Denhardt's solution, 0.1% sodium pyrophosphate, 100 mg/ml denatured sperm DNA), hybridized with HC56 probes, and shown by X-ray autoradiography.
The results were shown in
10 μg DNA extracted from human HC tissues and surrounding noncancerous tissues was completely digested by EcoRI and prepared into Southern membrane according to the method described in “Molecule Clone: A Laboratory Manual”. The membrane was conventionally pre-hybridized, hybridized, and washed.
As shown in
As shown in
In this example, the liposome kit was used to in vitro transform hepatocarcinoma cell.
(1) Cell line: primary hepatocellular carcinoma cell line 7721
(2) DNA: DNA from expression plasmid pBK-CMV-HC56
(3) Liposome: LIPOFECT AMINE™ Reagent Kit (BRL Co.)
(4) Medium: non-serum medium SF-DMEM
(5) Preparation of DNA-liposome complex: 50 μl lipofectin was mixed with 50 μl SF-DMEM. DNA (20 μg, in 20 μl TE) was added into 80 μl SF-DMEM, and mixed. The diluted DNA was added into diluted lipofectin solution, mixed for 5–10 mins at room temperature. 1.3 ml SF-DMEM was added into DNA-lipofectin complex and the final volume was 1.5 ml.
(6) Transfecting cells: It was preferred that cells were grown to 80–90% confluence. Before experiment, the culture medium was changed once. 1.5 ml lipofectin-DNA complex was added onto the cell surface, gently shaken and mixed homogeneously, incubated at 37° C. for 1–3 hrs. 1.5 ml SF-DMEM was added and mixed homogeneously, and cells were incubated at 37° C. for 1–3 hrs. The culture medium was changed and cells were incubated at 37° C. overnight. When the confluence of cell was 70% , the medium was changed by the medium containing G418. Then the medium was changed as before until clones appeared.
The results were shown in
The nested PCR primers and sequencing primers were constructed by using ABI3948 oligo synthesizer.
DNA was prepared conventionally from HC tissues, surrounding noncancerous tissues, and peripheral blood from patients. Meanwhile, the DNA from normal peripheral blood was prepared as control. The reaction system having a total volume of 50 μl contained 50 ng genomic DNA. The PCR was conducted in standard condition. The sequencing was carried on ABI 377 plate gel using ABI big Dye Terminator Chemistry, or using ABI3700 capillary sequencer. The SNPs or mutations were collected by Sequence Navigator™.
80 pairs of HC tissues and the surrounding noncancerous tissues were analyzed for SNPs in the exons of HC56 gene. There were 13 SNPs in the exons, 8 of which was accompanied by amino acid changes. Further, once position 3085 had a SNP, it was sure 3043, 4404, and 4602 also had SNPs. The results were shown in Table 3.
70 patients' peripheral blood DNA and 100 normal persons' peripheral blood DNA were detected for SNPs on positions 3043 and 3085. It was found that the frequencies of SNPs in HC patients' peripheral blood DNA were higher than those in the normal person and had sufficient statistical difference (p<0.01), indicating HC56 was a susceptibility gene for The results were shown in Table 4.
The hepatocarcinoma susceptibility detection kit was prepared, which comprised a pair of primers amplifying the SNPs on positions 3043 and 3085.
Upstream primer: 5′-tgtggagggt tttcaggaag-3′ (SEQ ID NO: 7) (corresponding to positions 2861–2880 in SEQ ID NO: 1)
Downstream primer: 5′-tggagccagt attcctctcg-3′ (SEQ ID NO: 8) (corresponding to 3357–3376 in SEQ ID NO: 1).
Further, the kit comprised restriction enzymes SecI and EarI, which were useful for detect the SNPs on positions 3043 and 3085. For the SNP on position 3043, once G was changed into C, the product could not be digested with SecI while the unchanged amplification product could be digested with SecI. For the SNP on position 3085, once A was changed into T, the product could not be digested with EarI while the unchanged amplification product could be digested with EarI. Therefore, the kit could easily detect the SNPs on positions 3043 and 3085 by digesting the amplification products.
All the documents cited herein are incorporated into the invention as reference, as if each of them is individually incorporated. Further, it would be appreciated that, in the above teaching of the invention, the skilled in the art could make certain changes or modifications to the invention, and these equivalents would still be within the scope of the invention defined by the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 01 1 42628 | Dec 2001 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN02/00182 | 3/21/2002 | WO | 00 | 6/10/2004 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO03/050279 | 6/19/2003 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5976790 | Pinkel et al. | Nov 1999 | A |
| Number | Date | Country |
|---|---|---|
| 1281899 | Jan 2001 | CN |
| Number | Date | Country | |
|---|---|---|---|
| 20050042616 A1 | Feb 2005 | US |
