BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage

Human DNA mismatch repair (MMR) is involved in the response to certain chemotherapy drugs, including 6-thioguanine (6-TG). Consistently, MMR-deficient human tumor cells show resistance to 6-TG damage as manifested by a reduced G(2)-M arrest and decreased apoptosis. In this study, we investigate the role of the BRCA1 protein in modulating a 6-TG-induced MMR damage response, using an isogenic human breast cancer cell line model, including a BRCA1 mutated cell line (HCC1937) and its transfectant with a wild-type BRCA1 cDNA. The MMR proteins MSH2, MSH6, MLH1, and PMS2 are similarly detected in both cell lines. BRCA1-mutant cells are more resistant to 6-TG than BRCA1-positive cells in a clonogenic survival assay and show reduced apoptosis. Additionally, the mutated BRCA1 results in an almost complete loss of a G(2)-M cell cycle checkpoint response induced by 6-TG. Transfection of single specific small interfering RNAs (siRNA) against MSH2, MLH1, ATR, and Chk1 in BRCA1-positive cells markedly reduces the BRCA1-dependent G(2)-M checkpoint response. Interestingly, ATR and Chk1 siRNA transfection in BRCA1-positive cells shows similar levels of 6-TG cytotoxicity as the control transfectant, whereas MSH2 and MLH1 siRNA transfectants show 6-TG resistance as expected. DNA MMR processing, as measured by the number of 6-TG-induced DNA strand breaks using an alkaline comet assay (+/-z-VAD-fmk cotreatment) and by levels of iododeoxyuridine-DNA incorporation, is independent of BRCA1, suggesting the involvement of BRCA1 in the G(2)-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR.     

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.     

Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2 and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) represents 1-3% of all colorectal cancers. HNPCC is caused by a constitutional defect in a mismatch repair (MMR) gene, most commonly affecting the genes MLH1, MSH2 and MSH6. The MMR defect results in an increased cancer risk, with the greatest lifetime risk for colorectal cancer and other cancers associated to HNPCC. The HNPCC-associated tumor phenotype is generally characterized by microsatellite instability (MSI) and immunohistochemical loss of expression of the affected MMR protein. The aim of this study was to determine the sensitivity of IHC for MLH1, MSH2 and MSH6, and MSI analysis in tumors from known MMR gene mutation carriers. Fifty-eight paired normal and tumor samples from HNPCC families enrolled in our high-risk colorectal cancer registry were studied for the presence of germline mutations in MLH1, MSH2 and MSH6 by DGGE and direct sequencing. MSI analysis and immunostaining for MLH1, MSH2 and MSH6 were evaluated. Of the 28 patients with a real pathogenic mutation, loss of immunohistochemical expression for at least 1 of these MMR proteins was found, and all except 1 have MSI-H. Sensitivity by MSI analysis was 96%. IHC analysis had a sensitivity of 100% in detecting MMR deficiency in carriers of a pathogenic MMR mutation, and can be used to predict which gene is expected to harbor the mutation for MLH1, MSH2 and MSH6. This study suggests that both analyses are useful for selecting high-risk patients because most MLH1, MSH2 and MSH6 gene carriers will be detected by this 2-step approach. This practical method should have immediate application in the clinical work of patients with inherited colorectal cancer syndromes.     

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen

Purpose and methods: The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. Results: We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O⁶-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. Conclusion: Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance. Received: 13 December 1999 / Accepted: 27 July 2000     

A homozygous germ-line mutation in the human MSH2 gene predisposes to hematological malignancy and multiple café-au-lait spots.

Individuals with a germ-line mutation in one of the DNA mismatch repair (MMR) genes are at significant risk for colorectal cancer and other tumors. Three families have previously been reported with individuals homozygous for mutations in the MMR gene MLH1 that are predicted to compromise MMR. These individuals develop hematological malignancies and/or neurofibromatosis type 1 at an early age. Here, in an individual, we demonstrate that a homozygous novel mutation in the MMR gene MSH2 is associated with leukemia and multiple café-au-lait spots, a feature of neurofibromatosis type 1. Because the hematological malignancies observed in the individuals homozygous for the loss of MMR are reflective of the lymphomas seen in mice lacking MMR, the mice may provide a useful model for human neoplasia.     

Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain

HNPCC is an autosomal dominantly inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and endometrial cancer at a young age. It also entails an increased risk of a variety of other tumors, such as ovarian, gastric, uroepithelial and biliary tract cancers. The underlying pathogenic mutation lies in 1 of the 5 known DNA MMR genes (MSH2, MLH1, PMS1, PMS2 and MSH6). We screened a total of 140 individuals from 56 Spanish families with suspected HNPCC for mutations in the DNA mismatch repair genes MLH1 and MSH2, using DGGE and direct DNA sequencing. Families were selected on the basis of a history of HNPCC-related tumors or the occurrence of other associated tumors in members besides the index case affected with colorectal cancer. We detected 14 definite pathogenic germline mutations, 9 in MLH1 and 5 in MSH2 in 13 unrelated families selected by the Amsterdam criteria and Bethesda guidelines (1 family carries 2 mutations) and 3 missense mutations in 3 unrelated families selected by the Amsterdam criteria. Among the 17 germline mutations noted in the Spanish cohort, 10 are novel, 7 in MLH1 and 3 in MSH2, perhaps demonstrating different mutational spectra in the Spanish population, where no founder mutation has been identified. Based on our results, we suggest that in the Spanish population not only HNPCC families fulfilling the Amsterdam criteria but also those following Bethesda guidelines should undergo genetic testing for MSH2 and MLH1 mutations.     

Role of MutSalpha in the recognition of iododeoxyuridine in DNA

We have previously demonstrated that both the MLH1 and MSH2 status impact the DNA levels of the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd), and thereby radiosensitization induced by these analogues, indirectly implicating both mismatch repair (MMR) proteins in the removal of these bases from DNA. More recent data from our group demonstrate that base excision repair (BER) also impacts IdUrd-DNA levels, supporting a role for the BER pathway in IdUrd removal as well. In this study, we have examined more direct interactions between the MSH2 protein and the processing of IdUrd incorporated in DNA. Our data demonstrate that the MutSalpha (MSH2/MSH6) complex binds specifically to DNA containing an IdUrd-G mismatch, using both purified human MutSalpha as well as nuclear extracts from Msh2-proficient and-deficient mouse cell lines. MutSalpha binding to a IdUrd-G is better recognized than a G-T mismatch in the same sequence context. In addition, MSH2 protein can be found colocalized with IdUrd-DNA using confocal microscopy in G(1) synchronized cells after treatment with IdUrd. Consistent with our recent publication, coadministration of IdUrd and a chemical inhibitor of BER, methoxyamine (MX), also increases the extent of MSH2 nuclear colocalization with IdUrd. Furthermore, we show that the extent of MSH2 colocalization with IdUrd in G(1)-synchronized human tumor cells varies with MLH1 status, suggesting a role for the MLH1 protein in stabilizing the interaction between the MSH2 protein and DNA containing IdUrd. These data, both in vitro and in vivo, suggest direct involvement of MSH2 in processing IdUrd in DNA.     

Non-truncating hMLH1 variants identified in Slovenian gastric cancer patients are not associated with Lynch Syndrome: a functional analysis report

Hereditary non-polyposis colorectal cancer is the most common known genetic syndrome that predisposes to various types of cancer including gastric cancer and occures mainly due to pathogenic germline mutations in DNA mismatch repair (MMR) genes, such as MLH1, MSH2 and MSH6. Impaired MMR activity can lead to microsatellite instability (MSI) in tumor tissues. Interpreting the pathogenic significance of identified mutations in MMR genes, especially missense alterations and short in-frame deletions and insertions is challenging and functional analysis is often needed to accurately assess their pathogenicities. The purpose of this study was to evaluate functional significance of MLH1 missense mutations, previously identified in unrelated Slovenian patients with MSI-positive gastric carinomas. A novel in vivo yeast-based approach and in silico predictions were used. Variant E433Q was characterized for the first time and was shown to have no effect on MLH1 protein function. Functional analysis of amino acid rearrangement K618A, with previously reported contradictory results of its pathogenicity, suggests that the variant is a neutral polymorphism. Results of our study imply that there is either germline mutation or an epigenetic inactivation of another MMR gene, which causes MSI phenotype in the referred gastric cancer cases.     

Alterations in PMS2, MSH2 and MLH1 expression in human prostate cancer

DNA mismatch repair (MMR) is involved in the post-replication correction of errors due to misincorporated nucleotides or DNA slippage during DNA synthesis. We previously reported the reduction or loss of MMR protein expression in human prostate cancer cell lines and some primary tumors. In the present report, we further demonstrate the involvement of defects of MMR in the pathogenesis of prostate cancer. Immunohistochemical analysis of 39 formalin-fixed, paraffin-embedded human prostate tumors, showed reduction or absence of MMR protein expression (MLH1, MSH2, PMS2) in the epithelium of prostate tumor foci compared to normal adjacent prostate tissue. The reduction or absence of the PMS2 and MSH2 (but not MLH1) protein was correlated to the differentiation of the tumor. Poorly differentiated tumors showed greater loss of these two proteins than the well differentiated tumors (P<0.05). We previously reported that microsatellite instability was detectable by a beta-galactosidase restoration mutation assay in the prostate cancer cell lines DU145, PC3, LNCaP, p67SV40T, M2182, and M12. In this study, we detected the insertion or deletion of one nucleotide in the mononucleotide repeats located within the coding regions of BAX gene in DU145, and TGFbetaRII in M12 cells. In addition, we used an in vitro model of defective MMR to demonstrate that microsatellite instability can be induced in an otherwise stable cancer cell line by transfection with a dominant negative fragment of PMS2. These results suggest that defects in MMR may result in MSI in the secondary genes in prostate cancer. From these results, we conclude that loss of MMR function can produce MSI and target some secondary genes containing microsatellites in their coding regions. These series of events may play important roles in the development of human prostate cancer.     

Microsatellite instability and mutation analysis among southern Italian patients with colorectal carcinoma: detection of different alterations accounting for MLH1 and MSH2 inactivation in familial cases.

BACKGROUND: Microsatellite instability (MSI) is due to defective DNA mismatch repair (MMR) and has been detected at various rates in colorectal carcinoma (CRC). The role of MSI in colorectal tumorigenesis was assessed further in this study by both microsatellite analysis of two CRC subsets [unselected patients (n = 215) and patients <50 years of age (n = 95)], and mutation screening of the two major MMR genes MLH1 and MSH2 among familial CRC cases. PATIENTS AND METHODS: PCR-based microsatellite analysis was performed on paraffin-embedded tissues. In CRC families, MLH1/MSH2 mutation analysis and MLH1/MSH2 immunostaining were performed on germline DNA and MSI+ tumour tissues, respectively. RESULTS: The MSI+ phenotype was detected in 75 (24%) patients, with higher incidence in early-onset or proximally located tumours. Among 220 patients investigated for family cancer history, MSI frequency was markedly higher in familial [18/27 (67%)] than in sporadic [32/193 (17%)] cases. Three MLH1 and six MSH2 germline mutations were identified in 14 out of 36 (39%) CRC families. Prevalence of MLH1/MSH2 mutations in CRC families was significantly increased by the presence of: (i) fulfilled Amsterdam criteria; (ii) four or more CRCs; or (iii) one or more endometrial cancer. While MSH2 was found mostly mutated, almost all [8/9 (89%)] familial MSI+ cases with loss of the MLH1 protein were negative for MLH1 germline mutations. CONCLUSIONS: Both genetic (for MSH2) and gene-silencing (for MLH1) alterations seem to be involved in CRC pathogenesis.     

Identification and characterization of a novel MLH1 genomic rearrangement as the cause of HNPCC in a Tunisian family: evidence for a homologous Alu-mediated recombination

High rates of early colorectal cancers are observed in Tunisia suggesting high genetic susceptibility. Nevertheless, up to now no molecular studies have been performed. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent cause of inherited colorectal cancer. It is caused by constitutional mutations in the DNA mismatch repair (MMR) genes. Here, we investigated a Tunisian family highly suspected of hereditary nonpolyposis colorectal cancer (HNPCC). Six patients were diagnosed with a colorectal or an endometrial cancer at an early age, including one young female who developed a colorectal cancer at 22 years and we tested for germline mutations in MMR genes. MMR genes were tested for rearrangements by MLPA (MLH1, MSH2) and the presence of point mutations by sequencing (MLH1, MSH2, MSH6). Moreover, tumors were analyzed for microsatellite instability and expression of MMR proteins, as well as for somatic rearrangements in MLH1 and MSH2 by MLPA. MMR gene analysis by MLPA revealed the presence of a large deletion in MLH1 removing exon 6. Sequence analysis of the breakpoint region showed that this rearrangement resulted from a homologous unequal recombination mediated by a repetitive Alu sequence. Moreover, tumors harbored biallelic deletion of MLH1 exon 6 and loss of heterozygosity at MLH1 intragenic markers, suggesting duplication of the rearranged allele in the tumor. This germline MLH1 rearrangement was associated to a severe phenotype in this family. This is the first report of a molecular analysis in a Tunisian family with HNPCC.     

Microsatellite instability markers for identifying early-onset colorectal cancers caused by germ-line mutations in DNA mismatch repair genes.

PURPOSE: Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes. EXPERIMENTAL DESIGN: Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel. RESULTS: The National Cancer Institute five-marker panel system scored 31 (29%) as (NCI)MSI-High, 13 (12%) as (NCI)MSI-Low, and 63 (59%) as (NCI)MS-Stable. The 10-marker panel classified 18 (17%) as (10)MSI-High, 17 (16%) as (10)MSI-Low, and 72 (67%) as (10)MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all (10)MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored (10)MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored (10)MSI-High). CONCLUSIONS: In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.     

Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome

Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.     

Parallel high-throughput RNA interference screens identify PINK1 as a potential therapeutic target for the treatment of DNA mismatch repair-deficient cancers

Synthetic lethal approaches to cancer treatment have the potential to deliver relatively large therapeutic windows and therefore significant patient benefit. To identify potential therapeutic approaches for cancers deficient in DNA mismatch repair (MMR), we have carried out parallel high-throughput RNA interference screens using tumor cell models of MSH2- and MLH1-related MMR deficiency. We show that silencing of the PTEN-induced putative kinase 1 (PINK1), is synthetically lethal with MMR deficiency in cells with MSH2, MLH1, or MSH6 dysfunction. Inhibition of PINK1 in an MMR-deficient background results in an elevation of reactive oxygen species and the accumulation of both nuclear and mitochondrial oxidative DNA lesions, which likely limit cell viability. Therefore, PINK1 represents a potential therapeutic target for the treatment of cancers characterized by MMR deficiency caused by a range of different gene deficiencies.     

Genome-wide copy neutral LOH is infrequent in familial and sporadic microsatellite unstable carcinomas

Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.     

缺氧对人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2表达的影响及其机制探讨

背景与目的:缺氧对DNA错配修复系统(mismatch repair, MMR)活性的调控是肿瘤细胞遗传不稳定的重要原因,但其机制尚不完全清楚.本研究拟观察缺氧状态下人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2的表达变化,初步探讨DNA甲基化在其中的作用.方法:应用RT-PCR、Western blot等方法检测H446细胞在缺氧状态下以及甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理后MLH1、MSH2基因的表达水平,同时,采用甲基化特异性PCR(MSP)方法检测MLH1、MSH2基因启动子CpG岛甲基化状态.结果:缺氧状态下,H446细胞MLH1、MSH2基因在转录和翻译水平均显著性降低.同时,随着缺氧时间延长,MLH1基因启动子逐渐由非甲基化状态、部分甲基化状态转变为完全甲基化状态,而MSH2基因启动子则直接由非甲基化状态转变为完全甲基化状态.甲基转移酶抑制剂5-Aza-CdR可使MLH1、MSH2基因表达水平有所恢复,但去除5-Aza-CdR后其表达再次下调.结论:启动子甲基化可能是缺氧诱导H446细胞显著性下调MLH1、MSH2基因表达的重要机制,甲基转移酶抑制剂5-Aza-CdR可恢复其表达.     

Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n = 614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.     

Brostallicin (PNU-166196)--a new DNA minor groove binder that retains sensitivity in DNA mismatch repair-deficient tumour cells

Defects in DNA mismatch repair (MMR) are associated with a predisposition to tumorigenesis and with drug resistance owing to high mutation rates and failure to engage DNA-damage-induced apoptosis. DNA minor groove binders (MGBs) are a class of anticancer agents highly effective in a variety of human cancers. Owing to their mode of action, DNA MGB-induced DNA damage may be a substrate for DNA MMR. This study was aimed at investigating the effect of loss of MMR on the sensitivity to brostallicin (PNU-166196), a novel synthetic alpha-bromoacrylic, second-generation DNA MGB currently in Phase II clinical trials and structurally related to distamycin A. Brostallicin activity was compared to a benzoyl mustard derivative of distamycin A (tallimustine). We report that the sensitivities of MLH1-deficient and -proficient HCT116 human colon carcinoma cells were comparable after treatment with brostallicin, while tallimustine resulted in a three times lower cytotoxicity in MLH1-deficient than in -proficient cells. MSH2-deficient HEC59 parental endometrial adenocarcinoma cells were as sensitive as the proficient HEC59+ch2 cells after brostallicin treatment, but were 1.8-fold resistant after tallimustine treatment as compared to the MSH2-proficient HEC59+ch2 counterpart. In addition, p53-deficient mouse fibroblasts lacking PMS2 were as sensitive to brostallicin as PMS2-proficient cells, but were 1.6-fold resistant to tallimustine. Loss of neither ATM nor DNA-PK affected sensitivity to brostallicin in p53-deficient mouse embryonic fibroblasts, indicating that brostallicin-induced cytotoxicity in a p53-deficient genetic background does not seem to require these kinases. These data show that, unlike other DNA MGBs, MMR-deficient cells retain their sensitivity to this new alpha-bromoacrylic derivative, indicating that brostallicin-induced cytotoxicity does not depend on functional DNA MMR. Since DNA MMR deficiency is common in numerous types of tumours, brostallicin potentially offers the advantage of being effective against MMR-defective tumours that are refractory to several anticancer agents.     

IGF1 is a modifier of disease risk in hereditary non-polyposis colorectal cancer

Patients diagnosed with HNPCC harbouring a confirmed germline mutation in DNA mismatch repair (MMR) genes have an 80% lifetime risk of developing an epithelial malignancy. There is, however, considerable variation in the age of disease onset in these patients. Insulin-like growth factor-I (IGFI) has been implicated in colorectal cancer (CRC), and elevated plasma IGFI levels are associated with both sporadic and hereditary CRC risk. In this study, we further investigate the cytosine-adenine (CA) dinucleotide repeat polymorphism located near the promoter region of IGF1 and its relation to early onset CRC risk in 443 Australian and Polish MMR gene mutation carriers using DNA sequencing, Kaplan-Meier survival curves and Cox proportional hazard regression analysis. A significantly smaller number of IGF1 CA repeats was observed in the Polish patient population, which was associated with an earlier age of disease onset compared to the Australian patients. The threshold for the observed modifying effect was again shown to be in patients with 17 or less CA repeats compared to those with 18 or more. Furthermore, when MMR mutation group (i.e., MLH1 or MSH2), gender and family clustering were included in the final Cox model we observed a more robust trend for the role of the IGF1 CA repeat in predicting age of disease onset in HNPCC patients. In addition, this effect was shown to be equal in both MLH1 and MSH2 mutation carrier groups and not restricted to a particular MMR subgroup (p = 0.001). We conclude that the IGF1 CA repeat is an important modifier of disease onset in HNPCC and the first polymorphism to yield consistent results across different populations.