WO2004083399A2 - Aberrantly methylated genes in pancreatic cancer - Google Patents
Aberrantly methylated genes in pancreatic cancer Download PDFInfo
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- WO2004083399A2 WO2004083399A2 PCT/US2004/008061 US2004008061W WO2004083399A2 WO 2004083399 A2 WO2004083399 A2 WO 2004083399A2 US 2004008061 W US2004008061 W US 2004008061W WO 2004083399 A2 WO2004083399 A2 WO 2004083399A2
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Definitions
- the present invention relates generally to the regulation of gene expression and more specifically to a method of determining the DNA methylation status of CpG sites in a given locus and correlating the methylation status with the presence of a cell proliferative disorder.
- DNA methylases transfer methyl groups from the universal methyl donor
- Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R.D. eds. in DNA Methylation Biochemistry and Biological Significance. Springer-Nerlag, New York, 1984).
- methylation of cytosine residues that are immediately 5' to a guanosine occurs predominantly in CG poor regions (Bird, A., Nature, 321:209, 1986).
- CpG islands remain unmethylated in normal cells, except during X- chromosome inactivation (Migeon, et al., supra) and parental specific imprinting (Li, et al., Nature, 366:362, 1993) where methylation of 5' regulatory regions can lead to transcriptional repression.
- De novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas (Sakai, et al., Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the NHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al., Proc. ⁇ atl. Acad.
- Human cancer cells typically contain somatically altered nucleic acid, characterized by mutation, amplification, or deletion of critical genes.
- the nucleic acid from human cancer cells often displays somatic changes in DNA methylation (E.R. Fearon, et al., Cell, 61:759, 1990; P. A. Jones, et al., Cancer Res., 46:461, 1986; R. Holliday, Science, 238:163, 1987; A. De Bustros, et al., Proc. Natl. Acad. Sci., USA, 85:5693, 1988); P.A. Jones, et al., Adv. Cancer Res., 54:1, 1990; S.B.
- CRC colorectal cancers
- TSG tumor suppressor genes
- the cancers from these patients show a characteristic mutator phenotype which causes microsatellite instability (MI), and mutations at other gene loci such as TGF- ⁇ -R-Q (Markowitz et al., Science, 268(5215): 1336-8, 1995) and BAX.
- MI microsatellite instability
- TGF- ⁇ -R-Q Markowitz et al., Science, 268(5215): 1336-8, 1995
- BAX a characteristic mutator phenotype which causes microsatellite instability (MI), and mutations at other gene loci such as TGF- ⁇ -R-Q (Markowitz et al., Science, 268(5215): 1336-8, 1995) and BAX.
- MMR mismatch repair
- CGI CpG island
- CGIs are short sequences rich in the CpG dinucleotide and can be found in the 5' region of about half of all human genes. Methylation of cytosine within 5' CGIs is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting. Aberrant methylation of CGIs has been detected in genetic diseases such as the fragile-X syndrome, in aging cells and in neoplasia.
- TSG methylation in cancer is usually associated with (Antequera, et al., Proc. ⁇ atl. Acad. Sci. USA, 90: 11995-11999, 1993) lack of gene transcription and (Baylin, et al., Adv. Cancer Res., 72:141-196, 1998) absence of coding region mutation.
- CGI methylation serves as an alternative mechanism of gene inactivation in cancer.
- Mapping of methylated regions in DNA has relied primarily on Southern hybridization approaches, based on the inability of methylation-sensitive restriction enzymes to cleave sequences which contain one or more methylated CpG sites. This method provides an assessment of the overall methylation status of CpG islands, including some quantitative analysis, but is relatively insensitive and requires large amounts of high molecular weight DNA.
- Another method utilizes bisulfite treatment of DNA to convert all unmethylated cytosines to uracil.
- the altered DNA is amplified and sequenced to show the methylation status of all CpG sites.
- this method is technically difficult, labor intensive and without cloning amplified products, it is less sensitive than Southern analysis, requiring approximately 10% of the alleles to be methylated for detection.
- Pancreatic cancer is the fourth leading cause of cancer death in men and in women and each year -28,000 Americans die of the disease (Greenlee, et al., CA Cancer J. Clin. 50:7-33, 2000). Frequent genetic changes such as mutational activation of the K- ras oncogene and inactivation of the pl6, DPC4, p53, MKK4, ST 11, TGFBR2, and TGFBR1 tumor suppressor genes have been described in pancreatic cancer (Goggins, et al., Ann. Oncol. 10:4-8, 1999, Rozenblum, et al, Cancer Res. 57:1731-1734, 1997).
- MCA methylated CpG island amplification
- the present invention is based on the finding that several genes are newly identified as being differentially methylated in pancreatic cancer. This seminal discovery is useful for cancer screening, risk-assessment, prognosis, minimal-residual disease identification, staging and identification of therapeutic targets.
- the identification of genes that are methylated in cancer, aging or diseases associated with aging increases the likelihood of finding genes methylated in a particular cancer; increases the sensitivity and specificity of methylation detection; allows methylation profiling using multiple genes; and allows identification of new targets for therapeutic intervention.
- a method for detecting pancreatic carcinoma in a subject comprising contacting a nucleic acid-containing specimen from the subject with an agent that provides a determination of the methylation state of at least one gene or associated regulatory region of the gene selected from Tables 1-3 and combinations thereof; and identifying aberrant methylation of regions of the gene or regulatory region, wherein aberrant methylation is identified as being different when compared to the same regions of the gene or associated regulatory region in a subject not having the pancreatic carcinoma, thereby detecting pancreatic carcinoma in the subject.
- the regions of the gene or regulatory region are contained within CpG rich regions. Illustrative genes are listed in Tables 1-3.
- the aberrant methylation is hypermethylation when compared to the same regions of the gene or associated regulatory regions in a subject not having pancreatic carcinoma.
- a pair of primers that hybridize with a target sequence in the gene or associated regulatory region of the gene are utilized to identify genes or regulatory regions associated with pancreatic cancer.
- the nucleic acid-containing specimen includes tissue such as pancreatic ductal epithelium, pancreatic tissue, stool, blood, or pancreatic fluid.
- Pancreatic carcinoma includes, but is not limited to, pancreatic ductal adenocarcinoma, chronic pancreatitis, islet cell tumor, or serus cystadenoma.
- a method of identifying at least one gene silenced by DNA methylation associated with pancreatic carcinoma includes contacting an array of nucleotide sequences representative of a genome with nucleic acid molecules corresponding to RNA expressed in cancer cells contacted with at least one agent that reactivates expression of silenced genes but not RNA expressed in normal cells corresponding to the cancer cells, under conditions suitable for selective hybridization of nucleic acid subtraction products to complementary nucleotide sequences of the array; and detecting selective hybridization of nucleic acid to a subpopulation of nucleotide sequences of the array, wherein nucleic acid molecules corresponding to RNA expressed in the normal cells corresponding the cancer cells do not hybridize to the subpopulation of nucleotide sequences under the conditions suitable for selective hybridization, whereby the nucleic acid molecules that selectively hybridize to the subpopulation of nucleotide sequences of the array represent epigenetically silenced genes of the cancer cells, thereby identifying at least one epige
- Determining the methylation state of the gene includes contacting the nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying a CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and detecting the methylated nucleic acid based on the presence or absence of amplification products produced in the amplifying step.
- the method includes optionally contacting the amplification products with a methylation sensitive restriction endonuclease.
- the methylating agent 5 aza 2' deoxycytidine (5Aza-dC) is used to treat cells for further determination of the methylation status.
- the histone deacetylase inhibitor trichostatin (TSA) is used to treat cells for further determination of the methylation status.
- TSA histone deacetylase inhibitor
- a combination of 5 aza 2' deoxycytidine (5 Aza-dC) and trichostatin (TSA) is utilized. Genes associated with pancreatic carcinoma are listed in Tables 1-3 by way of example.
- genes include CDH3, reprimo, CLDN5, DR3, FOXE1, LDOC1, LHX1, NEFH, NPTX2, PIG11, SARP2, ST14, SMARCA1, TJP2, UCHL1, WNT7A, or a combination thereof.
- an amplification primer pair in another aspect of the invention, includes a forward primer and a reverse primer as set forth in SEQ ID NOS: 1 to 64, wherein the amplification primer pair amplifies a portion of a gene of Tables 1-3. In one aspect, the amplification primer pair specifically amplifies a methylated 5' regulatory region of the nucleic acid molecule.
- these amplification primer pairs include SEQ ID NOS: 3 and 4, SEQ ID NOS: 7 and 8, SEQ ID NOS: 11 and 12, SEQ ID NOS: 15 and 16, SEQ ID NOS: 19 and 20, SEQ ID NOS: 23 and 24, SEQ ID NOS: 27 and 28, SEQ ID NOS: 31 and 32, SEQ ID NOS: 35 and 36, SEQ ID NOS: 39 and 40, SEQ ID NOS: 43 and 44, SEQ ID NOS: 47 and 48, SEQ ID NOS: 51 and 52, SEQ ID NOS: 55 and 56, SEQ ID NOS: 59 and 60 or SEQ ID NOS: 63 and 64.
- the amplification primer pair specifically amplifies a unmethylated 5' regulatory region of the nucleic acid molecule. More specifically, these amplification primer pairs include SEQ ID NOS: 1 and 2, SEQ ID NOS: 5 and 6, SEQ ID NOS: 9 and 10, SEQ ID NOS: 13 and 14, SEQ ID NOS: 17 and 18, SEQ ID NOS: 21 and 22, SEQ ID NOS: 25 and 26, SEQ ID NOS: 29 and 30, SEQ ID NOS: 33 and 34, SEQ ID NOS: 37 and 38, SEQ ID NOS: 41 and 42, SEQ ID NOS: 45 and 46, SEQ ID NOS: 49 and 50, SEQ ID NOS: 53 and 54, SEQ ID NOS: 57 and 58, or SEQ ID NOS: 61 and 62.
- a method for monitoring a therapeutic regimen for treating a subject having pancreatic cancer includes a) obtaining a nucleic acid-containing specimen from the subject prior to therapy; b) contacting the nucleic acid-containing specimen with an agent that provides a determination of the methylation state of at least one gene or associated regulatory region of the gene; c) identifying aberrant methylation of regions of the gene or regulatory region, wherein aberrant methylation is identified as being different when compared to the same regions of the gene or associated regulatory region in a subject not having the pancreatic cancer; and d) determining a change in the methylation state of the gene during therapy, wherein the change is determined by comparing the methylation state of the gene with the methylation state of the same gene from the nucleic acid-containing sample from the same subject prior to therapy, thereby monitoring a therapeutic regimen for treating a pancreatic cancer subject.
- the kit further includes a reagent that modifies methylated cytosine residues.
- the kit further comprises a methylation sensitive restriction endonuclease.
- the kit comprises reagents for performing an amplification reaction.
- the kit includes 5 -aza-2' -deoxycytidine and/or trichostatin A.
- kits useful for the detection of pancreatic carcinoma in a subject including a carrier means compartmentalized to receive a sample therein; one or more containers comprising a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of a CpG-containing nucleic acid, wherein the primers are pairs from SEQ ID NO: 1-64.
- the kit includes a third container containing a methylation sensitive restriction endonuclease.
- the modifying reagent is typically bisulfite.
- the primer hybridizes with a target sequence as set forth in Tables 1-3.
- Another aspect of the invention provides a gene associated with pancreatic carcinoma as listed in Tables 1-3, wherein associated regulatory sequences contain CpG- rich regions.
- the state of methylation of the CpG-rich regions is determinative of the presence of pancreatic carcinoma in a subject from which the nucleic acid molecule is isolated.
- hypermethylation of the CpG-rich regions is indicative of the presence of pancreatic carcinoma in a subject from which the nucleic acid is isolated.
- Figure la shows RT-PCR analysis of five genes (CDH3, NPTX2, SARP2,
- UCHL1, and WNT7A pancreatic cancer cell lines in pancreatic cancer cell lines (AsPCl, and MiaPaCa2).
- Cells were treated with 5Aza-dC alone, TSA alone, or a combination of both and subjected to RNA extraction.
- Glyceraldehyde-3-phospate dehydrogenase (GAPDH) serves as a RNA control.
- Figure lb shows MSP analysis of five genes (CDHS, CLDN5, NPTX2,
- Figure 2 is a graphical diagram summarizing the methylation profiles of
- Figure 3 shows graphical expression patterns of 11 genes aberrantly methylated in pancreatic cancer after treatment with 5Aza-dC, TSA, or a combination of both in four pancreatic cancer cell lines.
- Cells were treated with 5Aza-dC alone, TSA alone, or a combination of both, and subjected to oligonucleotide microarray hybridization.
- FIG. 4 shows MSP analysis of three genes (CLDN5, NPTX2, and
- SARP2 in a series of normal pancreatic ductal epithelia, primary pancreatic carcinomas, and pancreatic juice samples.
- an aberrant methylation state and/or histone deacetylase (HDAC) activity (with the methylation being predominant) of nucleic acids in certain genes, particularly regulatory sequences, is diagnostic for the presence or potential development of a cellular proliferative disorder in subjects bearing the aberrantly methylated nucleic acids. More particularly, the hypermethylation of certain nucleotides localized in CpG islands has been shown to affect the expression of genes associated with the CpG islands; typically such hypermethylated genes have reduced or abolished expression, primarily due to down-regulated transcription. Using a well known technique called methylation specific PCR (MSP), several nucleic acid molecules aberrantly methylated in pancreatic cancer cells were identified.
- MSP methylation specific PCR
- the presently disclosed microarray based strategy obviates the disadvantages of previous methods by coupling gene expression status to epigenetic regulation. Furthermore, the approach exploits the observation that global changes in gene expression in cancer genes can be dependent on both dense CpG island methylation and HDAC activity (Cameron et al., Nature Genet. 21:103-107, 1999, which is incorporated herein by reference).
- the disclosed methods robustly identify genes for which transcriptional repression can have a key role in tumorigenesis.
- the disclosed genomic screening method allowed an identification of gene hypermethylation events that cluster to specific tumor types, and can simultaneously involve multiple members of a single gene family.
- a global change in gene expression refers to a change in any function normally attributed to a cell containing the gene
- Such global changes in gene expression include, but are not limited to, reactivation of a gene that is epigenetically silenced.
- the term "epigenetically silenced” or “epigenetic silenced”, when used in reference to a gene, means that the gene is not being transcribed, or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (e.g., a normal cell), due to a mechanism other than a genetic change.
- Epigenetic mechanisms of gene silencing include, for example, hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of a gene, and structural changes in chromatin due, for example, to histone acetylation, such that gene transcription is reduced or inhibited.
- Methods for detecting epigenetic silencing of a gene include, for example, detecting re- expression (reactivation) of the gene following contact of a cell with an agent that relieves the epigenetic silencing, for example, with a demethylating agent where the silencing is due to hypermethylation.
- Methylated nucleic acid sequences are also provided.
- methylation or “hypermethylation”, when used in reference to a gene, means that cytosine residues of CpG dinucleotides in a CpG island associated with the gene are methylated at the 5'-position, i.e., 5'-methylcytosine.
- methylation status is used herein to refer to a relative abundance, including the presence or absence, of methylated cytosine residues of CpG dinucleotides in a CpG island.
- cytosine residues in a CpG island are not methylated in a transcriptionally active gene and, therefore, the detection of methylated cytosine residues in a CpG island indicates that expression of the gene is reduced or inhibited.
- reference herein to a "methylation silenced" gene means that the gene is not being transcribed, or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (generally a normal cell) due to hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of the gene.
- a consequence of methylation silenced gene expression is that a cell containing the gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene (i.e., the gene product) such that any function normally attributed to the gene product in the cell is reduced or absent.
- a method of identifying an epigenetically silenced gene associated with a cancer can be performed, for example, by contacting an array of nucleotide sequences representative of a genome with nucleic acid subtraction products (i.e., nucleic acid molecules corresponding to RNA expressed in cancer cells contacted with at least one agent that reactivates expression of epigenetically silenced genes, but not RNA expressed in normal cells corresponding to the cancer cells) under conditions suitable for selective hybridization of nucleic acid subtraction products to complementary nucleotide sequences of the array; and detecting selective hybridization of nucleic acid subtraction products to a subpopulation of nucleotide sequences of the array, wherein nucleic acid molecules corresponding to RNA expressed in the normal cells corresponding to the cancer cells do not hybridize to the subpopulation of nucleotide sequences under such conditions suitable for selective hybridization, whereby the nucleic acid subtraction products that selectively hybridize to the subpopulation of nucleotide
- nucleic acid molecules corresponding to RNA of a cell means RNA such as mRNA or polyA+ RNA, cDNA generated using RNA from the cell as a template, or cRNA generated using RNA or cDNA as a template.
- the nucleic acid molecules corresponding to RNA of a cell generally are detectably labeled, for example, with a radioisotope, a paramagnetic isotope, a luminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, an enzyme, a substrate for an enzyme, a receptor, or a ligand for a receptor; or are capable of being detected, for example, using a detectably labeled probe, such that hybridization of the nucleic acid molecules to nucleotide sequences of the array can be detected.
- array of nucleotide sequences representative of a genome means an organized group of nucleotide sequences that are linked to a solid support, for example, a microchip or a glass slide, wherein the sequences can hybridize specifically and selectively to nucleic acid molecules expressed in a cell.
- the array is selected based on the organism from which the cells to be examined are derived and/or on a tissue or tissues that are to be examined.
- the array is representative of the genome of a eukaryotic cell or cell type, particularly a mammalian cell or cell type, and preferably a human cell, including a cell of one or more tissues, as desired (e.g., pancreatic epithelial cells).
- an array of probes that is "representative" of a genome will identify at least about 10% of the expressed nucleic acid molecules in a cell, generally at least about 20% or 40%, usually about 50% to 70%, typically at least about 80% or 90%, and particularly 95% to 99% or more of the expressed nucleic acid molecules of a cell or organism. It should be recognized that the greater the representation, the more likely that a method of the invention can identify all genes that are epigenetically silenced in a cancer.
- Arrays containing nucleotide sequences representative of specified genomes can be prepared using well known methods, or obtained from a commercial source (e.g., Invitrogen Corp.; Affymetrix), as exemplified by a Human Genome U133A chip (Affymetrix, Santa Clara, CA) used in the present studies.
- a commercial source e.g., Invitrogen Corp.; Affymetrix
- Affymetrix Human Genome U133A chip
- the agent that reactivates expression of epigenetically silenced genes can be a methyltransferase inhibitor(e.g., 5 aza 2' deoxycytidine; 5Aza-dC), a histone deacetylase inhibitor (e.g., trichostatin A; TSA), or a combination of agents such as a combination of 5 Aza-dC and TSA.
- a methyltransferase inhibitor e.g., 5 aza 2' deoxycytidine; 5Aza-dC
- a histone deacetylase inhibitor e.g., trichostatin A; TSA
- TSA histone deacetylase inhibitor
- a combination of agents such as a combination of 5 Aza-dC and TSA.
- RNA can be isolated from cells such as cancer cells treated with such an agent or agent, and the RNA, or a cDNA product of the RNA can be contacted with RNA molecules from corresponding cells (e.g., cancer cells) that were not treated with the agent(s) under conditions such that RNA (or cDNA) expressed only in the treated cells can be isolated, thus obtaining nucleic acid subtraction products.
- Methods for performing a nucleic acid subtraction reaction are well known (Hedrick et al., Nature 308: 149-155, 1984, which is incorporated herein by reference), and kits for performing such methods are available from commercial sources (e.g., Gibco/BRL).
- the methods of the invention identify potential targets for aberrant methylation in pancreatic cancer by analyzing gene expression profiles of cancer cells after exposure to 5Aza-dC and/or TSA.
- This embodiment includes, in part, a comparison of the methylation status of a gene in a test cell or sample with the methylation status of a corresponding gene in a corresponding cell exhibiting regulated growth.
- corresponding means a reference material, with which a test material is being compared. Generally, the reference material provides a control or standard with which the test material is compared.
- reference to a corresponding unmethylated SFRP gene with respect to an SFRP gene being examined for methylation status, means that the unmethylated SFRP gene is the same type of gene as the a SFRP gene being examined for methylation status, e.g., the test gene and the corresponding unmethylated gene are both human a SFRP1 genes.
- Reference to a corresponding cell exhibiting regulated growth, with respect to a test cell generally refers to a normal cell, i.e., a cell that has a cell cycle and growth pattern characteristic of a population of such cells in a healthy individual, for example, a normal pancreatic epithelial cell where the test cell being examined is suspected of being a pancreatic cancer cell.
- a test cell examined according to a method of the invention also can be a primary cell that has been obtained from a subject and placed in culture, for example, for the purpose of establishing a primary cell culture that exhibits substantially the same growth characteristics as the cells from which the culture was established, or for the purpose of treating and/or expanding the cells for readministration to the subject.
- pancreatic ductal epithelial cells can be obtained from a cancer patient suffering from pancreatic cancer, wherein the cells exhibit methylation silenced expression of one or more genes associated with the cancer.
- the cells can be treated in culture using one or more agents to be tested for an ability to restore expression of the silenced gene(s), thus providing a means to identify an agent that can be useful for treating the cancer patient, or another patient having a pancreatic cancer characterized by methylation silencing of one or more of the same genes.
- a test cell can be obtained from a subject in any way typically used in clinical setting for obtaining a sample containing the cells.
- the test cells (or a sample comprising the test cells) can be obtained by a biopsy procedure such as needle biopsy of an organ or tissue containing the cells to be tested.
- the test cells can be obtained from a gastrointestinal tract sample (e.g., a biopsy of a polyp), a liver sample, a pancreatic tissue sample, a bone marrow sample, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, a brain sample, or the like.
- the test cell also can be a component of a biological fluid, for example, blood, lymph, cerebrospinal fluid, pancreatic juice, saliva, sputum, stool, urine, or ejaculate.
- the test cells also can be obtained by lavage, for example, for obtaining test cells from the colon, uterus, abdominal cavity, or the like, or using an aspiration procedure, for example, for obtaining a bone marrow sample.
- the method for identifying a cell that exhibits or is predisposed to exhibiting unregulated growth is performed by detecting methylation of one or more target genes in the cell.
- Methylation of a CpG dinucleotide in a CpG island of a gene can be detected using any of various well known methods for detecting CpG methylation of a nucleic acid molecule.
- Such methods include contacting the gene with one or a series of chemical reagents that selectively modify either unmethylated cytosine residues or methylated cytosine residues, but not both, such that the presence or absence of the modification can be detected; contacting the gene sequence with a methylation sensitive restriction endonuclease, which has a recognition site that includes a CpG dinucleotide, and that cleaves a recognition site either having a methylated cytosine residue of the CpG or lacking a methylated cytosine residue of the CpG, but not both, such that the presence or absence of cleavage of the sequence can be detected; or contacting a nucleic acid molecule comprising the gene with an oligonucleotide probe, primer, or amplification primer pair that selectively hybridizes to the gene sequence and allows a determination to made as to whether the CpG methylation is present.
- Methylation of a target gene can be detected, for example, by contacting a region comprising a 5' regulatory region of a nucleic acid molecule comprising the gene with a methylation sensitive restriction endonuclease, which cleaves a recognition site in the 5' regulatory region comprising a methylated cytosine residue of a CpG dinucleotide, whereby cleavage of the nucleic acid molecule is indicative of methylation and, therefore, methylation silencing of the gene of the test cell.
- Methylation sensitive restriction endonucleases are well known and include, for example, Ace HI, Ban I, BstN I, Msp I, and Xma I.
- methylation silencing can be detected by contacting a region comprising a 5' regulatory region of a nucleic acid molecule comprising the gene with a methylation sensitive restriction endonuclease, which cleaves a recognition site in the 5' regulatory region comprising a methylated cytosine residue of a CpG dinucleotide, provided the cytosine residue of the CpG dinucleotide is unmethylated, whereby a lack of cleavage of the nucleic acid molecule is indicative of methylation silencing of the gene of the test cell.
- Such methylation sensitive restriction endonucleases are exemplified by Ace If, Ava I, BssH ⁇ , BstU I, Hpa TJ, and Not I.
- cleavage of a nucleic acid molecule comprising a target gene sequence by a methylation sensitive restriction endonuclease can be identified using any method useful for detecting the length or continuity of a polynucleotide sequence.
- cleavage of the target gene sequence can be detected by Southern blot analysis, which allows mapping of the cleavage site, or using any other electrophoretic method or chromatographic method that separates nucleic acid molecules on the basis of relative size, charge, or a combination thereof.
- Cleavage of a target gene also can be detected using an oligonucleotide ligation assay, wherein, following contact with the restriction endonuclease, a first oligonucleotide that selectively hybridizes upstream of and adjacent to a restriction endonuclease cleavage site and a second oligonucleotide that selectively hybridizes downstream of and adjacent to the cleavage site are contacted with the target gene sequence, and further contacted with a ligase such that, in the absence of cleavage the oligonucleotides are adjacent to each other and can be ligated together, whereas, in the absence of cleavage, ligation does not occur.
- ligated oligonucleotides By determining the size or other relevant parameter of the oligonucleotides following the ligation reaction, ligated oligonucleotides can be distinguished from unligated oligonucleotides, thereby providing an indication of restriction endonuclease activity.
- Methylation silencing of a gene also can be detected by contacting a 5' regulatory region of the nucleic acid molecule comprising the gene of the test cell with a chemical reagent that selectively modifies either an unmethylated cytosine residue or a methylated cytosine residue, and detecting a product generated due to the contacting, wherein the product is indicative of methylation of a cytosine residue in a CpG dinucleotide of the gene, thereby detecting methylation silencing of the gene of the test cell.
- the product can be detected using an electrophoresis method, a chromatography method, a mass spectrometry method, or a combination of such methods.
- a nucleic acid molecule comprising the target gene is contacted with a chemical reagent comprising bisulfite ions, for example, sodium bisulfite, which converts unmethylated cytosine residues to bisulfite modified cytosine residues, then the bisulfite ion treated gene sequence is exposed to alkaline conditions, which convert bisulfite modified cytosine residues to uracil residues.
- bisulfite ions for example, sodium bisulfite
- alkaline conditions which convert bisulfite modified cytosine residues to uracil residues.
- Sodium bisulfite reacts readily with the 5,6 double bond of cytosine (but poorly with methylated cytosine) to form a sulfonated cytosine reaction intermediate that is susceptible to deamination, giving rise to a sulfonated uracil.
- the sulfonate group can be removed by exposure to alkaline conditions, resulting in the formation of uracil.
- the DNA then can amplified, for example, by PCR, and sequenced to determine the methylation status of all CpG sites.
- Uracil is recognized as a thymine by Taq polymerase and, upon PCR, the resultant product contains cytosine only at the position where 5 methylcytosine was present in the starting template DNA.
- the amount or distribution of uracil residues also can be detected by contacting the bisulfite ion treated target gene sequence, following exposure to alkaline conditions, with an oligonucleotide that selectively hybridizes to a nucleotide sequence of the target gene that either contains uracil residues or that lacks uracil residues, but not both, and detecting selective hybridization (or the absence thereof) of the oligonucleotide.
- selective hybridization or “selectively hybridize” or “specific hybridization” refers to an interaction of two nucleic acid molecules that occurs and is stable under moderately stringent or highly stringent conditions.
- selective hybridization preferentially occurs, for example, between an oligonucleotide and a target nucleic acid molecule, and not substantially between the oligonucleotide and a nucleic acid molecule other than the target nucleic acid molecule, including not with nucleic acid molecules encoding related but different members of a gene family.
- an oligonucleotide useful as a probe or primer that selectively hybridizes to a target nucleic acid molecule is at least about 12 to 15 nucleotides in length, generally at least about 18 to 20 nucleotides in length, usually at least about 21 to 25 nucleotides in length, and particularly about 26 to 35 nucleotides in length or. Examples of oligonucleotides useful in practicing the methods of the invention are disclosed herein in Table 4.
- Conditions that allow for selective hybridization can be determined empirically, or can be estimated based, for example, on the relative GC:AT (or GC:AU) content of the hybridizing oligonucleotide and the target nucleic acid molecule, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and target sequence to which it is to hybridize (see, for example, Sambrook et al., "Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989)). As such, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the hybridizing nucleic acid molecules.
- An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
- An example of progressively higher stringency conditions is as follows: 2X SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2X SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2X SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1X SSC at about 62°C (high stringency conditions).
- Hybridization and/or washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed.
- oligonucleotide with a target gene can be detected, for example, by performing the method using an oligonucleotide that includes a detectable label.
- the detectable label can be any molecule that conveniently can be linked to the oligonucleotide and detected using readily available equipment.
- the detectable label can be a fluorescent compound such a Cy3, Cy5, Fam, fluorescein, rhodamine, or a green fluorescent protein or enhanced or modified form thereof;, a radionuclide such as sulfur-35, technicium-99, phosphorus-32, tritium or iodine 125; a paramagnetic spin label such as carbon-13, Gd- 157, Mn-55 s Dy-162, Cr 52, or Fe 56; a luminescent compound such as an aequorin; a ehemiluminescent compound; a metal chelate; an enzyme such as luciferase or J- galactosidase, or a substrate for an enzyme; or a receptor or a ligand for a receptor, for example, biotin.
- a fluorescent compound such as Cy3, Cy5, Fam, fluorescein, rhodamine, or a green fluorescent protein or enhanced or modified form thereof
- a radionuclide such as sulfur
- the means for detecting the detectable label will be selected based on the characteristics of the label, as will the means for linking the label to an oligonucleotide (see, for example, Hermanson, “Bioconjugate Techniques” (Academic Press 1996), which is incorporated herein by reference).
- Selective hybridization also can be detected, for example, by utilizing the oligonucleotide as a substrate for a primer extension reaction, further contacting the sample with deoxyribonucleotides (dNTPs), including, if desired, a detectable dNTP (e.g., a fluorescently labeled dNTP, a digoxigenin labeled dNTP, or a biotin labeled dNTP), and a DNA dependent DNA polymerase under conditions sufficient for the primer extension reaction to proceed, and detecting a product of the primer extension reaction.
- dNTPs deoxyribonucleotides
- a detectable dNTP e.g., a fluorescently labeled dNTP, a digoxigenin labeled dNTP, or a biotin labeled dNTP
- a DNA dependent DNA polymerase under conditions sufficient for the primer extension reaction to proceed, and detecting a product of the primer extension reaction.
- the amount or distribution of uracil residues in a bisulfite ion treated nucleic acid molecule comprising a target gene sequence following exposure to alkaline conditions also can be detected using an amplification reaction such as PCR.
- An amplification reaction is performed under conditions that allow selective hybridization of the forward and reverse primers of an amplification primer pair to the target nucleic acid molecule.
- the reaction is performed in a buffered aqueous solution, at about pH 7-9, usually about pH 8.
- the reaction generally is performed in a molar excess of primers to target nucleic acid molecule, for example, at a ratio of about 100:1 primer: genomic DNA.
- a range of primer amounts can be used in samples run in parallel, although generally even the addition of a small amount of primers will result in a sufficient molar excess such that the amplification reaction can proceed.
- the deoxyribonucleoside triphosphates can be added to the synthesis mixture either separately or as a mixture, which can further include the primers, in adequate amounts and the resulting solution is heated to about 90°-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to room temperature, which is preferable for the primer hybridization.
- an appropriate agent for effecting the primer extension reaction generally a polymerase, and the reaction is allowed to occur under conditions as disclosed herein (see Example 1) or otherwise known in the art. Where the polymerase is heat stable, it can be added together with the other reagents.
- the polymerase can be any enzyme useful for directing the synthesis of primer extension products, including, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes, as are well known in the art and commercially available.
- the amplification products can be identified as methylated or non-methylated by a sequencing method, oligomer restriction (Saiki et al., BioTechnology 3:1008-1012, 1985), allele-specific oligonucleotide probe analysis (Conner et al., Proc. Natl. Acad. Sci.
- a methylation-specific amplification reaction such as methylation-specific PCR (MSP) is used alone, or in combination with bisulfite treatment, to detect the methylation status, of a nucleic acid molecule (see U.S. Pat. Nos. 6,265,171; 6,200,756; and 6,017,704, each of which is incorporated herein by reference; see, also, Example 1).
- MSP methylation-specific PCR
- MSP is a particularly sensitive method that allows detection of low numbers of methylated alleles and the use of small amounts of a nucleic acid sample, including paraffin-embedded materials, and also can be conveniently adapted to a multiplex analysis, including, for example, simultaneous detection of unmethylated and methylated products in a single sample, thus providing an internal control.
- the amplification primer pairs used in an MSP reaction are designed to specifically distinguish between bisulfite untreated or unmodified DNA, and methylated and unmethylated DNA.
- MSP primer pairs for unmethylated DNA generally have a thymidine residue in the 3' CpG pair to distinguish it from the cytosine residue retained in methylated DNA, and the complement is designed for the antisense primer.
- MSP primer pairs usually contain relatively few cytosine or guanine residues in the sequence because cytosine is absent in the sense (forward) primer and the guanine is absent in the antisense (reverse) primer; cytosine becomes modified to uracil, which is amplified as thymidine in the amplification product.
- MSP unmethylation (MSP(U)) specific primer pairs and MSP methylation (MSP(M)) specific are exemplified in Table 4.
- amplification primer pairs useful for such a method include, for example, methylation specific primer pairs as set forth in SEQ ID NO: 3 and 4 for cadherin3 (CDH3); SEQ ID NOS:7 and 8 for reprimo; SEQ ID NOS:l 1 and 12 for claudin 5 (CLDN5); SEQ ID NOS: 15 and 16 for death receptor 3 (DR3); SEQ ID NOS: 19and 20 for forkhead box El (FOXE1); SEQ ID NOS:23 and 24 for leucine zipper down-regulated in cancer 1 (LDOC1); SEQ ID NOS:27 and 28 for LIM homeobox protein 1 (LHX1); SEQ ID NOS:31 and 32 for neuro ⁇ lament heavy polypeptide(NEFH); SEQ ID NOS:35 and 36 for neuronal pentraxin II (NPTX2); SEQ ID NOS:39 and 40 for 5 -induced protein (PIG11); SEQ ID NOS:43 and 44 for secreted apoptos
- Amplification primer pairs useful for such a method include, for example, unmethylation specific primer pairs as set forth in SEQ ID NO:l and 2 for cadherinS (CDH3); SEQ ID NOS:5 and 6 for reprimo; SEQ ID NOS:9 and 10 for claudin 5 (CLDN5); SEQ ID NOS: 13 and 14 for death receptor 3 (DR3); SEQ ID NOS : 17 and 18 for forkhead box El (FOXE1); SEQ ID NOS :21 and 22 for leucine zipper down-regulated in cancer 1 (LDOC1); SEQ ID NOS:25 and 26 for LIM homeobox protein 1 (LHX1); SEQ ID NOS:29 and 30 for neuro ⁇ lament heavy polypeptide(NEFH); SEQ ID NOS:33 and 34 for neuronal pentraxin II (NPTX2); SEQ ID NOS:37 and 38 for p5 -induced protein (PIG11); SEQ ID NOS:41 and 42 for secreted apoptos
- MSP is used for detecting the amount or distribution of uracil residues in a bisulfite ion treated target genes following alkaline treatment.
- Such a method can be performed by contacting the gene sequence with a first amplification primer pair and a second amplification primer pair under conditions suitable for amplification, wherein the first amplification primer pair comprises a forward primer and a reverse primer, and at least one primer of the first primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the target gene that contains uracil residues, and wherein the second amplification primer pair comprises a forward primer and a reverse primer, and both primers of the second primer pair selectively hybridize to a target gene containing cytosine residues, but not to a target gene sequence containing uracil residues, and wherein an amplification product, if any, generated by the first primer pair has a first length, and an amplification product, if any, generated by the second primer pair has a second length, which is different from the first length, whereby the length of the amplification products is indicative of the amount or distribution of uracil residues and,
- the amount or distribution of uracil residues also can be detected by contacting the 5' regulatory region of the gene with a first amplification primer pair and a second amplification primer pair under conditions suitable for amplification, wherein the first amplification primer pair comprises a forward primer and a reverse primer, wherein at least one primer of the first primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the 5' regulatory region of the gene containing uracil residues, and wherein the second amplification primer pair comprises a forward primer and a reverse primer, wherein both primers of the second primer pair selectively hybridize to a nucleotide sequence of the 5' regulatory region of the gene containing cytosine residues, but not to a corresponding nucleotide sequence of the 5' regulatory region of the gene containing uracil residues, and wherein an amplification product, if any, generated by the first primer pair has a first length, and wherein an amplification product,
- Methylation silencing of a gene in a cell exhibiting or suspected of exhibiting unregulated growth also can be identified by contacting a test cell with a demethylating agent, and detecting increased expression of an RNA encoded by the gene as compared to a level of expression of the RNA in a test cell not contacted with a demethylating agent.
- Such a method can further include detecting methylation, if any, of cytosine residues in a CpG dinucleotide in a CpG island of the 5' regulatory region of the gene in a corresponding cell exhibiting regulated growth, or an extract of the corresponding cell
- the demethylating agent can be a methyltransferase inhibitor such as DAC.
- Increased expression of an RNA can be detected using any method for detecting RNA, including, for example, northern blot analysis, a reverse transcription-polymerase chain reaction assay, or selective hybridization to an array of nucleotide sequences as disclosed herein.
- test cell, or extract of the test cell comprises one of a plurality of test cells, or extracts of the test cells, or a combination thereof; and each of the test cells, or extracts of the test cells, of the plurality is the same or different, or a combination thereof.
- test cells, or extracts of the test cell can be arranged in an array, which can be an addressable array, on a solid support such as a microchip, a glass slide, or a bead, and the cells (or extracts) can be contacted serially or in parallel with an oligonucleotide probe or primer (or primer pair) as disclosed herein.
- Samples arranged in an array or other reproducible pattern can be assigned an address (i.e., a position on the array), thus facilitating identification of the source of the sample.
- An additional advantage of arranging the samples in an array, particularly an addressable array is that an automated system can be used for adding or removing reagents from one or more of the samples at various times, or for adding different reagents to particular samples.
- an automated system can be used for adding or removing reagents from one or more of the samples at various times, or for adding different reagents to particular samples.
- high throughput assays provide a means for examining duplicate, triplicate, or more aliquots of a single sample, thus increasing the validity of the results obtained, and for examining control samples under the same conditions as the test samples, thus providing an internal standard for comparing results from different assays.
- cells or extracts at a position in the array can be contacted with two or more oligonucleotide probes or primers (or primer pairs), wherein the oligonucleotides are differentially labeled or comprise a reaction that generates distinguishable products, thus providing a means for performing a multiplex assay.
- Such assays can allow the examination of one or more, particularly 2, 3, 4, 5, 10, 15, 20, or more genes to identify epigenetically silenced genes in a test cell.
- the present invention also provides oligonucleotides, which can be useful as probes or primers for identifying an epigenetic silenced gene (or the absence thereof).
- oligonucleotide polynucleotide
- nucleic acid molecule is used broadly to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond.
- an "isolated polynucleotide” is a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
- an isolated polynucleotide may include a coding region with its associated regulatory sequences.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
- nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. Specifically, methylated forms of nucleotides in a polynucleotide sequence are also included. The term includes single and double forms of DNA.
- the term "gene” also is used herein to refer to a polynucleotide sequence contained in a genome. It should be recognized, however, that a nucleic acid molecule comprising a portion of a gene can be isolated from a cell or can be examined as genomic DNA, for example, by a hybridization reaction or a PCR reaction. Thus, while in a genome, it may not always be clear as to a specific nucleotide position where a gene begins or ends, for purposes of the present invention, a gene is considered to be a discrete nucleic acid molecule that includes at least the nucleotide sequence set forth in the GenBank Accession Numbers shown in Tables 1-3 for various genes identified and/or examined herein.
- RNA the deoxynucleotides A, G, C, and T of DNA are replaced by ribonucleotides A, G, C, and U, respectively.
- fragments of the above- described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the polypeptides.
- the nucleic acid sequence includes the disclosed sequence and sequences that encode conservative variations of the polypeptides encoded by polynucleotides provided herein.
- conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like.
- conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
- Nucleic acid sequences of the invention can be expressed in vitro by DNA transfer into a suitable host cell.
- "Host cells” are cells in which a vector can be propagated and its DNA expressed.
- the cell may be prokaryotic or eukaryotic.
- the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cells” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
- the nucleic acid sequences may be inserted into an expression vector.
- expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the sequence of interest genetic sequences.
- Polynucleotide sequence which encode sequence of interest can be operatively linked to expression control sequences.
- “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
- An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the regulatory or expression control sequences.
- regulatory sequences and “expression control sequences” refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
- expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
- regulatory sequences and "expression control sequences” are intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
- An example of an expression control sequence includes a promoter.
- a "promoter” is a minimal sequence sufficient to direct transcription.
- promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene.
- constitutive and inducible promoters are included in the invention (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).
- inducible promoters such as pL of bacteriophage plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
- promoters derived from the genome of mammalian cells e.g., metallothionein promoter
- mammalian viruses e.g., the retro virus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
- Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.
- the polynucleotide sequences may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
- the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
- Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.
- the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedron promoters).
- Polynucleotide sequences of the invention can be expressed in either prokaryotes or eukaryotes.
- Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
- Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
- "Transformation" means a genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, the genetic change is generally achieved by introduction of the DNA into the genome of the cell (i.e., stable).
- a "transformed cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding sequence of interest. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art. Alternatively, MgCl or RbCl can be used. Transformation can also be performed after foraiing a protoplast of the host cell if desired.
- Eukaryotic cells can also be cotransformed with DNA sequences encoding the sequence of interest, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
- Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
- a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
- Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
- the invention provides substantially purified polypeptides.
- substantially purified refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
- One skilled in the art can purify a polypeptide sequence using standard techniques for protein purification.
- the substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
- the purity of the polypeptide can also be determined by amino-terminal amino acid sequence analysis.
- the polypeptides of the invention also include dominant negative forms of the invention polypeptide which do not have the biological activity of invention polypeptide sequence.
- a "dominant negative form" of invention is a polypeptide that is structurally similar to the invention polypeptide but does not have wild-type invention function.
- a dominant-negative invention polypeptide may interfere with wild-type invention function by binding to, or otherwise sequestering, regulating agents, such as upstream or downstream components, that normally interact functionally with the invention polypeptide.
- the method includes assaying, in nucleic acid- containing specimen taken from the subject, the methylation state of a gene or its associated regulatory regions, wherein the expression state of the gene or its associated regulatory regions is associated with the presence of the cellular proliferative disorder, and identifying as having a cellular proliferative disorder a subject that has aberrant methylation of regions of the gene.
- the method provides for detecting a cellular proliferative disorder in a subject having or at risk for the cellular proliferative disorder by identifying aberrantly methylation of regions of a gene when compared to the same regions of the gene in a subject not having the cellular proliferative disorder.
- the aberrant methylation comprises hypermethylated CpG rich regions
- the CpG rich regions are associated with the invention genes, and affect the expression thereof in a methylation state-dependent manner.
- a "cell proliferative disorder” or “cellular proliferative disorder” is any disorder in which the proliferative capabilities of the affected cells is different from the normal proliferative capabilities of unaffected cells.
- An example of a cell proliferative disorder is neoplasia. Malignant cells (i.e., cancer) develop as a result of a multistep process.
- cell proliferative disorders associated with increased methylation of CpG-islands are low grade astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric cancer, colorectal cancer, colorectal adenoma, acute myelogenous leukemia, lung cancer, renal cancer, pancreatic cancer, leukemia, breast cancer, prostate cancer, endometrial cancer and neuroblastoma.
- the illustrative example of the present invention is pancreatic cancer.
- a cell proliferative disorder as described herein may be a neoplasm.
- neoplasms are either benign or malignant.
- the term “neoplasm” refers to a new, abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal.
- a neoplasm creates an unstructured mass (a tumor) which can be either benign or malignant.
- the neoplasm may be a head, neck, lung, esophageal, stomach, small bowel, colon, bladder, kidney, or cervical neoplasm.
- benign refers to a tumor that is noncancerous, e.g. its cells do not proliferate or invade surrounding tissues.
- a cell proliferative disorder may be an age-associated disorder.
- age-associated disorders which are cell proliferative disorders include colon cancer, lung cancer, breast cancer, prostate cancer, and melanoma, amongst others.
- a "nucleic acid containing specimen” includes any type of material containing a nucleic acid to be subject to invention methods.
- the nucleic acid may be contained in a biological sample.
- samples include but are not limited to any bodily fluid, such as a serum, urine, saliva, blood, cerebrospinal fluid, pleural fluid, ascites fluid, sputum, stool, or a biopsy sample.
- Samples or specimens include any CpG-rich DNA sequence, whatever the origin, as long as the sequence is detectably present in a sample. While routine diagnostic tests may not be able to identify cancer cells in these samples, the method of the present invention identifies neoplastic cells derived from the primary tumor or from a metastases.
- the method includes non-invasive sampling (e.g., bodily fluid) as well as invasive sampling (e.g., biopsy).
- the sample of DNA of the subject may be serum, plasma, lymphocytes, urine, sputum, bile, stool, cervical tissue, saliva, tears, pancreatic juice, duodenal juice, cerebral spinal fluid, regional lymph node, histopathologic margins, and any bodily fluid that drains a body cavity or organ. Therefore, the method provides for the non-invasive detection of various tumor types including head and neck cancer, lung cancer, esophageal cancer, stomach cancer, small bowel cancer, colon cancer, bladder cancer, kidney cancers, pancreatic cancers, cervical cancer and any other organ type that has a draining fluid accessible to analysis. For example, neoplasia of regional lymph nodes associated with a primary mammary tumor can be detected using the method of the invention.
- Regional lymph nodes for head and neck carcinomas include cervical lymph nodes, prelaryngeal lymph nodes, pulmonary juxta-esophageal lymph nodes and submandibular lymph nodes.
- Regional lymph nodes for mammary tissue carcinomas include the axillary and intercostal nodes.
- Samples also include urine DNA for bladder cancer or plasma or saliva DNA for head and neck cancer patients.
- Any nucleic acid sample, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids in accordance with the present invention, provided it contains, or is suspected of containing, a nucleic acid sequence containing a target locus (e.g., CpG-containing nucleic acid).
- a target locus e.g., CpG-containing nucleic acid
- the CpG-containing nucleic acid is DNA.
- invention methods may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample.
- a mixture of nucleic acids may also be employed.
- the specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA.
- the nucleic acid-containing sample used for detection of methylated CpG may be from any source including, but not limited to, brain, colon, urogenital, lung, renal, pancreas, liver, esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian, and uterine tissue, and may be extracted by a variety of techniques such as that described by Maniatis, et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, NY, pp 280, 281, 1982).
- the nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a differentially methylated CpG island.
- the CpG island comprises a CpG island located in a gene or regulatory region for a gene.
- a "CpG island” is a CpG rich region of a nucleic acid sequence.
- the nucleic acid sequence may include, for example, MICP 1-42.
- any gene or nucleic acid sequence of interest containing a CpG sequence can provide diagnostic information (i.e., via its methylation state) using invention methods.
- markers can also be multiplexed in a single amplification reaction to generate a low cost, reliable cancer screening test for many cancers simultaneously.
- a combination of DNA markers for CpG-rich regions of nucleic acid may be amplified in a single amplification reaction.
- the markers are multiplexed in a single amplification reaction, for example, by combining primers for more than one locus.
- DNA from a urine sample can be amplified with three different randomly labeled primer sets, such as those used for the amplification of the MICP38-42 loci, in the same amplification reaction.
- the reaction products are separated on a denaturing polyacrylamide gel, for example, and then exposed to film for visualization and analysis.
- the sample may be treated before amplification with a reagent effective for lysing the cells contained in the fluids, tissues, or animal cell membranes of the sample, and for exposing the nucleic acid(s) contained therein.
- a reagent effective for lysing the cells contained in the fluids, tissues, or animal cell membranes of the sample.
- Methods for purifying or partially purifying nucleic acid from a sample are well known in the art (e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press, 1989, herein incorporated by reference).
- invention methods include any means known in the art for detecting such differential methylation.
- detecting the differential methylation may include contacting the nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying a CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and detecting the methylated nucleic acid based on the presence or absence of amplification products produced in the amplifying step.
- This embodiment includes the PCR-based methods described in U.S. Patent No. 5,786,146, incorporated herein in its entirety. [0089] For the first time, the methylation state of a number of genes has been correlated with cell proliferative disorders, and more specifically pancreatic cancers. Examples of such genes and their NCBI accession numbers, including the location of the clone, are set out in Tables 1-3.
- detection of differential methylation is accomplished by contacting a nucleic acid sample suspected of comprising a CpG- containing nucleic acid with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid.
- the sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease, that cleaves both methylated and unmethylated CpG-sites, under conditions and for a time to allow cleavage of methylated nucleic acid.
- Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified by conventional methods such as PCR wherein primers complementary to the oligonucleotides are employed.
- the methylated CpG-containing nucleic acid can be cloned, using method well known to one of skill in the art (see Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, 1989).
- a "methylation sensitive restriction endonuclease” is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated.
- the methylation sensitive restriction endonuclease has inhibited activity when the C is methylated (e.g., Sma ⁇ ).
- Specific non-limiting examples of a methylation sensitive restriction endonucleases include Sma I, BssHII, or HpalL Such enzymes can be used alone or in combination.
- methylation sensitive restriction endonucleases will be known to those of skill in the art and include, but are not limited to SacII, Eagl, and BstUI, for example.
- An "isoschizomer" of a methylation sensitive restriction endonuclease is a restriction endonuclease which recognizes the same recognition site as a methylation sensitive restriction endonuclease but which cleaves both methylated and unmethylated CGs.
- One of skill in the art can readily determine appropriate conditions for a restriction endonuclease to cleave a nucleic acid (see Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, 1989). Without being bound by theory, actively transcribed genes generally contain fewer methylated CGs than in other genes.
- a nucleic acid of interest is cleaved with a methylation sensitive endonuclease.
- cleavage with the methylation sensitive endonuclease creates a sufficient overhang on the nucleic acid of interest.
- the cleavage product can still have a sufficient overhang.
- An "overhang” refers to nucleic acid having two strands wherein the strands end in such a manner that a few bases of one strand are not base paired to the other strand.
- a "sufficient overhang” refers to an overhang of sufficient length to allow specific hybridization of an oligonucleotide of interest.
- a sufficient overhang is at least two bases in length. In another embodiment, the sufficient overhang is four or more bases in length.
- An overhang of a specific sequence on the nucleic acid of interest may be desired in order for an oligonucleotide of interest to hybridize. In this case, the isoschizomer can be used to create the overhang having the desired sequence on the nucleic acid of interest.
- the cleavage with a methylation sensitive endonuclease results in a reaction product of the nucleic acid of interest that has a blunt end or an insufficient overhang.
- an isoschizomer of the methylation sensitive restriction endonuclease can create a sufficient overhang on the nucleic acid of interest.
- Boilt ends refers to a flush ending of two stands, the sense stand and the antisense strand, of a nucleic acid.
- an oligonucleotide is ligated to the nucleic acid cleaved of interest which has been cleaved by the methylation specific restriction endonuclease.
- “Ligation” is the attachment of two nucleic acid sequences by base pairing of substantially complementary sequences and/or by the formation of covalent bonds between two nucleic acid sequences.
- an "oligonucleotide” is a nucleic acid sequence of about 2 up to about 40 bases in length. It is presently preferred that the oligonucleotide is from about 15 to 35 bases in length.
- an adaptor is utilized to create DNA ends of desired sequence and overhang.
- An "adaptor” is a double-stranded nucleic acid sequence with one end that has a sufficient single-stranded overhang at one or both ends such that the adaptor can be ligated by base-pairing to a sufficient overhang on a nucleic acid of interest that has been cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme.
- Adaptors can be obtained commercially, or two oligonucleotides can be utilized to form an adaptor.
- two oligonucleotides are used to form an adaptor; these oligonucleotides are substantially complementary over their entire sequence except for the region(s) at the 5' and/or 3' ends that will form a single stranded overhang.
- the single stranded overhang is complementary to an overhang on the nucleic acid cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme, such that the overhang on the nucleic acid of interest will base pair with the 3' or 5' single stranded end of the adaptor under appropriate conditions.
- the nucleic acid of interest is amplified using a primer complementary to the oligonucleotide.
- primer refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight, wherein the sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a nucleic acid such as an adaptor or a ligated oligonucleotide.
- the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
- the primer is an oligodeoxyribo-nucleotide.
- the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition.
- the oligonucleotide primer typically contains 12-20 or more nucleotides, although it may contain fewer nucleotides.
- Primers of the invention are designed to be “substantially" complementary to each strand of the oligonucleotide to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with a 5' and 3' oligonucleotide to hybridize therewith and permit amplification of CpG containing nucleic acid sequence.
- Primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction or PCR).
- one primer is complementary to the negative (-) strand of the locus and the other is complementary to the positive (+) strand.
- Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides results in newly synthesized + and - strands containing the target locus sequence.
- the oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphos- phoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. (Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
- the CpG-containing nucleic acid sequence of interest contains two strands
- Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products.
- This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word "denaturing" includes all such means.
- One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105°C for times ranging from about 1 to 10 minutes.
- Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA.
- an enzyme from the class of enzymes known as helicases or by the enzyme RecA which has helicase activity, and in the presence of riboATP, is known to denature DNA.
- the reaction conditions suitable for strand separation of nucleic acids with helicases are described by Kuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
- the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands.
- This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, generally at a pH of about 7-9. Preferably, a molar excess (for genomic nucleic acid, usually about 108:1 primer:template) of the two oligonucleotide primers is added to the buffer containing the separated template strands.
- the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty.
- the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands, a large molar excess is preferred to improve the efficiency of the process.
- the deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to approximately room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable.
- This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
- the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
- the agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
- Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation such as Taq DNA polymerase, and the like).
- Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each locus nucleic acid strand.
- the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
- agents for polymerization may be agents for polymerization, however, which initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above.
- the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art.
- alternative methods of amplification have been described and can also be employed.
- PCR techniques and many variations of PCR are known. Basic PCR techniques are described by Saiki et al. (1988 Science 239:487-491) and by U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, which are incorporated herein by reference.
- the conditions generally required for PCR include temperature, salt, cation, pH and related conditions needed for efficient copying of the master-cut fragment.
- PCR conditions include repeated cycles of heat denaturation (i.e. heating to at least about 95 °C.) and incubation at a temperature permitting primer: adaptor hybridization and copying of the master-cut DNA fragment by the amplification enzyme.
- Heat stable amplification enzymes like the pwo, Thermus aquaticus or Thermococcus litoralis DNA polymerases are commercially available which eliminate the need to add enzyme after each denaturation cycle.
- the salt, cation, pH and related factors needed for enzymatic amplification activity are available from commercial manufacturers of amplification enzymes.
- an amplification enzyme is any enzyme which can be used for in vitro nucleic acid amplification, e.g. by the above-described procedures.
- amplification enzymes include pwo, Escherichia coli DNA polymerase I, Klenow fragment of E.
- coli DNA polymerase I T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney Murine Leukemia Virus reverse transcriptase, T4 DNA ligase, E. coli DNA ligase or Q.beta. replicase.
- Preferred amplification enzymes are the pwo and Taq polymerases. The pwo enzyme is especially preferred because of its fidelity in replicating DNA.
- the nucleic acid can be attached to a solid support, such as a membrane, and can be hybridized with any probe of interest, to detect any nucleic acid sequence.
- a solid support such as a membrane
- membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose (NITROPURE) or other membranes used in for detection of gene expression such as polyvmylchloride, diazotized paper and other commercially available membranes such as GENESCREEN, ZETAPROBE (Biorad), and NYTRAN Methods for attaching nucleic acids to these membranes are well known to one of skill in the art. Alternatively, screening can be done in a liquid phase.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
- An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization of the probe of interest.
- the probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.
- a radioisotope for example, with a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.
- Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.
- representational difference analysis RDA
- MCA utilizes kinetic and subtractive enrichment to purify restriction endonuclease fragments present in one population of nucleic acid fragments but not in another.
- RDA enables the identification of small differences between the sequences of two nucleic acid populations.
- RDA uses nucleic acid from one population as a "tester” and nucleic acid from a second population as a "driver” in order to clone probes for single copy sequences present in (or absent from) one of the two populations.
- nucleic acid from a "normal” individual or sample, not having a disorder such as a cell-proliferative disorder is used as a "driver”
- nucleic acid from an "affected” individual or sample, having the disorder such as a cell proliferative disorder is used as a "tester.”
- the nucleic acid used as a "tester” is isolated from an individual having a cell proliferative disorder such as low grade astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric cancer, colorectal cancer, colorectal adenoma, acute myelogenous leukemia, leukemia, lung cancer, renal cancer, breast cancer, prostate cancer, endometrial cancer and neuroblastoma.
- the nucleic acid used as a "driver” is thus normal asfrocytes, normal glial cells, normal brain cells, normal gastric cells, normal colorectal cells, normal leukocytes, normal lung cells, normal kidney cells, normal breast cells, normal prostate cells, normal uterine cells, and normal neurons, respectively.
- the nucleic acid used as a "driver” is isolated from an individual having a cell proliferative disorder such as low grade astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric cancer, colorectal cancer, colorectal adenoma, acute myelogenous leukemia, leukemia, lung cancer, renal cancer, breast cancer, prostate cancer, endometrial cancer and neuroblastoma.
- a cell proliferative disorder such as low grade astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric cancer, colorectal cancer, colorectal adenoma, acute myelogenous leukemia, leukemia, lung cancer, renal cancer, breast cancer, prostate cancer, endometrial cancer and neuroblastoma.
- the nucleic acid used as a "tester” is thus normal asfrocytes, normal glial cells, normal brain cells, normal gastric cells, normal colorectal cells, normal leukocytes, normal lung cells, normal kidney cells, normal breast cells, normal prostate cells, normal uterine cells, and normal neurons, respectively.
- tester nucleic acid useful with to identify methylated nucleic acid sequences in given "driver" population.
- kits it useful for the detection of a cellular proliferative disorder in a subject having or at risk for the cellular proliferative disorder.
- kits include a carrier means compartmentalized to receive a sample in close confinement therein, one or more containers comprising a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of a CpG-containing nucleic acid, wherein the primers distinguish between modified methylated and nonmethylated nucleic acid, and optionally, a third container containing a methylation sensitive restriction endonuclease.
- Primers contemplated for use in accordance with the invention include those that would amplify sequences or fragments thereof as set forth in SEQ ID NOS: 1-64.
- the kit is ideally suited for high throughput automated analysis.
- the kit further comprises a plurality of nucleotides arranged in an array on a solid support such as a microchip, a glass slide, or a bead, which can be contacted serially or in parallel with test samples as prepared above.
- Carrier means are suited for containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
- container means such as vials, tubes, and the like
- each of the container means comprising one of the separate elements to be used in the method.
- the container means can comprise a container containing an oligonucleotide for ligation to nucleic acid cleaved by a methylation sensitive restriction endonuclease.
- One or more container means can also be included comprising a primer complementary to the oligonucleotide.
- container means can also be included which comprise a methylation sensitive restriction endonuclease.
- container means can also be included containing an isoschizomer of the methylation sensitive restriction enzyme.
- pancreatic duct epithelial cells were selectively microdissected from resected pancreata from 10 patients (mean age, 64.3 years; range, 36-83 years) with various pancreatic disorders using a laser capture microdissection system.
- Pancreatic juice samples were collected from 37 patients (mean age, 62.9 years; range 31-81 years) undergoing pancr ⁇ aticduodenectormy for pancreatic ductal adenocarcinoma (24 patients), chronic pancreatitis (8 patients), islet cell tumor (4 patients), and serous cystadenoma (1 patient).
- Pancreatic juice was retrieved by direct aspiration from the transected pancreatic duct at the time of surgical resection.
- pancreatic cancer cell lines (AsPCl, Hs766T, MiaPaCa2, and Panel) were treated with 5Aza-dC (sigma, St. Louis, MO) and TSA (Sigma), wither alone or in combination.
- Cells were exposed continuously to 5Aza-dC (1 ⁇ M) for 4 days or to TSA (1 ⁇ M) for 24h. It was observed that treatment of the cell lines with 5Aza-dC (1 ⁇ M) for 4 days resulted in marked induction of several genes silenced by aberrant methylation without evidence for cell death.
- Mock-treated cells were cultured with the equivalent volume of PBS alone. For combined treatment, these cells were cultured in the presence of 5Aza-dC (1 ⁇ M) for 3 days and then treated for another 24h with TSA (0.5 ⁇ M).
- Second-stranded cDNA was synthesized from 10 ⁇ g of total RNA using T7- dT) primer (Genset Corp., La Jolla, CA) and Superscript Choice system (Invifrogen). Labeled cRNA was synthesized from the purified cDNA by in vitro transcription reaction using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Inc., Farmingdale, NY) at 37°C for 6h. The cRNA was fragmented at 94°C for 35 min.
- the fragmented cRNA was then hybridized to the Human Genome U133A chips (Affymetrix, Snata Clara, CA) with 18462 unique gene/EST transcripts at 45°C for 16h. The washing and staining procedure was performed in the Affymetrix Fluidics Station according to the manufacturer's instructions. The probes were then scanned using a laser scanner, and signal intensity for each transcript (background-subtracted and adjusted for noise) was calculated using Microarray Suite Software 5.0 (Affymetrix).
- PCR conditions were as follows: (a) 95°C for 5 min.; (b) 40 cycles of 95°C for 20 s, 60°C-62°C for 20 s, and 72°C for 30 s; and (c) a final extension of 4 min. at 72°C. 5 ⁇ l of each PCR product were loaded onto 3% agarose gels and visualized by ethidium bromide staining.
- the 707 genes induced by the combined freatment include several genes known to be aberrantly methylated in cancers (e.g., pi 6 and MLH1), supporting a previous notion that some of the genes with densely methylated CpG islands are reexpressed by a combined freatment with 5 Aza-dC and TSA.
- pi 6 and MLH1 genes known to be aberrantly methylated in cancers
- MLH1 e.g., pi 6 and MLH1
- some of the genes with densely methylated CpG islands are reexpressed by a combined freatment with 5 Aza-dC and TSA.
- treatment of all of the four pancreatic cancer cell lines with 5 Aza-dC alone or TSA alone did not result in apparent changes in their phenotypes during the treatment period, combined freatment of certain pancreatic cancer cell lines with 5 Aza-dC and TSA induced cell death in a small fraction of cells.
- Novel targets for aberrant methylation in pancreatic cancer were identified using 16 candidate genes that have been reported to be cancer associated or considered functionally important from the list of 457 genes identified as markedly (> 5-fold) up- regulated by 5 Aza-dC treatment in one or more of pancreatic cancer cell lines but not in the nonneoplastic HPDE cells.
- the genes are listed in Table 4.
- a literature search using PubMed revealed that 14 of the 16 genes have not been implicated for aberrant methylation in any tumor type, whereas SARP2 (also tenned (SFRP1) and TJP2 (alos termed ZO-2) have been recently reported to be frequently methylated in colorectal and pancreatic cancers, respectfully. All of the 16 genes were identified as having CpC-rick sequences fulfilling the criteria of CpG island [GC content > 50%, CpG:GpC ratio > 0.6, and minimum length (200 bp)] in their 5' regions.
- RT-PCR was performed on 5 (CDH3, NPTX2, SARP2, UCHL1, and
- WNT7A WNT7A of these 16 genes in two pancreatic cancer cell lines (AsPCl and MiaPaCa2) to compare the results with the corresponding microarray data and found concordant results (Figure 1A).
- the methylation status of the 16 genes was then determined in the nonneoplastic HPDE cells.
- MSP five of these genes (DR3, LDOC1, NEFH, PIG11, and SMARCA1) showed partial methylation in HPDE and were excluded from further analysis.
- the remaining 11 genes were completely unmethylated in HPDE, and the methylation status of these 11 genes was determined in a panel of 22 pancreatic cancer cell lines. Hypermethylation of all 11 of these genes was found in varying frequencies as depicted in Figure IB.
- methylated was UCHL1 (methylated in 100%), followed by CLDN5 (95%), SARP2 (91%), reprimo (91%), LHXl (11%), FOXEl (64%), TJP2 (59%), WNT7A (59%), CE>H3(18%),a nd ST14 (9%).
- the number of abenantly methylated genes varied among individual cell lines, with an average number of loci of 7.6 (range 4-11) per cell line ( Figure 2). There was no significant correlation between the number of methylated loci and the genetic profile of cell lines with regard to the presence or absence of mutations in the K-ras, p53, and/or SMADA4 genes.
- pancreatic cancer cell lines To test whether the aberrant methylation of the genes identified in pancreatic cancer cell lines also occurred in primary pancreatic cancers, the methylation status of the 11 genes in 20 primary pancreatic cancers and 10 normal pancreatic ductal epithelia was analyzed.
- NPTX2, SARP2, and CLDN5 were selected. These three genes were found to be frequently methylated in pancreatic cancer and not methylated in any of the normal ductal epithelia studied.
- the methylation status of the genes in an expanded series of 43 surgically resected, primary pancreatic cancers was analyzed. Aberrant methylation of NPTX2, SARP2, and CLDN5 was detected in 42 (98%), 41 (95%), and 35 (81%) of these 43 primary pancreatic cancers ( Figure 4), and hypermethylation of at least one of these loci was found in 100% of the primary tumors tested.
- pancreatic juice samples collected from patients with pancreatic cancer as described above was then subject to analysis to determine whether aberrant hypermethylation of NPTX2, SARP2, and CLDN5 could be detected.
- MSP MSP
- a total of 37 pancreatic juice samples including 24 samples from patients with pancreatic cancer and 13 samples from patients with benign pancreatic disorders were analyzed for the methylation patterns of the three genes.
- pancreatic cancer To identify potential targets for aberrant methylation in pancreatic cancer, gene expression profiles of four pancreatic cancer cell lines were analyzed after exposure to 5 Aza-dC and/or TSA. A substantial number of genes were identified as having expression that was markedly induced by 5 Aza-dC and TSA, either alone or in combination. Using MSP, the abnormal methylation patterns of 11 selected genes were confirmed in pancreatic cancer cells and in a series of resected primary pancreatic carcinomas. Most these genes have not been implicated as sites of aberrant methylation in any tumor type. These results demonstrate that gene expression profiling can be used to identify novel target genes that display aberrant methylation in pancreatic cancer.
- pancreatic ductal epithelia Although slight methylation was occasionally noted in only a small number of samples from normal pancreatic ductal epithelia, most of the genes identified as aberrantly methylated in pancreatic cancer were completely unmethylated in a panel of normal pancreatic ductal epithelia.
- An advantage of using high-throughput oligonucleotide microarray data from multiple cell lines is the ability to identify a substantial number of candidates genes targeted for aberrant methylation in human cancers. Such data also provides the ability to conservatively estimate the number of genes directly affected by aberrant methylation in pancreatic cancers.
- Treatment of pancreatic cancer cell lines with 5 Aza-dC induced an average of -200 franscripts (range 116-251 transcripts) per cell line.
- Sixteen genes with CpG islands were selected from the list of genes induces by 5 Aza-dC in pancreatic cancer cell lines but not in nonneoplastic HPDE cells, and it was confirmed that -70% (11 of 16) of these genes were aberrantly methylated in pancreatic cancer.
- an average of 140 genes (70% of 200 genes) may be aberrantly methylated in a pancreatic cancer cell line, of which 60 would be expected to be CpG islands [one previous study has estimated that 60% of genes induced by 5 Aza-dC do not have CpG islands within their 5' regions].
- RLGS also identifies methylated CpG islands that are unrelated to genes (-22% of CpG islands in their study). Treatment with 5 Aza-dC induced the expression of only one-third of the CpG islands they identified as hypermethylated in tumors. In addition, RLGS may also identify methylated CpG islands in tumors when corresponding normal tissue has a low level methylation.
- TSA alone could induce the expression of 4 of the 11 genes whose CpG islands were identified as aberrantly methylated in pancreatic cancer.
- genes previously characterized as having methylated CpG islands such asp57KIP2 and CACNAIG
- TSA alone is not sufficient to induce the expression of genes with densely methylated CpG islands, although it can facilitate induction of gene expression when combined with 5Aza-dC.
- El-Osta et al. Mol. Cell.
- reprimo apoptosis
- CDH# cell adhesion
- CLDN5 and TJP2 tight junction barrier
- SARP2 is a frequent target for aberrant methylation in pancreatic cancer.
- SARP2 is a member of SARP gene families that counteract the Wnt oncogenetic signaling pathway, and this gene is considered to be involved in the regulation of apoptosis.
- Breast cancer cells transfected with SARP2 show an increased sensitivity to different proapoptotic stimuli. Therefore, inactivation of SARP2 by aberrant methylation may provide a growth advantage to cancer cells through increasing the cellular resistance to apoptosis.
- SARP2 ahs recently been identified as frequently hypermethylated in colorectal and gastric cancer, thus suggesting general involvement of this gene in tumorigenesis of digestive organs.
- Genes that are aberrantly methylated at a high frequency in a given cancer are particularly suitable for early cancer detection strategies.
- Several studies have addressed the diagnostic utility of epigenetic markers in detection of cancer. Methylation abnormalities have been detected in blood or sputum of patients with lung cancer, in serum of patients with head and neck cancer, in ductal lavage fluid of patients with breast cancer, and in urine from patients with prostate and bladder cancer.
- the inclusion of multiple genes in these analyses appears to provide a highly sensitive and specific marker for cancer diagnosis. Using three markers, it was possible to detect aberrantly methylated DNA in 75% of pancreatic juice samples from patients with pancreatic cancer.
- GAGE1 G antigen 1
- NPTX2 neuronal pentraxin II
- PIGI1 No Yes NM_006034 p53 induced protein
- SARP2 apoptosis related protem 2
- TNF rumor necrosis factor
- CACNAIG voltage-dependent calcium channel alpha IG subunit a isoform
- MMP1 matrix metal ⁇ oproteinase 1 (interstitial collagenase)
- N _002507 nerve growth factor receptor TNFR superfamily, member 16
- CSF3 colony stimulating factor 3
- DAPK2 death-associated protein kinase 2
- E coh homolog 1 (colon cancer, nonpolyposis type 2) (MLHI)
- SARP2 apoptosis related protein 2
- WISP2 No No N _003881 WNTI inducible signaling pathway protein 2
- Methylated F TATTAA GCG TTTTCG TGG ATA C
- LIM homeobox protein 1 (LHXl ) Unmethylated F GTG TTTTTT TTG TAATTTGAG TTT G
- SWI/SNF -related gene (SMARCAl ) Unmethylated F TGTGGATGTGATTGTTATTATTG
Abstract
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Also Published As
Publication number | Publication date |
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CA2519456A1 (en) | 2004-09-30 |
WO2004083399A3 (en) | 2007-10-25 |
AU2004221394A1 (en) | 2004-09-30 |
EP1604013A4 (en) | 2009-02-11 |
EP1604013A2 (en) | 2005-12-14 |
US8785614B2 (en) | 2014-07-22 |
US7485418B2 (en) | 2009-02-03 |
US20110003284A1 (en) | 2011-01-06 |
US20070015156A1 (en) | 2007-01-18 |
JP2007524369A (en) | 2007-08-30 |
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