CHD8 is a chromatin remodeling ATPase of the SNF2 family. We found that depletion of CHD8 impairs cell proliferation. In order to identify CHD8 target genes, we performed a transcriptomic analysis of CHD8-depleted cells, finding out that CHD8 controls the expression of cyclin E2 ( CCNE2 ) and thymidylate synthetase ( TYMS ), two genes expressed in the G1/S transition of the cell cycle. CHD8 was also able to co-activate the CCNE2 promoter in transient transfection experiments. Chromatin immunoprecipitation experiments demonstrated that CHD8 binds directly to the 5′ region of both CCNE2 and TYMS genes. Interestingly, both RNA polymerase II (RNAPII) and CHD8 bind constitutively to the 5′ promoter-proximal region of CCNE2 , regardless of the cell-cycle phase and, therefore, of the expression of CCNE2 . The tandem chromodomains of CHD8 bind in vitro specifically to histone H3 di-methylated at lysine 4. However, CHD8 depletion does not affect the methylation levels of this residue. We also show that CHD8 associates with the elongating form of RNAPII, which is phosphorylated in its carboxy-terminal domain (CTD). Furthermore, CHD8-depleted cells are hypersensitive to drugs that inhibit RNAPII phosphorylation at serine 2, suggesting that CHD8 is required for an early step of the RNAPII transcription cycle.
A plethora of preclinical evidences suggests that pharmacological targeting of epigenetic dysregulation is a potent strategy to combat human diseases. Nevertheless, the implementation of epidrugs in clinical practice is very scarce and mainly limited to haematological malignancies. In this review, we discuss cutting-edge strategies to foster the chemical design, the biological rationale and the clinical trial development of epidrugs. Specifically, we focus on the development of dual hybrids to exploit multitargeting of key epigenetic molecules deregulated in cancer; the study of epigenetic-synthetic lethality interactions as a mechanism to address loss-of-function mutations, and the combination of epidrugs with other therapies such as immunotherapy to avoid acquired chemoresistance and increase therapy sensitivity. By exploring these challenges, among others, the field of epigenetic chemical biology will increase its potential for clinical benefit, and more effective strategies targeting the aberrant epigenome in cancer are likely to be developed both in haematological and solid tumours.
The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the O6 position of guanine. MGMT expression is decreased in some tumor tissues, and lack of activity has been observed in some cell lines. Loss of expression is rarely due to deletion, mutation, or rearrangement of the MGMT gene, but methylation of discrete regions of the CpG island of MGMT has been associated with the silencing of the gene in cell lines. We used methylation-specific PCR to study the promoter methylation of the MGMT gene. All normal tissues and expressing cancer cell lines were unmethylated, whereas nonexpressing cancer cell lines were methylated. Among the more than 500 primary human tumors examined, MGMT hypermethylation was present in a subset of specific types of cancer. In gliomas and colorectal carcinomas, aberrant methylation was detected in 40% of the tumors, whereas in non-small cell lung carcinomas, lymphomas, and head and neck carcinomas, this alteration was found in 25% of the tumors. MGMT methylation was found rarely or not at all in other tumor types. We also analyzed MGMT expression by immunohistochemistry in relation to the methylation status in 31 primary tumors. The presence of aberrant hypermethylation was associated with loss of MGMT protein, in contrast to retention of protein in the majority of tumors without aberrant hypermethylation. Our results suggest that epigenetic inactivation of MGMT plays an important role in primary human neoplasia.
It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI. phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC. displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%. cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases. It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI. phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC. displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%. cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases. Endometrial carcinoma is the most common gynecological malignancy and is the fourth most common cancer of women in the United States.1Parker SL Tong T Bolden S Wingo PA Cancer statistics.CA Cancer J Clin. 1997; 47: 5-27Crossref PubMed Scopus (2333) Google Scholar Uterine endometrioid carcinoma (UEC) is the most common histological type. UEC often precedes or coexists with endometrial hyperplasias, which have been proposed as possible precursor lesions.2Sherman A Brown S The precursors of endometrial carcinoma.Am J Obstet Gynecol. 1979; 135: 947-954Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar Atypical endometrial hyperplasia (AEH) is most associated with progression to carcinoma, and women with AEH without concurrent invasive carcinoma have a 30% risk of developing UEC.3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar UEC and AEH share cytological atypia and a monoclonal pattern,4Jovanovic AS Boynton KA Mutter GL Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability.Cancer Res. 1996; 56: 1917-1921PubMed Google Scholar, 5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar but UEC are distinguished by the presence of invasion.3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar A spectrum of hyperplasia without atypia, including simple and complex hyperplasia, also exists. These lesions show a low rate of progression to UEC3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar and a putative polyclonal pattern.4Jovanovic AS Boynton KA Mutter GL Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability.Cancer Res. 1996; 56: 1917-1921PubMed Google Scholar, 5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar Very little is known about the genetic alterations underlying the biology of the precursor lesions of UEC. Only mutations in the oncogene K-ras and the tumor suppressor gene PTEN have been described in AEH.6Enomoto T Inoue M Perantoni AO Buzard GS Miki H Tanizawa O Rice JM K-ras activation in premalignant and malignant epithelial lesions of the human uterus.Cancer Res. 1991; 51: 5308-5314PubMed Google Scholar, 7Levine R Cargile CB Blazes MS van Rees B Kurman RJ Ellenson LH PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma.Cancer Res. 1998; 58: 3254-3258PubMed Google Scholar PTEN mutations may even precede the appearance of an evident atypia in the endometrial hyperplasia.8Maxwell GL Risinger JI Gumbs C Shaw H Bentley RC Barret JC Berchuck A Futreal PA Mutations of the PTEN tumor suppressor gene in endometrial hyperplasias.Cancer Res. 1998; 58: 2500-2503PubMed Google Scholar However, a subset of AEH displays the microsatellite instability (MSI. phenotype.5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar, 7Levine R Cargile CB Blazes MS van Rees B Kurman RJ Ellenson LH PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma.Cancer Res. 1998; 58: 3254-3258PubMed Google Scholar, 9Duggan B Felix J Muderspach L Tourgeman D Zheng J Shibata D Microsatellite instability in sporadic endometrial carcinoma.J Natl Cancer Inst. 1994; 86: 1216-1221Crossref PubMed Scopus (189) Google Scholar, 10Mutter GL Boynton KA Faquin WC Ruiz RE Jovanovic AS Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.Cancer Res. 1996; 56: 4483-4486PubMed Google Scholar MSI was first detected in tumors from patients with hereditary non-polyposis colorectal carcinoma (HNPCC). In this setting, the cause of MSI has been attributed to germline defects in DNA mismatch repair (MMR) genes, mainly involving hMLH1 and hMSH2.11Aaltonen L Peltomaki P Leach F Sistonen P Pylkkanen L Mecklin J Jarvinen H Powell S Jen J Hamilton S Petersen G Kinzler K Vogelstein B de la Chapelle A Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2559) Google Scholar, 12Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2773) Google Scholar, 13Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2384) Google Scholar, 14Liu B Parsons RE Hamilton SR Petersen GM Lynch HT Watson P Markowitz S Willson JKV Green J de la Chapelle A Kinzler KW Vogelstein B hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindred's.Cancer Res. 1994; 54: 4590-4594PubMed Google Scholar, 15Kolodner RD Hall NR Lipford J Kane MF Morrison PT Finan PJ Burn J Chapman P Earabino C Merchant E Bishop T Structure of the human hMLH1 locus and analysis of a large hereditary nonpolyposis colorectal cancer kindred for MLH1 mutations.Cancer Res. 1995; 55: 242-248PubMed Google Scholar, 16Fishel R Lescoe MK Rao MRS Copeland NG Jenkins NA Garber J Kane M Kolodner R The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer.Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2578) Google Scholar, 17Leach FS Nicolaides NC Papadopoulos N Liu B Jen J Parsons R Peltomaki P Sistnen P Aaltonen LA Nystrom-Lahi M Guan X-Y Zhang J Meltzer PS Yu J-W Kao F-T Chen DJ Cerosaletti KM Fournier REK Todd S Lewis T Leach RJ Naylor SL Weissenbach J Mecklin J-P Jarvinen H Petersen GM Hamilton SR Green I Jass J Watson P Lynch HT Tent JM de la Chapelle A Kinzler KW Vogelstein B Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.Cell. 1993; 75: 1215-1225Abstract Full Text PDF PubMed Scopus (2087) Google Scholar, 18Papadopoulos N Nicolaides NC Wei Y-F Ruben SM Carter KC Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Hamilton SR Petersen GM Watson P Lynch HT Peltomaki P Mecklin J-P de la Chapelle A Kinzler KW Vogelstein B Mutation of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1628Crossref PubMed Scopus (1761) Google Scholar, 19Bronner CE Baker SM Morrison PT Warren G Smith LG Lescoe MK Kane M Earabino C Lipford J Lindblom A Tannergard P Bollag RJ Godwin AR Ward DC Nordenskjld MA Kolodner R Liskay RM Mutations in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary nonpolyposis colon cancer.Nature. 1994; 368: 258-261Crossref PubMed Scopus (1913) Google Scholar Other MMR genes identified include hPMS1, hPMS2, hMSH3, and hMSH6.20Nicolaides NC Papadopoulos N Liu B Wei Y-F Carter KC Ruben SM Rosen CA Hasentine WA Fleischmann RD Fraser CM Adams MD Venter JC Dunlop MG Hamilton SR Petersen GM de la Chapelle A Vogelstein B Kinzler KW Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 75-80Crossref PubMed Scopus (1429) Google Scholar, 21Nicolaides NC Papadopoulos N Liu B Parsons R Lengauer C Palombo F D'Arrigo A Markowitz S Willson JKV Kinzler KW Jiricny J Vogelstein B Mutations of GTBP in genetically unstable cells.Science. 1995; 268: 1915-1917Crossref PubMed Scopus (473) Google Scholar Endometrial carcinoma (EC) is the most common noncolorectal carcinoma occurring in women with HNPCC.22Watson P Lynch HT Extracolonic cancer in hereditary nonpolyposis colorectal cancer.Cancer. 1993; 7: 677-685Crossref Scopus (670) Google Scholar MSI has also been observed in approximately 20. of sporadic EC.9Duggan B Felix J Muderspach L Tourgeman D Zheng J Shibata D Microsatellite instability in sporadic endometrial carcinoma.J Natl Cancer Inst. 1994; 86: 1216-1221Crossref PubMed Scopus (189) Google Scholar, 23Risinger JI Berchuck A Kohler MF Watson P Lynch HT Boyd J Genetic instability of microsatellites in endometrial carcinoma.Cancer Res. 1993; 53: 1-4PubMed Google Scholar, 24Burks RT, Kessis TD, Cho KR, Hedrick L: Microsatellite instability in endometrial carcinoma. Oncogene 9:1163–1166Google Scholar, 25Kobayashi K Sagae S Kudo R Koi S Saito H Nakamura Y Microsatellite instability in endometrial carcinomas.Genes Chromosomes Cancer. 1995; 14: 128-132Crossref PubMed Scopus (97) Google Scholar, 26Caduff RF Johnston CM Svodoba-Newman SM Poy EL Merajver SD Frank TS Clinical and pathological significance of microsatellite instability in sporadic endometrial carcinoma.Am J Pathol. 1996; 148: 1671-1678PubMed Google Scholar, 27Katabuchi H van Rees B Lambers AR Ronnett BM Blazes MS Leach FS Cho KR Hedrick L Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.Cancer Res. 1995; 55: 5556-5560PubMed Google Scholar, 28Lim PC Tester D Cliby W Ziesmer SC Roche PC Hartmann L Thibodeau SN Podratz KC Jenkins RB Absence of mutations in DNA mismatch repair genes in sporadic endometrial tumors with microsatellite instability.Clin Cancer Res. 1996; 2: 1907-1911PubMed Google Scholar, 29Kobayashi K Matsushima M Koi S Saito H Sagae S Kudo R Nakamura Y Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.Jpn J Cancer Res. 1996; 87: 141-145Crossref PubMed Scopus (61) Google Scholar, 30Kowalski LD Mutch DG Herzog TJ Rader JS Goodfellow PJ Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers.Genes Chromosomes Cancer. 1997; 18: 219-227Crossref PubMed Scopus (107) Google Scholar, 31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar It was expected that UECs with MSI+ would have mutations in one of the known DNA MMR genes. However, hMSH2 or hMLH1 mutations have been found in less than 10% of UECs with MI+.27Katabuchi H van Rees B Lambers AR Ronnett BM Blazes MS Leach FS Cho KR Hedrick L Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.Cancer Res. 1995; 55: 5556-5560PubMed Google Scholar, 28Lim PC Tester D Cliby W Ziesmer SC Roche PC Hartmann L Thibodeau SN Podratz KC Jenkins RB Absence of mutations in DNA mismatch repair genes in sporadic endometrial tumors with microsatellite instability.Clin Cancer Res. 1996; 2: 1907-1911PubMed Google Scholar, 29Kobayashi K Matsushima M Koi S Saito H Sagae S Kudo R Nakamura Y Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.Jpn J Cancer Res. 1996; 87: 141-145Crossref PubMed Scopus (61) Google Scholar, 30Kowalski LD Mutch DG Herzog TJ Rader JS Goodfellow PJ Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers.Genes Chromosomes Cancer. 1997; 18: 219-227Crossref PubMed Scopus (107) Google Scholar Several studies have described a different pathway for inactivation of hMLH1 in colorectal tumors with MSI associated with silencing of the gene by promoter hypermethylation.32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines.Cancer Res. 1997; 57: 808-811PubMed Google Scholar, 33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 34Cunningham JM Christensen ER Tester DJ Kim CY Roche PC Burgart LJ Thibodeau SN Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.Cancer Res. 1998; 58: 3455-3460PubMed Google Scholar, 35Veigl ML Kasturi L Olechnowicz J Ma AH Lutterbaugh JD Periyasamy S Li GM Drummond J Modrich PL Sedwick WD Markowitz SD Biallelic inactivation of hMLH1 by epigenetic silencing, a novel mechanism causing human MSI cancers.Proc Natl Acad Sci USA. 1998; 95: 8698-8702Crossref PubMed Scopus (550) Google Scholar These studies demonstrate that hMLH1 promoter hypermethylation is associated with the loss of hMLH1 expression and the MSI phenotype in colorectal cancer cell lines and sporadic colorectal carcinomas. Recent data also strongly suggest that the epigenetic silencing of hMLH1 is associated with MSI in gastric cancer.36Fleisher AS Esteller M Wang S Tamura G Suzuki H Yin J Zou TT Abraham JM Kong D Smolinski KN Shi YQ Rhyu MG Powell SM James SP Wilson KT Herman JG Meltzer SJ Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.Cancer Res. 1999; 59: 1090-1095PubMed Google Scholar, 37Leung SY Yuen ST Chung LP Chu KM Chan AS Ho JC hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability.Cancer Res. 1999; 59: 159-164PubMed Google Scholar In addition, the demethylation of the hMLH1 gene in cell lines, including one endometrial carcinoma cell line, using the agent 5-azacytidine results in reactivation of hMLH1 expression and restoration of the MMR activity.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar We have also recently described the strong association between hMLH1 promoter hypermethylation and the presence of a MSI phenotype in sporadic cases of UEC.38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar This finding has also been corroborated lately in two independent studies.39Gurin CC Federici MG Kang L Boyd J Causes and consequences of microsatellite instability in endometrial carcinoma.Cancer Res. 1999; 59: 462-466PubMed Google Scholar, 40Simpkins SB Bocker T Swisher EM Mutch DG Gersell DJ Kovatich AJ Palazzo JP Fishel R Goodfellow PJ MLH1 promoter methylation, and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers.Hum Mol Genet. 1999; 8: 661-666Crossref PubMed Scopus (265) Google Scholar Methylation of normally unmethylated CpG islands in the promoter regions of tumor suppressor genes including p16, p15, VHL, and E-cadherin correlates with loss of transcription and suggests an alternative mechanism for tumor suppressor gene inactivation.41Baylin SB Herman JG Graff JR Vertino PM Issa JP Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar All of the above findings led us to investigate promoter methylation status in relation to the MSI phenotype in a large series of endometrial hyperplasias and carcinomas. Our results suggest that hMLH1 promoter hypermethylation is an early event in endometrial tumorigenesis, being present in AEH, and preceding in some cases a detectable MSI phenotype. Twenty-seven endometrial carcinomas and 116 endometrial hyperplasias were obtained from the Departments of Pathology of Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) and Brigham and Women's Hospital (Boston, MA). All tumors and hyperplasias were examined and classified according to the criteria of the World Health Organization. Concerning our series of hyperplasias, 62 cases of simple endometrial hyperplasia without atypia, 33 cases of complex endometrial hyperplasia without atypia, and 21 cases of complex hyperplasia with atypia were included in the study. No cases of simple endometrial hyperplasia with atypia were found in this set of samples. DNA was isolated from tumors and hyperplasias and matching normal DNA by standard methods from fresh frozen biopsies, paraffin-embedded material, and peripheral blood. Microdissection was performed on some sections to separate neoplastic and nonneoplastic tissues. For MSI analysis five dinucleotide microsatellite DNA sequences (D5S107, D10S197, D12S79, D12S95, and D18S58) on chromosomes 5,10, 12, and 18 were amplified by the PCR using MapPairs (Research Genetics Inc.) according to the same conditions previously described.31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar Reconfirmation of MSI in endometrial cancers and determination of MSI in hyperplasias were performed using primer BAT-26. BAT-26 is a repeat of 26 deoxyadenosines localized within intron 5 of the MSH2 gene. BAT-26 was amplified by polymerase chain reaction (PCR) and analyzed by single-strand conformation polymorphism (SSCP) analysis as described.42Hoang JM Cottu PH Thuille B Salmon RJ Thomas G Hamelin R BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res. 1997; 57: 300-303PubMed Google Scholar DNA bands were visualized by silver staining method. DNA methylation patterns in the CpG islands of hMLH1, hMSH2, hMSH6, and hMSH3 genes were determined by MSP.43Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5174) Google Scholar MSP distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated, but not methylated, cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA.43Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5174) Google Scholar Primer sequences of hMLH1 and MSH2 for the MSP analysis have been previously described.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar Primer sequences of hMSH6 for the unmethylated reaction were 5′-TTT GGG TTT TTT TGG TGG AGT GT-3′ (sense) and 5′-CTT AAA AAA AAA ACT ATA CAA AAT ACT CTA TCA CA-3′ (antisense) and for the methylated reaction 5′-TTT TTT CGG CGG AGC GC-3′ (sense) and 5′-AAA AAA AAA CTA TAC AAA ATA CTC TAT CGC-3′ (antisense). Primer sequences of hMSH3 for the unmethylated reaction were 5′-GGT TTG TGT TTT TTG TTA GGT TTT GTT-3′ (sense) and 5′-CTA AAA ACA ACA AAA CCA CCC AAC A-3′ (antisense. and for the methylated reaction 5′-CGT TTT TCG TTA GGT TTT GTC GTC-3′ (sense) and 5′-AAC GAA ACC GCC CGA CG-3′ (antisense). SW48 DNA, which has previously been characterized as methylated at the hMLH1 locus,32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines.Cancer Res. 1997; 57: 808-811PubMed Google Scholar, 33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar was used as a positive control for methylated alleles of hMLH1. Because there are no previous reports for cell lines with hypermethylation of hMSH2, hMSH6, and hMSH3, placental DNA treated in vitro with Sss I methyltransferase was used as a positive control for methylated alleles of the three genes. DNA from normal lymphocytes was used as negative control for methylated genes. Ten microliters of each PCR reaction were loaded directly onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination. To check for a specific cytosine methylation within the hMLH1 promoter region studied, we digested with the restriction enzyme BstU I following bisulfite modification as described.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 44Sadri R Hornsby PJ Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.Nucleic Acids Res. 1996; 24: 5058-5059Crossref PubMed Scopus (72) Google Scholar The BstU I recognition site, CGCG, will remain CGCG if both C's are methylated after bisulfite treatment and amplification, but will become TGTG if they are unmethylated. The colorectal carcinoma cell lines RKO and SW480 were used as positive and negative controls for hMLH1 promoter hypermethylation.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar To determine whether the locus containing the hMLH1 gene was deleted in tumors, LOH was assessed. Five different (CA)n repeats (Research Genetics, Inc., Huntsville, AL) localized on chromosome 3 (D3S1314, D3S1286, D3S1283, D3S1298, and D3S1611) were used to screen endometrial cancers for allelic loss in hMLH1 gene. D3S1611 is actually located within an intron of hMLH1.18Papadopoulos N Nicolaides NC Wei Y-F Ruben SM Carter KC Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Hamilton SR Petersen GM Watson P Lynch HT Peltomaki P Mecklin J-P de la Chapelle A Kinzler KW Vogelstein B Mutation of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1628Crossref PubMed Scopus (1761) Google Scholar PCR amplification of marker loci was carried out on paired normal-tumor DNAs in 25-μl reaction volume containing 100 ng template DNA, 10 pmol each forward and reverse primers, 0.2 mmol/L each dNTP, 1.5 units of Taq DNA polymerase (Ecotaq, Ecogen, Langhorne, PA) and 1.5 to 3 mmol/L MgCl2, dependent on primer. Thirty PCR cycles were used, with each cycle consisting of 30 seconds at 95 °C, 30 seconds at 58 °C, and 1 minute at 72 °C. The denatured samples were loaded onto a 8. denaturing polyacrylamide gels containing 8.3 Mol/L urea, and run at 1800 V for 4.5 hours at room temperature. PCR products were capillary-blotted onto Hybond-N membrane (Amersham, Buckinghamshire, UK) and DNA was fixed by alkali treatment. The membranes were analyzed using the ECL Gene Detection System (ECL Amersham) according to the specifications of the Amersham kit. Allelic loss of heterozygosity (LOH) was defined as a visible reduction of 50. or more in the band intensity of the tumor sample when compared to the corresponding normal band. We investigated 27 UECs previously characterized for the MSI phenotype10Mutter GL Boynton KA Faquin WC Ruiz RE Jovanovic AS Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.Cancer Res. 1996; 56: 4483-4486PubMed Google Scholar, 31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar for promoter hypermethylation of hMLH1 using MSP.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar Methylation of the promoter region studied for hMLH1 correlates with the loss of hMLH1 expression in primary sporadic colorectal cancer using MSP33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar and another PCR-based approach.32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hML
