Patent Application
20080160520
The invention relates to generally to the detection and treatment of cancer.
Human growth and development requires the spatial and temporal regulation of cell differentiation, cell proliferation, and apoptosis. These processes coordinately control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and maintenance. At the cellular level, growth and development is governed by the cell's decision to enter into or exit from the cell division cycle and by the cell's commitment to a terminally differentiated state. These decisions are made by the cell in response to extracellular signals and other environmental cues it receives.
Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms. In multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Progression through the cell cycle is governed by the intricate interactions of protein complexes. This regulation depends upon the appropriate expression of proteins that control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including cyclins, cyclin-dependent protein kinases, growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, and tumor-suppressor proteins.
Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins. In the second event, mitosis, the nuclear material is divided and separates to opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm. The sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system that controls the process by positive or negative regulatory circuits at various checkpoints.
Mitosis marks the end of interphase and concludes with the onset of cytokinesis. There are four stages in mitosis, occurring in the following order: prophase, metaphase, anaphase and telophase. Prophase includes the formation of bi-polar mitotic spindles, composed of microtubules that originate from polar mitotic centers. Furthermore, structural rearrangements occur ensuring appropriate distribution of cellular components between daughter cells. Breakdown of interphase structures into smaller subunits is common. The nuclear envelope breaks into vesicles, and nuclear lamins are disassembled. Subsequent phosphorylation of these lamins occurs and is maintained until telophase, at which time the nuclear lamina structure is reformed. During prophase, the nuclear material condenses and develops kinetochore fibers that aid in its physical attachment to the mitotic spindles. The ensuing movement of the nuclear material to opposite poles along the mitotic spindles occurs during anaphase. Telophase includes the disappearance of the mitotic spindles and kinetochore fibers from the nuclear material. Mitosis depends on the interaction of numerous proteins.
All key cell cycle transitions, including the entry and exit of a cell from mitosis, are dependent upon the activation and inhibition of cyclin-dependent kinases (Cdks). The Cdks are composed of a kinase subunit, Cdk, and an activating subunit, cyclin, in a complex that is subject to many levels of regulation. There appears to be a single Cdk in Saccharomyces cerevisiae and Schizosaccharomyces pombe whereas mammals have a variety of specialized Cdks. Cyclins act by binding to and activating cyclin-dependent protein kinases which then phosphorylate and activate selected proteins involved in the mitotic process. The Cdk-cyclin complex is both positively and negatively regulated by phosphorylation, and by targeted degradation involving molecules such as CDC4 and CDC53. In addition, Cdks are further regulated by binding to inhibitors and other proteins such as Suc1 that modify their specificity or accessibility to regulators (Patra, D. and W. G. Dunphy (1996) Genes Dev. 10: 1503-1515; and Mathias, N. et al. (1996) Mol. Cell Biol. 16:6634-6643).
Cyclins are degraded through the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. The UCS is implicated in the degradation of mostif not all cyclins, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra).
The process of ubiquitin conjugation and protein degradation occurs in five principle steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a small, heat stable protein is activated by a ubiquitin-activating enzyme (E1) in an ATP dependent reaction which binds the C-terminus of Ub to the thiol group of an internal cysteine residue in E1. Second, activated Ub is transferred to one of several Ub-conjugating enzymes (E2). Different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits that direct them to proteins carrying a particular degradation signal. Third, E2 transfers the Ub molecule through its C-terminal glycine to a member of the ubiquitin-protein ligase family, E3. Fourth, E3 transfers the Ub molecule to the target protein. Additional Ub molecules may be added to the target protein forming a multi-Ub chain structure. Fifth, the ubiquinated protein is then recognized and degraded by the proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for re-utilization.
Prior to activation, Ub is usually expressed as a fusion protein composed of an N-terminal ubiquitin and a C-terminal extension protein (CEP) or as a polyubiquitin protein with Ub monomers attached head to tail. CEPs have characteristics of a variety of regulatory proteins; most are highly basic, contain up to 30% lysine and arginine residues, and have nucleic acid-binding domains (Monia, B. P. et al. (1989) J. Biol. Chem. 264:4093-4103). The fusion protein is an important intermediate that appears to mediate co-regulation of the cell's translational and protein degradation activities, as well as localization of the inactive enzyme to specific cellular sites. Once delivered, C-terminal hydrolases cleave the fusion protein to release a functional Ub (Mania et al., supra).
Abnormal activities of the UCS are implicated in a number of diseases and disorders. These include, e.g., cachexia (Llovera, M. et al. (1995) Int. J. Cancer 61:138-141), degradation of the tumor-suppressor protein, p53 (Ciechanover, supra), and neurodegeneration such as observed in Alzheimer's disease (Gregori, L. et al. (1994) Biochem. Biophys. Res. Commun. 203:1731-1738). Since ubiquitin conjugation is a rate-limiting step in antigen presentation, the ubiquitin degradation pathway may also have a critical role in the immune response (Grant, E. P. et al. (1995) J. Immunol. 155:3750-3758).
Cell cycle regulation not only involves proteins that replicate DNA and segregate it to the daughter cells but also involves numerous proteins whose function it is to ensure the precise order of cell cycle events. These control mechanisms are called checkpoints. For example, DNA damage (G2) and DNA replication (S-phase) checkpoints arrest eukaryotic cells at the G2/M transition. This arrest provides time for DNA repair or DNA replication to occur before entry into mitosis. Thus, the G2/M checkpoint ensures that mitosis only occurs upon completion of DNA replication and in the absence of chromosomal damage. The Hus1 gene of Schizosaccharomyces pombe is a cell cycle checkpoint gene, as are the rad family of genes (e.g., rad1 and rad9) (Volkmer, E. and L. M. Kamitz (1999) J. Biol. Chem. 274:567-570; Kostrub C. F. et al. (1998) EMBO J. 17:2055-2066). These genes are involved in the mitotic checkpoint, and are induced by either DNA damage or blockage of replication. Induction of DNA damage or replication block leads to loss of function of the Hus1 gene and subsequent cell death. Human homologs have been identified for most of the rad genes, including ATM and AIR, the human homologs of rad3p. Mutations in the ATM gene are correlated with the severe congenital disease ataxia-telagiectasia (Savitsky, K. et al. (1995) Science 268: 1749-1753). The human Hus1 protein has been shown to act in a complex with rad1 protein which interacts with rad9, making them central components of a DNA damage-responsive protein complex of human cells (Volkner and Kamitz, supra). Examples of additional cell cycle regulatory proteins that are regulated by the DNA damage checkpoint include the histone deacetylases (HDACs). HDACs are involved in cell cycle regulation, and modulate chromatin structure. Human HDAC1 has been found to interact in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G2/M checkpoint control (Cai, R. L. et al. (2000) J. Biol. Chem 275:27909-27916).
Cell cycle regulatory proteins play an important role in cell proliferation and cancer. For example, failures in the proper execution and timing of cell cycle events can lead to chromosome segregation defects resulting in aneuploidy or polyploidy. This genomic instability is characteristic of transformed cells (Luca, F. C. and M. Winey (1998) Mol. Biol. Cell. 9:2946) and a hallmark of solid tumors as virtually no solid tumor exists that does not show some alterations of the genome. With the vast majority of tumors this instability is expressed at the level of the chromosomal complement, and thus is detectable by cytogenetic approaches (Mitelman, F., Catalog of Chromosome Aberrations in Cancer, 5th Edition (New York: Wiley-Liss) (1994)). Further, cancers are characterized by continuous or uncontrolled cell proliferation. Strategies for treatment may involve either reestablishing control over cell cycle progression, or selectively stimulating apoptosis in cancerous cells (Nigg, E. A. (1995) BioEssays 17:471-480).
While a substantial amount of work has been performed in further understanding the cell cycle and how cancer is affected, no work has been done to understand why the problems occur. For example, cancer cells are known to be aneuploid. However, those of skill in the art have determined that aneuploidy or chromosomal rearrangement per se is not indicative of malignancy and many benign tumors can have an aberrant karyotype (Mitelman, 1994). It would be useful to determine the role aneuploidy plays in cancer progression and torn determine how this role can be monopolized to affect new cancer treatments.
The invention provides biological markers to monitor the diagnosis and prognosis of cancer.
Cancer or a predisposition thereof is diagnosed in a subject by detecting the presence or an absence of a mutation in tumorgenesis-associated gene or polypeptide. The presence of a mutation indicates the presence of a cancer or a predisposition thereto in the subject. Whereas, the absence of a mutation indicates absence of a cancer or a predisposition thereto in the subject. Optionally, the level of the mutation in tumorgenesis-associated gene in the subject is compared to a control (i.e. standard) value. A higher level of mutation in tumorgenesis-associated gene or polypeptide in the test sample compared to the control sample indicates cancer in the subject. By higher level is meant at least a 2, 4, 5, 10-fold or higher value in the test sample compared to the control sample.
A tumorgenesis-associated gene or polypeptide include for Ubiquitin-specific protease, DNA helicase, a Subunit of the Set3C deacetylase complex and ORF YJL213W or a human homologue thereof. An Ubiquitin-specific protease includes for example UBP6 or a human homologue thereof such as USP14. A DNA helicase includes for example RAD3 or a human homologue thereof such as ERCC2. A Subunit of the Set3C deacetylase complex includes for example is SNT1 or a human homologue thereof such as FRS2 or SNTA1.
The mutation in the gene encoding an Ubiquitin-specific protease polypeptide is a stop codon which results in a less then full length polypeptide being expressed. For example, the mutation results in a C-terminal truncation of the polypeptide causing a decrease of peptidase activity of the polypeptide. The mutation in the gene encoding a DNA helicase polypeptide or a subunit of the Set3C deacetylase complex polypeptide is a point mutation. The point mutation results in an alteration, e.g., increase or decrease of polypeptide activity. The point mutation in DNA helicase polypeptide results in an amino acid change at position 148 resulting in an aspartic acid being changed to an asparagine. The point mutation in the Subunit of the Set3C deacetylase polypeptide results in an amino acid change at position 431 resulting in a leucine being changed to an arginine.
The mutation of ORF YJL213W is a mutation in the promoter region resulting in increased expression of the polypeptide encode by ORF YJL213W or the human homologue.
The sample is a biological sample obtained from the subject. The sample is for example, serum, blood plasma, ascites fluid, urine, a vaginal secretion or a tissue biopsy.
The tumorgenesis-associated polypeptide or gene is detected by any means known in the art. For example the tumorgenesis-associated polypeptide is detected electrophoretically or immunochemically. Immunochemical detection includes for example, radio-immunoassay, immunofluorescence assay, or enzyme-linked immunosorbant assay. The tumorgenesis-associated nucleic acid or gene is detected by the polymerase chain reaction, single nucleotide polymorphism (SNP) arrays, or interphase fluorescent in situ hybridization (FISH) analysis.
The subject has not been previously diagnoses as having cancer. Alternatively, the subject has been diagnosed with cancer. Optionally, the subject has been previously treated for cancer.
Also included in the invention is a method of decreasing cancer cell growth by contacting the tumor cell with a glucose transporter inhibitor, an RNA polymerases inhibitor, a protein synthesis inhibitor or a HSP90 inhibitor (e.g. geldanamycin). Protein synthesis inhibitors include for example, cycloheximide, hygromycin or rapamycin.
Alternatively, cancer cell growth decreased contacting the cancer cell with a compound that increases the expression or activity of a non-mutated UBP6, RAD3 or SNT1 polypeptide or human homologue thereof, decrease intracellular protein degradation; or decreases the expression of activity of the polypeptide encoded by ORF YJL213W or a human homologue thereof.
The cell is an aneuploid cell. The cell is contacted in vivo, in vitro or ex vivo. The methods treat or alleviate a symptom of cancer in a subject.
In a further aspect the invention provides a method for screening for an inhibitor of tumor cell proliferation or viability by contacting an aneuploid cell with a candidate compound. Cell proliferation or viability is measured. A decrease in aneuploid cell proliferation or viability in the presence of the compound, as compared to the absence of the compound, indicates that the compound is an inhibitor of tumor cell proliferation or viability. Alternatively, a diploid cell is also contacted with the candidate compared. A decrease in aneuploid cell proliferation or viability, as compared to the diploid cell indicates that the compound is a specific inhibitor of tumor cell proliferation or viability.
The aneuploid cell displays wild-type physiology. For example, the aneuploid cell has at least one mutation in a Ubiquitin-specific protease gene; a DNA helicase gene; a Subunit of the Set3C deacetylase complex gene or a promoter of ORF YJL213W or a human homologue thereof.
Also included in the invention are the compounds identified by the methods.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
(A) Nop1 protein and RNA levels are shown in WT (A11311), Dis IV (A12687) and Dis XIII (A12695).
(B) Pup3 protein and RNA levels are shown in WT (A11311), Dis V (A14479) and Dis XI (A13771).
(C) Pre6 protein and RNA levels are shown in WT (A11311), Dis IV (A12687) and Dis XV (A12697).
(D) Hht1 protein and RNA levels are shown in WT (A11311), Dis I (A12683) and II (A12685).
(E) Rpt1 protein and RNA levels are shown in WT (A11311), Dis V (A14479) and XI (A13771).
(F) Lcb4 protein and RNA levels are shown in WT (A11311), Dis XV (A12697) and XVI (A12700).
(G) Elp3 protein and RNA levels are shown in WT (A11311), Dis XV (A12697) and XVI (A12700).
(H) Eaf3 protein and RNA levels are shown in WT (A11311), Dis XV (A12697) and XVI (A12700).
(I) Yaf9 protein and RNA levels are shown in WT (A11311), Dis IV (A12687) and XIV (A13979).
(J) Fcy1 protein and RNA levels are shown in WT (A11311), Dis VIII (A13628) and XVI (A12700).
(A) Wild type haploid (A11311) and diploid (A702) strains plated on YPD at 30° C. and 37° C. Also plated on YPD at 30° C. containing 20 μg/ml of Thiolutin, 10 nM Rapamycin and 0.05 μg/ml Cycloheximide.
(B-I) Wild type strains and aneuploid strains were plated on medium containing increasing concentrations of Hydroxyurea (B), 3% glycerol (YPG) or 2% raffinose and galactose (Raf/Gal) as the sole carbon source (C), medium containing 1 mM chloroquine (D), 0.5 mg/ml azetidine 2-carboxylic acid (E), 10 and 20 μg/ml benomyl (F), 1 mM hydrogen peroxide (G), increasing concentrations of Thiolutin (H), 60 μg/ml of 6-Azauracil, YEP medium containing glycerol and ethanol as the sole carbon source (YPGE) and increasing concentrations of Oligomycin (I).
The invention is based in part upon the discovery that aneuploid yeast strains share a number of phenotypes that are distinct from diploid cells and are independent of the identity of the individual extra chromosomes. Specifically, aneuploid cells have defects in cell cycle progression, increased glucose uptake and increased sensitivity to conditions interfering with protein synthesis and protein folding. More specifically, the invention is based upon the identification of specific mutations in aneuploid cells which allows the cells to tolerate aneuploidy.
Aneuploidy is a condition frequently found in cancer cells and share several properties with yeast cells carrying additional chromosomes. For example, proliferation of both types of cells is impaired in the presence of protein synthesis inhibitors (27) and geldanamycin (28) and both exhibit increased glucose uptake (29). Until now it has been thought that the proliferative advantage of tumor cells which makes them independent of growth control was the direct result of the aneuploidy which leads to the cell carrying extra copies of oncogenes. In contrast, the data described herein demonstrate that aneuploidy results in a proliferative disadvantage and that this disadvantage needs to be overcome during cancer formation. This proliferative disadvantage is overcome in part by acquiring specific mutations in the genes encoding ubiquitin-specific protease, DNA helicase, a subunit of the Set3C deacetylase complex and in the promoter region of yeast ORF YJL213W. These mutations are summarized in Table A and are collectively referred to herein as “tumorgenesis-associated mutations” and the corresponding genes and polypeptides in which these mutation are found are referred to herein as “tumorgenesis-associated genes”, tumorgenesis-associated nucleic acids” “tumorgenesis-associated polynucleotides”, “tumorgenesis-associated polypeptides” or “tumorgenesis-associated proteins.” The genes have been previously described and are presented along with a database accession numbers.
Accordingly, the invention provides methods of detecting and evaluating cancer in a subject by the detection of a mutation in a tumorgenesis-associated gene or tumorgenesis-associated polypeptide. Also provided by the inventions are methods of decreasing tumor cell growth and treating or alleviating a symptom of cancer by contacting a cell or administering to a subject a compound that modulates the expression of a tumorgenesis-associated gene, a tumorgenesis-associated polypeptide, a glucose transporter inhibitor, an RNA polymerase inhibitor, a protein synthesis inhibitor, or a HSP90 inhibitor. The methods disclosed herein are employed with subjects suspected of having cancer, to monitor subjects who have been previously diagnosed as having cancer, and to screen subjects who have not been previously diagnosed as having cancer.
The cell division cycle is a highly controlled process that generates two daughter cells of identical genetic make-up. Surveillance mechanisms known as checkpoints ensure that this process occurs with high fidelity. However, despite these surveillance mechanisms, chromosome mis-segregation occurs once every 5×105 cell divisions in yeast (1) and on the order of once every 104-105 divisions in mammalian cells (2), producing a condition known as aneuploidy. More than a century ago, aneuploidy was postulated to be a common characteristic of cancer cells (3). Since then, it has been proposed that aneuploidy contributes to tumorigenesis by providing a mechanism by which oncogenes are gained or tumor suppressor genes are lost (4). Studies examining the effects of aneuploidy on cell proliferation in S. pombe (5), Drosophila (6), and of trisomy on cell proliferation in humans (7) suggest that aneuploidy can also interfere with cell proliferation. To address how aneuploidy affects the proliferation and the physiology of normal cells, a set of yeast strains in which each strain bears an extra copy of one or more of almost all of the yeast chromosomes were generated. Their characterization represents a comprehensive analysis of the effects of aneuploidy on cellular physiology and revealed that in addition to chromosome-specific phenotypes, aneuploid strains share a number of traits, pointing towards the existence of a general cellular response to aneuploidy.
Specifically, the 20 aneuploid yeast strains that were analyzed shares several phenotypes. In contrast, diploid yeast cells did not exhibit these phenotypes (
These studies indicated that most phenotypes common to aneuploids are caused by the additional yeast gene products. These findings together with the observation that disomy for the small chromosome VI is lethal (10) indicate that likely both the total amount of additional RNA and protein produced by aneuploids as well as specific gene products present on individual chromosomes contribute to the phenotypes shared by aneuploids.
Striking among the phenotypes shared by aneuploid yeast strains are those indicative of protein degradation and folding distress. These observations suggest that proteins synthesized from the additional chromosomes disrupt cellular physiology, interfering with metabolic pathways and other basic cellular processes. It is hypothesized the cells respond to this state of imbalance in a multi-layered fashion not dissimilar to that of a stress response. The cell's attempt to restore wild type physiology is reflected by the fact that although most genes present on the additional chromosomes are transcribed, the amounts of many proteins are not increased. Carbohydrate uptake is increased which could provide the energy needed to degrade protein and induce mechanisms that shield the cell from the effects of excess proteins or compensate for their effects. The delay in G1 might also be part of a response to this disruption in cellular homeostasis.
Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. UBPs help regulate the ubiquitin/26S proteolytic pathway by generating free ubiquitin monomers from their initial translational products, recycling ubiquitins during the breakdown of ubiquitin-protein conjugates, and/or by removing ubiquitin from specific targets and thus presumably preventing target degradation.
A point mutation in the yeast gene UBP6 was identified in aneuploid yeast cells that have overcome the proliferation disadvantage of aneuoploid cells. UBP6 is located on yeast chromosome VI from coordinates 165060 to 166559. The point mutation resulted in nucleotide change (G→T) at position 165623, resulting in a stop codon. This stop codon results in a truncated version of the ubiquitin-specific protease polypeptide. Specifically, the protein lacks the C-terminal peptidase activity, while retaining the N-terminal ubiquination-like domain.
DNA helicase is an enzyme that aids in DNA synthesis by ‘unzipping’ the two strands of a DNA helix so that DNA polymerase can access the DNA to add nucleotides and effect copying.
Many cellular processes such as DNA replication, RNA transcription, DNA recombination, DNA repair, Ribosome biogenesis involve the separation of nucleic acid strands. Helicases are often utilized to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP or GTP hydrolysis. They move incrementally along one nucleic acid strand of the duplex with directionality specific to each particular enzyme. There are many helicases (e.g., 24 in human cells) resulting from the great variety of processes in which strand separation must be catalyzed.
The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology; they all possess common sequence motifs located in the interior of their primary sequence. These are thought to be specifically involved in ATP binding, ATP hydrolysis and translocation on the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.
Based on the presence and the form of helicase motifs, helicases have been separated in 4 superfamilies and 2 smaller families.
A point mutation in the yeast gene RAD3 was identified in aneuploid yeast cells that have overcome the proliferation disadvantage of aneuoploid cells. Rad3 is located on yeast chromosome V from coordinates 527077 to 529413. The point mutation resulted in nucleotide change (G→A) at position 527517, resulting D→N amino acid change at position 148.
SET3C is a NAD-dependent histone deacetylase. The Set3 complex (Set3C) includes two potential histone deacetylases, Hos2 and Hst1. Hos2 is a class I histone deacetylase like Rpd3 and the mammalian HDACs 1-3 (Rundlett et al. 1996). Hst1 is a member of the recently identified Sir2 class of NAD-dependent deacetylases (Imai et al. 2000; Landry et al. 2000; Smith et al. 2000) and has been linked previously to repression of sporulation genes (Xie et al. 1999; Lindgren et al. 2000). The other components of the complex are Snt1, YIL112w, Sif2 and Cpr1. IN budding the yeast Set3C has been implicated in establishing silenced chromatin and the repression of meiosis-specific genes. The human homologs of this deacetlyase are also involved in silencing transcription.
A point mutation in the yeast gene SNT1 was identified in aneuploid yeast cells that have overcome the proliferation disadvantage of aneuoploid cells. SNT1 is located on yeast yeast chromosome III from coordinates 186485 to 190165. The point mutation resulted in nucleotide change (T→G) at position 187773, resulting amino L→R acid change at position 148.
YJL213W is a protein of unknown function that may interact with ribosomes; periodically expressed during the yeast metabolic cycle; and is phosphorylated in vitro by the mitotic exit network (MEN) kinase complex, Dbf2p/Mob1p.
A point mutation in the yeast gene the upstream (e.g, in the promoter region) of YJL213W of was identified in aneuploid yeast cells that have overcome the proliferation disadvantage of aneuoploid cells. YJL213W is located on yeast yeast chromosome X from coordinates 32163 to 33158. The point mutation resulted in nucleotide change (C→G) at position 31906, resulting in the upregulation of the expression of the YJL213W protein.
The invention provides diagnostic and prognostic methods for identifying a subject with cancer or a predisposition thereto.
Cancers or a predisposition of developing cancer are detected by examining the presence or absence of a mutation in tumorgenesis-associated gene or tumorgenesis-associated polypeptide in a test population of cells (i.e., a patient derived sample). The presence of one or more tumorgenesis-associated mutation indicates that the subject has is predisposed to developing cancer. Whereas, the absence of a tumorgenesis-associated mutation indicates that the subject does not have cancer or predisposed to developing cancer.
In some aspects mutation in tumorgenesis-associated gene or tumorgenesis-associated polypeptide is determined in the test sample to provide a test value and the test value is compared to a standard value. By standard value is meant the level tumorgenesis-associated gene or tumorgenesis-associated polypeptide typically found in a population not having cancer. The standard value can be a range or an index. Alternatively, the standard value can be a database of test values from previously tested samples.
The difference in the standard value compared to the test value is statistically significant. By statistically significant is meant that the alteration is greater than what might be expected to happen by chance alone. Statistical significance is determined by method known in the art. For example statistical significance is determined by p-value. The p-values is a measure of probability that a difference between groups during an experiment happened by chance. (P(z≧zobserved)). For example, a p-value of 0.01 means that there is a 1 in 100 chance the result occurred by chance. The lower the p-value, the more likely it is that the difference between groups was caused by treatment. An alteration is statistically significant if the p-value is at least 0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.
Cancer includes solid tumors and hematologic tumors. Cancers include for example, lung cancer, head or neck cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, liver cancer, colon cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, or leukemia.
The patient derived sample can be any tissue or fluid. Tissue samples include for example, paraffin imbedded tissue, frozen tissue, surgical fine needle aspirations, cells of the skin, muscle, lung, head and neck, esophagus, kidney, pancreas, mouth, throat, pharynx, larynx, esophagus, facia, brain, prostate, breast, endometrium, small intestine, blood cells, liver, testes, ovaries, uterus, cervix, colon, stomach, spleen, lymph node, bone marrow or kidney. Fluid samples include of example, bronchial brushes, bronchial washes, bronchial ravages, peripheral blood lymphocytes, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, esophageal washes, and stool or urinary specimens such as bladder washing and urine.
Mutation in the genes disclosed herein is determined at the protein or nucleic acid level using methods known in the art. Mutation in nucleic acids are detected for example by Hybridization-based Assays and Amplification-based Assays. Mutations are is also determined at the protein level, i.e., by measuring the levels of polypeptides encoded by the gene products described herein. Such methods are well known in the art and include, e.g. immunoassays based on antibodies to proteins encoded by the genes.
The subject is preferably a mammal. The mammal is, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The subject has been previously diagnosed as having cancer, and possibly has already undergone treatment for the cancer. Alternatively, the subject has not been previously diagnosed as having cancer. The present invention is useful with all patients at risk for cancer.
Diagnosis of cancer is made through methods known in the art for a particular cancer such as patient history and physical examination along with diagnostic testing such as blood test, urinalysis, and tumor markers (e.g., PSA, PAP, CA125, CES, AFP and HCG)
The invention provides a method for decreasing tumor cell growth, treating or alleviating a symptom of cancer in a subject by contacting the cell or administering to a subject a compound that increasing the expression or activity of a non-mutated ubiquitin-specific protease polypeptide, DNA helicase polypeptide, or a subunit of the Set3C deacetylase complex polypeptide, decreases intracellular protein degradation or decreases the expression or activity of the polypeptide encoded by ORFYJL213W or human homologue thereof. Alternatively, tumor cell growth is decreased and/or cancer is treated by contacting the cell or administering to a subject a glucose transporter inhibitor, an RNA polymerase inhibitor, a transcription inhibitor, a protein synthesis inhibitor or a HSP90 inhibitor.
Therapeutic compounds are administered prophylactically or therapeutically to subject suffering from, or at risk of or susceptible to developing, cancer. Such subjects are identified using standard clinical methods.
The therapeutic method includes increasing the expression, or function, or both of one or more gene products of a non-mutated ubiquitin-specific protease polypeptide, DNA helicase polypeptide, or a subunit of the Set3C deacetylase in a subject or cell relative to a normal subject or cells of the same tissue type. In these methods, the subject is treated with an effective amount of a compound, which increases the amount of one of more of the genes or polypeptides in the subject. Administration can be systemic or local. Therapeutic compounds include a polypeptide product (e.g., a ubiquitin-specific protease polypeptide, DNA helicase polypeptide, or a subunit of the Set3C deacetylase), or a biologically active fragment thereof, and a nucleic acid encoding polypeptide and having expression control elements permitting expression in the cell or subject. Administration of such compounds counters the effects of mutated genes in the subject and improves the clinical condition of the subject
The method also includes decreasing the expression, or function, or both, of the polypeptide encoded by ORF YJL213W or human homologue thereof whose expression is aberrantly increased (“overexpressed gene”) in cancer cells. Expression is inhibited in any of several ways known in the art. For example, expression is inhibited by administering to the subject a nucleic acid that inhibits, or antagonizes, the expression of the ORF YJL213W, e.g., an antisense oligonucleotide or siRNA which disrupts expression of the cancer-associated gene or genes.
Alternatively, function of one or more gene product of ORF YJL213W is inhibited by administering a compound that binds to or otherwise inhibits the function of the ORF YJL213W gene products. For example, the compound is an antibody which binds to the overexpressed gene product.
The method further includes decreasing intracellular protein degradation. Intracellular protein degradation is decreased by methods known in the art such as by decreasing ubiquination. For example, ubiquination is inhibited by administering to the subject a nucleic acid that inhibits, or antagonizes, the expression of ubiquitin or a polypeptide in the ubiquination pathways, e.g., an antisense oligonucleotide or siRNA which disrupts expression of the genes encoding these polypeptides.
Once the cancer cells have been modified to react as normal aneuploid cells the cells become susceptible to the normal sensitivities of aneuploid cells. Such as the aneuploid cells are sensitive to temperature, compounds that interfere with protein synthesis, geldanamycin (an inhibitor or HSP90, which is important to help fold misfolded proteins), hygromycin B, rapamycin, and cycloheximide. Accordingly in some aspects the cells or subjects are further contacted with a glucose transporter inhibitor, an RNA polymerase inhibitor, a transcription inhibitor, a protein synthesis inhibitor or a HSP90 inhibitor.
These modulatory methods are performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The method involves administering a protein or combination of proteins, a nucleic acid molecule or combination of nucleic acid molecules, or a combination of one or more nucleic acids and one or more proteins, as therapy to counteract aberrant expression or activity of the differentially expressed genes.
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity of the genes may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the overexpressed gene or genes. Therapeutics that antagonize activity are administered therapeutically or prophylactically.
Therapeutics that may be utilized include, e.g., (i) a polypeptide, or analogs, derivatives, fragments or homologs thereof, of the overexpressed or underexpressed sequence or sequences; (ii) antibodies to the overexpressed or underexpressed sequence or sequences; (iii) nucleic acids encoding the over or underexpressed sequence or sequences; (iv) antisense nucleic acids or nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences of one or more overexpressed or underexpressed sequences); or (v) modulators (i.e., inhibitors, agonists and antagonists that alter the interaction between an over/underexpressed polypeptide and its binding partner. The dysfunctional antisense molecule is utilized to “knockout” endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 244: 1288-1292 1989). The siRNA is designed by methods known in the art to bind to gene transcripts and prevent translation into proteins.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a polypeptide (or analogs, derivatives, fragments or homologs thereof) or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Therapeutic methods include contacting a cell with an agent that modulates one or more of the activities of the gene products. An agent that modulates protein activity includes a nucleic acid or a protein, a naturally-occurring cognate ligand of these proteins, a peptide, a peptidomimetic, or other small molecule. For example, the agent stimulates one or more protein activities.
The aneuploid yeast cells disclosed herein can also be used to identify candidate inhibitors of tumor cell proliferation. The method is based on screening a candidate therapeutic agent to determine whether the compound has an effect on cell proliferation and or cell viability.
In the method, a cell is exposed to a test agent or a combination of test agents (sequentially or consequentially) and cell proliferation or cell viability is measured. Cell proliferation or cell viability in the test population is compared cell proliferation in a reference cell population that is not exposed to the test agent.
Cell proliferation is measured by methods known in the art, such as bromodeoxyuridine incorporation. Cell viability is measured for example by trypan blue exclusion.
The aneuploid cell displays normal wild type physiology. By normal wild type physiology is mean that the cell does not display the aneuploid phenotype such as defects in cell cycle progression, increased glucose uptake and increased sensitivity to conditions interfering with protein synthesis and protein folding. For example, the cell has one or more tumorgenesis-associated mutations.
The cell population in the reference population is a cell population that is identical to the test population. For example, the reference cell population is an aneuploid cell displays normal wild type physiology. Alternatively, the reference cell population is a normal cell, i.e., diploid. This allows the identification of compounds that preferentially decreases cell proliferation and or viability in tumor cells that have one or more tumorgenesis-associated mutations compared to normal cells. Such compositions are particularly useful in treating cancer as they will be specific for tumor cells and have no deleterious effects on normal cells.
An agent effective in decreasing cell proliferation and/or increasing cell viability is deemed to lead to a clinical benefit such compounds are further tested for the ability to decrease tumor cell proliferation and/or viability in animals or test subjects.
Kits for Use in Diagnostic and/or Prognostic Applications
For use in diagnostic, research, and therapeutic applications suggested above, kits are also provided by the invention. In the diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, nucleic acids for detecting the target sequences and other hybridization probes and/or primers. A therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The phrases “pharmaceutical” and “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one composition or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required within the industry.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 μg/kg/body weight, about 5 μg/kg/body weight, about 10 μg/kg/body weight, about 50 μg/kg/body weight, about 100 μg/kg/body weight, about 200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 mg/kg/body weight, etc., can be administered, based on the numbers described above.
Gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype. The genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide, or peptide of therapeutic value. Alternatively, the genetic material of interest can encode a suicide gene. For a review see, in general, the text “Gene Therapy” (Advances in Pharmacology 40, Academic Press, 1997).
Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ [Culver, 1998]. These genetically altered cells have been shown to express the transfected genetic material in situ.
The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5′UTR and/or 3′UTR of the gene can be replaced by the 5′UTR and/or 3′UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5′UTR and/or 3′UTR of the actual gene to be transferred and only include the specific amino acid coding region.
The expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence that works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.
Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see U.S. Pat. No. 4,866,042 for vectors involving the central nervous system and also U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus-derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor that includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation can not occur.
Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally_This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector(s) to be used in the methods of the invention depends on desired cell type to be targeted and are known to those skilled in the art. For example, if breast cancer were to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system were to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.
Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles that are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection.
An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Those of skill in the art know such transfection vehicles.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Strains and plasmids. All strains except strain A5644 are derivatives of W303 (A2587) and are listed in Table S2. The CLN2-HA fusion is described in Tyers et a. (S1). Thepdr5:: TRP1 deletion was generated using the PCR-based methods described by Longetine et al. (S2). Aneuploids carrying a deletion of PDR5 or a CLN2-HA fusion were obtained by crossing aneuploid strains to strains carrying the deletion and fusion, respectively. Strains disomic for the chromosome of interest were readily recovered in such crosses. All aneuploid strains used in this study were subjected to comparative genomic hybridization (CGH) to ensure that the additional chromosome was present in its entirety (
Generation of aneuploid yeast strains. Strains carrying extra chromosome(s), henceforth referred to as aneuploid strains, were generated by a chromosome transfer strategy described by Hugerat et al. (S3). The strategy is outlined in
Two copies of the can1-100 allele no longer confer canavinine resistance (data not shown). Because the can1-100 allele was located on chromosome V, strains disomic for chromosome V had to be constructed by a different strategy. Instead of integrating HIS3 and kanMX6 at the same genomic location HIS3 was integrated at the CAN1 locus and kanMX6 was integrated at the intergenic region (187520-187620) between ORFs YERO15 W and YERO16W.
Cells disomic for chromosomes III and VII were not obtained because the MAT locus and the CYH2 locus are located on chromosome III and VII, respectively. Despite several attempts, we failed to obtained strains disomic for chromosome VI. This finding suggests that two copies of chromosome VI are lethal. ACT1 and TUB2, which encode actin and β-tubulin are located in chromosome VI. Cells are extremely sensitive to increased levels of actin or β-tubulin (S7-S9), which could explain our inability to obtain cells disomic for this chromosome. Consistent with this idea is the observation that we obtained strains disomic for chromosomes I, VI and XIII in two independent attempts to isolate strains disomic for chromosome VI. The gene encoding α-tubulin is located on chromosome XIII and increasing the amount of the α-tubulin has been shown to rescue the lethality associated with excess (β-tubulin (S9).
The CGH analysis revealed that several of our disomic candidate strains not only carried the chromosomes selected for but also an extra chromosome. Although we were not able to select for the presence of the additional chromosome, gene expression and CGH analyses showed that the karyotypes of these multiple disomic cells were stable enough to conduct the experiments described in this manuscript.
Generation of trisomic strains. The kanMX6 marker of disomic strains was replaced with URA3. The resulting disomes carrying a HIS3 and URA3 marker at the same genomic location were mated to a haploid containing the kanMX6 marker at the same locus and trisomic strains were recovered by selection on −His-Ura+G418 media.
Generation of strains carrying YACs. All strains containing yeast artificial chromosomes (YACs) were generated by the procedure used to transfer yeast chromosomes except cells were plated on medium lacking uracil to select for the presence of the YAC. kar1Δ15 strains carrying YAC-7 and YAC-1 were described in Huang et al. (S10). YAC-2, -3, -4, -5 and -6 contain regions of the human Y chromosome and were obtained through ATCC (cat#77393) and are described in Foote et al. (S11). The presence of the YAC was confirmed by Pulse Field Gel Electrophoresis and Southern blotting using a probe for URA3 as described in Huang et al. (S10).
CGH analysis of aneuploid strains. To prepare genomic DNA, cells were grown to saturation in selective media. 15 mls of culture were spun down, rinsed and incubated for 60 minutes at 37° C. in 1.5 mls of 1 M Sorbitol, 10 mM Na-phosphate, pH 7.0, 10 mM EDTA, 200 μg/ml zymolase and 150 μM (3-mercaptoethanol. Cells were pelleted and incubated in 1.5 mls of 50 mM EDTA, pH 8.0, 0.3% SDS, 200 μg/ml proteinase K and incubated for another 60 minutes at 65° C. 0.6 mls of 5 M KOAc was added and incubated on ice for 30 minutes. After centrifugation, the supernatant was subjected to a phenol/chloroform extraction and DNA was precipitated. The DNA was RNAse treated at 37° C. for 2 hours (10 mM Tri-HCl, 1 mM EDTA, pH 7.5, 1 mg/ml RNAse), followed by another phenol/chloroform extraction, and precipitated with ethanol.
1 μg HhaI digested DNA was labeled with Klenow polymerase and Cy3- or Cy5-dCTP according to the BioPrime CGH labeling kit (Invitrogen), using half volume reactions. Yield and dye incorporation were checked with a Nanodrop spectrophotometer. 200 ng differentially labeled DNA from the reference strain and the strain of interest were mixed, combined with control targets and hybridization buffer, boiled for 5 minutes, and applied to a microarray consisting of 60 mer probes for each yeast open reading frame (Agilent). Microarrays were rotated at 60° C. for 17 hours in a hybridization oven (Agilent). Arrays were then washed according to the Agilent SSPE wash protocol, and scanned on an Agilent scanner. The image was processed using the default settings with Agilent Feature Extraction software. All data analysis was performed using the resulting log2 ratio data, and filtered for spots called as significantly over background in at least one channel.
Gene expression arrays. Total RNA was isolated from cells frozen on filters. Filters were incubated for 1 hour at 65° C. in lysis buffer (10 mM EDTA, 0.5% SDS, and 10 mM Tris, pH 7.5) and acid phenol. The aqueous phase was further extracted twice with an equal volume of chloroform using phase lock gel (Eppendorf). Total RNA was then ethanol precipitated and further purified over RNeasy columns (Qiagen). RNA quality was checked using the Bioanalyzer RNA Nano kit, and 325 ng was used for microarray labeling with the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Reactions were performed as directed except using half the recommended reaction volume and one quarter the recommended Cy-CTP amount. Dye incorporation and yield were measured with a Nanodrop spectrophotometer. Equal amounts of differentially labeled control and sample cRNA were combined such that each sample contained at least 2.5 pmol dye. Samples were mixed with control targets, fragmented, combined with hybridization buffer, and hybridized as described above for the CGH analysis.
Data analysis. The Agilent normalization method assumes an average ratio of 1 between experimental strains and the wild type reference. This assumption is not accurate for strains carrying extra chromosomes. All expression data were therefore renormalized to account for the extra chromosome by averaging the log2 ratios of all genes not contained on the disomic chromosome. This number was then subtracted from all log2 ratios in order to make the average log2 ratios zero for genes not on the disomic chromosome. With this correction, genes contained on all disomic chromosomes over several replicate experiments increased in expression by an average of 1.8-fold. Replicate flasks of the wild-type strain were also compared in order to find the extent of noise in gene expression pattern under these conditions. A cutoff of two standard deviations from the mean (changes of 1.8-fold) was used to remove genes for which the experimental noise could explain the variation in gene expression. Genes were included in the further analysis if their expression exceeded this cutoff in at least one experiment in which gene dosage was not increased compared to wild type (3124 genes).
Expression data obtained from cells grown in the chemostat were filtered and renormalized as described for data obtained from exponentially growing cells. A comparison of two independently grown wild-type samples showed better reproducibility than the batch cultures, with half the standard deviation. Using a cutoff of two standard deviations over the mean (a 1.3 fold change), 4963 genes, most of the yeast genome, changed in at least one experiment in which the gene was not contained on a disome.
Hierarchical clustering was performed using the program WCluster (http://function.princeton.edu/WCluster/). WCluster takes both a data table and a weight table to allow individual measurements to be differentially considered by the clustering algorithm. Expression data were clustered by a Pearson correlation metric with equal weighting given to all data, or with no weight given to genes on the disomic chromosomes, as indicated in the text.
Batch culture growth conditions. Wild-type and disomic strains were grown to OD600=1 in −His G418 medium.
Chemostat growth conditions. ATR Sixfors fermenters were modified for use as chemostats. Chemostat cultures were run at 30° C. at a working volume of 300 mls, mixed at 400 rpm, and sparged at 5 standard liters per minute with humidified and filter-sterilized air. The dilution rate was set to 0.17 volumes/hour. Cultures were run in phosphate limited minimal defined medium containing the following (per liter): 100 mg calcium chloride, 100 mg sodium chloride, 500 mg magnesium sulfate, 5 g ammonium sulfate, 1 g potassium chloride, 500 μg boric acid, 40 μg copper sulfate, 100 μg potassium iodide, 200 μg ferric chloride, 400 μg manganese sulfate, 200 μg sodium molybdate, 400 μg zinc sulfate, 1 μg biotin, 200 μg calcium pantothenate, 1 μg folic acid, 1 mg inositol, 200 μg niacin, 100 μg p-aminobenzoic acid, 200 μg pyridoxine, 100 μg riboflavin, 200 μg thiamine, 50 mg adenine, 50 mg tryptophan, 20 mg uracil, 100 mg lysine, 20 mg methionine, 100 mg leucine, 100 mg G418, and 5 g glucose.
Chemostats were inoculated with 1 ml overnight culture grown in chemostat media. Cultures were maintained in batch for 24 hours, at which time the media flow was switched on. Cultures were sampled daily for cell density by Coulter count, klett, and absorbance, and were considered to be in steady state when all parameters were the same for two consecutive measurements, which occurred 4 days after inoculation for all cultures. 100 ml of cultures were harvested by vacuum filtration, flash-frozen in liquid nitrogen, and stored at −80° C. until RNA extraction. 50 ml cultures were harvested onto pre-weighed filters, baked overnight, and weighed for yield measurements. Samples of the filtrate were also saved for analysis.
Cell cycle analyses. Cells were arrested in G1 in YPD with 5 μg/ml α-factor for 3 hours. 1.5 hours into the arrest 2.5 μg/ml α-factor was readded. Cells were washed with 10 volumes of YEPD and released into medium lacking pheromone. In the analysis shown in
Cell viability analysis. Cells were grown in selective media overnight and counted using a Multizer 3 Coulter counter. 500 cells were plated on YPD plates and colonies were counted after 2-3 days. Viability is reported as the fraction of the cells plated able to form colonies. In the case of strains disomic for chromosomes IV, XI, XI+XV or I+VI+XIII 2,000-5,000 cells were plated. To determine the viability of cells grown to saturation the number of cells plated was increased as viability decreased. Only data from plates containing 100 colonies or more were included in the analysis.
Quantitative western blot analysis. For quantitative Western blot analysis cells were grown in −His G418 medium to OD600=1. 10 mls of culture were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM DTT, 2 mM EDTA plus protease inhibitors) using acid washed glass beads. 50, 25, 12.5 and 6.25 μg of lysate were loaded. Nop1 was detected using a mouse anti-Nop 1 antibody at 1:10,000 dilution (Abcam cat#ab4575). Rpa1 was detected using a mouse anti-Rpa1 antibody at 1:1,000 dilution (Genetex cat# GTX16850). Mre11 was detected using a rabbit anti-Mre11 antibody at 1:1,000 dilution (Abcam cat#ab12159). Rps2/Rp132 were detected using a rabbit anti-Rps2 antibody at 1:2,000 dilution (kindly provided by Dr. Jonathan R. Warner). Arp5 was detected using a rabbit anti-Arp5 antibody at 1:1,000 dilution (Abcam cat#ab12099). Tcp1 was detected using a rat anti-Tcp1 antibody at 1:5,000 dilution (Abcam cat#ab2797). Cdc28 was detected using a rabbit anti-Cdc28 antibody at 1:1,000 dilution (S12). Pgk1 was detected using a mouse anti-Pgk1 antibody at 1:25,000 dilution (Molecular Probes cat#A-6457), Pup3 was detected using a mouse anti-Pup3 antibody at 1:1000 dilution (Abcam cat#ab22672). Pre6 was detected using a mouse anti-Pre6 antibody at 1:1,000 dilution (Abcam cat#ab22667). Hht1 was detected using a mouse anti-Hht1 antibody at 1:20,000 dilution (Upstate cat#07-690). Rpt1 was detected using a mouse anti-Rpt1 antibody at 1:5,000 dilution (Abcam cat#ab22678), Lcb4 was detected using a goat anti-Lcb4 antibody at 1:200 dilution (Santa Cruz cat# sc-27723). Elp3 was detected using a goat anti-Elp3 antibody at 1:1,000 dilution (Santa Cruz cat# sc-26320). Eaf3 was detected using a rabbit anti-Eaf3 antibody at 1:1,000 dilution (Abcam cat#ab4467). Yaf9 was detected using a rabbit anti-Yaf9 antibody at 1:1,000 dilution (Abcam cat#ab4468). Fcy1 was detected using a sheep anti-Fcy1 antibody at 1:1,000 (AbD Serotec cat#2485-4906).
Other techniques. CLN2 RNA, Cln2 protein levels and DNA content were analyzed as described in (S13-S15). Glucose measurements were performed using the Glucose Assay kit (Sigma Cat#GAHK-20) according to the manufacture's instructions.
To generate yeast cells that contain an additional chromosome, a chromosome transfer strategy was used. During mating, if one of the mating partners lacks the karyogamy gene KAR1, nuclear fusion does not occur (8). However, occasionally individual chromosomes are transferred from one nucleus to the other during these abortive matings (8, 9). When the two mating partners carry different selectable markers at the same genomic location, these rare chromosome transfers can be selected for (
To ensure that strains with the correct marker combination were indeed disomic for the entire chromosome, comparative genomic hybridization (CGH) was performed, which allows for the quantification of gene copy number on a genome-wide scale. This analysis also revealed that some of the strains obtained from the chromosome transfer procedure carried one or two extra chromosomes in addition to the one that was selected for (
In order to characterize the effects of aneuploidy on gene expression, each aneuploid yeast strain was grown to mid-log phase in batch culture and genome wide gene expression relative to the wild type strain was measured by DNA microarrays. An approximate 2-fold increase in gene expression was observed along the entire length of the disomic chromosomes, indicating that most if not all genes are expressed proportionally to the number of DNA copies in the cell (
To reveal more subtle correlations masked by the strong chromosome-specific signals (FIG. S3A), a clustering program was applied that allows the assignment of a reduced weight to genes on disomic chromosomes (10)(
All aneuploid strains that were examined proliferated more slowly than did wild type cells (
To determine how aneuploidy affects cell physiology, the proliferation properties of strains carrying one or several extra chromosomes were characterized. The doubling time and cell size was slightly increased in most aneuploid strains in complete medium (YEPD;
To determine in which stage of the cell cycle the aneuploid yeast strains were delayed, cell cycle progression after release from a pheromone-induced G1 phase arrest was examined. Entry into the cell cycle, as judged by bud formation (
The molecular events underlying the G1 to S phase transition are well characterized in S. cerevisiae. The cyclin-dependent kinase (CDK) Cdc28 associated with the cyclin Cln3, inhibits Whi5, an inhibitor of the transcription factor complex SBF (15, 16). SBF in turn induces the transcription of genes encoding two other cyclins CLN1 and CLN2, which when complexed with Cdc28, promote entry into the cell cycle (17). The abundance of CLN2 RNA and Cln2 protein in strains disomic for chromosome IV, XIII or VIII+XIV was analyzed. Accumulation of CLN2 RNA and Cln2 protein was delayed and paralleled the delay in bud formation and DNA replication (
To further investigate the effects of aneuploidy on cell proliferation, the kinetics with which aneuploid cells enter stationary phase were examined. Most aneuploids reached saturation at a lower population size (measured by optical density at 600 nm [OD600],
To determine whether the lower OD600 at which aneuploids enter stationary phase was due to nutrient depletion, we simultaneously measured glucose uptake and accumulation of biomass. This comparison revealed that wild-type cells generated more biomass per internalized glucose molecule than did aneuploid cells. Whereas wild-type cells reached cell densities of OD600=9 having taken up ¾ of the glucose in the medium, cells disomic for chromosome IV only reached a cell density of OD600 of less than 4 (
Consistent with the idea that aneuploids take up more glucose was the observation that the gene loci encoding the high affinity glucose transporters Hxt6 and Hxt7 were amplified (FIG. S2B) and more highly expressed (
It was hypothesized that macromolecule biosynthesis from the additional chromosome present in aneuploid strains could be one reason why aneuploids need additional glucose. To test this hypothesis expression profile analysis of aneuploids was performed and showed that most genes present on the additional chromosomes were transcribed: 93% of genes carried on the chromosome that was present in two copies were overexpressed at least 1.3-fold over wild type, and expression of 83% of genes went up by 1.5-fold or more (
Interestingly, most of the proteins (13 out of 16) that were analyzed showed no change in abundance even though the amount of transcript was increased in accordance with the increase in gene copy number (
Consistent with the idea that increased protein degradation occurs in aneuploid yeast strains is the observation that proliferation of a number of aneuploid strains (IV, XII, XIII, XIV, and XVI) is inhibited by concentrations of the proteasome inhibitor MG 132 that wild-type cells grow at as judged by their ability to form colonies on plates containing the drug (
To determine whether the synthesis of proteins from the additional chromosomes and their presence in the cell represents an increased burden on the cell's protein production machinery, the ability of aneuploid strains to grow under conditions that interfere with transcription, protein synthesis and protein folding was examined. Proliferation of all aneuploids with the exception of strains disomic for chromosome I, X, or XIV was inhibited by high (20 μg/ml) concentration of the RNA polymerase inhibitor Thiolutin (
Proliferation of aneuploids was also decreased under conditions that led to the accumulation of unfolded proteins. All strains carrying an extra chromosome, with the exception of cells disomic for chromosome I, showed impaired proliferation at increased temperatures (37° C.;
Aneuploids did not exhibit increased sensitivity to any toxic agents. Aneuploids formed colonies as well as wild-type cells on medium containing the DNA replication inhibitor hydroxyurea (
The phenotypes shared by aneuploids might result from the mere presence of additional DNA or from the RNAs and proteins synthesized from these chromosomes. Thus, the effects of seven yeast artificial chromosomes (YACs) containing human or mouse DNA inserts ranging from approximately 350 kb to 1.6 MB in size were tested (Table 3). Although the possibility that some transcription and translation occurs from the mammalian DNA in yeast cannot be excluded, the YACs do not produce yeast proteins and it is highly likely that the amount of transcription and translation from the YACs is less than that occurring from yeast chromosomes, which are densely packed with mostly intron-less genes.
The gene expression profile shared by aneuploid strains grown under phosphate-limiting chemostat conditions was also observed in YAC-carrying strains (
15d
aFACS analysis revealed similar delays.
bNumber of experiments.
cThese delays were difficult to quantify due to severity of G1 delay.
dDelays were variable between different strains for disome XII.
eThese strains contain a small YAC (155 kb) that contains the left arm of chromosome III.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Ser. No. 60/815,692, filed Jun. 22, 2006, which is incorporated herein by reference in its entirety.
This invention was supported in part by National Institutes of Health grant GM56800. The United States government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60815692 | Jun 2006 | US |
