Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.     

Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin.

In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene.     

Selective sensitivity to carboxyamidotriazole by human tumor cell lines with DNA mismatch repair deficiency

We have previously reported that high-dose nifedipine had a selective antiproliferative effect on colon cancer cell lines deficient in DNA mismatch repair (MMR). We hypothesized that carboxyamidotriazole (CAI), a calcium channel blocker, would also have a selective inhibitory effect on colon cancer cell lines with DNA MMR deficiency. In addition, we speculated that this effect may also be seen in cell lines deficient in DNA MMR derived from other tumor types. Fourteen human cancer cell lines with and without DNA MMR derived from carcinomas of the colon, bladder, ovary and prostate were treated with CAI, vehicle or control drugs (nifedipine and 5-flurouracil). The effect of treatment on growth inhibition, invasion, apoptosis and cell cycle progression was assessed. Selective sensitivity to CAI was observed in all cancer cell lines deficient in MMR. Compared with the MMR-proficient cells, the matched deficient cells were significantly more sensitive to the growth inhibitory effect of CAI and nifedipine, but less sensitive to 5-flurouracil. CAI significantly inhibited the invasive ability of MMR-deficient cancer cells compared to 5-flurouracil. CAI induced more apoptosis but similar level of G(2)/M arrest in MMR (hMLH1- or hMSH6-)-deficient colon cancer cells than MMR-proficient counterparts. CAI selectively inhibits proliferation and invasion in MMR-deficient human cancer cell lines. The antitumor effect is at least partly explained by G2/M cell cycle arrest and induction of apoptosis. These findings may have clinical implications directing clinical trials in selectively targeted patients with DNA MMR tumors.     

Mutation rate at the hprt locus in human cancer cell lines with specific mismatch repair-gene defects

Spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus were measured in human cancer cell lines defective in the mismatch repair (MMR) genes hMLH1, hPMS2, or GTBP, as well as in a cell line carrying mutations in both hMLH1 and hPMS2. The mutation rate was determined by quantitating mutant frequency increases within a single culture as a function of cell division. These MMR- deficient cell lines exhibited a 50- to 750-fold increase in mutation rate relative to a MMR-proficient cancer cell line. From lowest to highest, the spontaneous mutation rates relative to the MMR-gene defects studied here are as follows: hMLH1- < GTBP- < hPMS2- < hMLH1- / hPMS2-. In addition, a cell line in which MMR was restored by chromosome transfer exhibited a mutation rate 12-fold below the MMR- deficient parental cell line. These data support the notion that MMR plays an important role in controlling the rate of spontaneous mutation and suggest that different MMR-gene defects may vary in their ability to repair different types of DNA mismatches, thus leading to measurable quantitative differences in spontaneous mutagenesis. Furthermore, a difference in mutation rates was observed between a hPMS2-defective cell line (3.1 x 10(-5) mutations/cell/generation) and two hMLH1- defective cell lines (4.0 x 10(-6) and 7.3 x 10(-6) mutations/cell/generation). Assuming the hPMS2- and hMLH1-gene products only function in the proposed hMutL alpha heterodimer, then defects in either gene should yield comparable mutation rates. These data suggest that hPMS2 plays a critical role in MMR, while additional hMLH1 homologues or hPMS2 alone may function to partially complement defects in hMLH1.      

Microsatellite mutations of transforming growth factor-beta receptor type II and caspase-5 occur in human precursor T-cell lymphoblastic lymphomas/leukemias in vivo but are not associated with hMSH2 or hMLH1 promoter methylation

In solid cancers, defective DNA mismatch repair (MMR) is most commonly caused by hMSH2 or hMLH1 mutations, or epigenetic silencing of hMLH1 by promoter hypermethylation, and results in the acquisition of characteristic frameshift microsatellite mutations of mononucleotide repeats located within the coding regions of defined target genes. We previously identified hMSH2 mutations in T-cell lymphoblastic lymphoma (T-LBL) patient tumor samples and others have reported coding region microsatellite mutations in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Thus, while MMR gene mutations are known to occur in some human T-lymphoblastic tumors in vivo, it is still unknown if the coding region microsatellite mutations detected in human cell lines also occur in vivo or if hMLH1 or hMSH2 promoter hypermethylation contributes to defective MMR in these tumors. We analyzed the TGFbetaRII (A)10 and caspase-5 (A)10 coding region repeats in 16 human T-LBL/ALL patient tumor samples and identified six with microsatellite mutations in one or both repeats. There was no evidence of hMSH2 or hMLH1 promoter methylation as assessed by standard methylation specific PCR or by a novel temporal temperature gradient electrophoresis (TTGE) method that analyzed 25 and 30 CpG sites in the hMLH1 and hMSH2 promoters, respectively. Our results indicate that coding region microsatellite mutations characteristic of defective MMR occur in some human T-LBL/ALL in vivo but not as a consequence of hMLH1 or hMSH2 promoter hypermethylation. Furthermore, the identification of TGFbetaRII and caspase-5 coding region mutations in vivo implicates these genes in the pathogenesis of human T-LBL/ALL.     

Hypersensitivity in DNA mismatch repair-deficient colon carcinoma cells to DNA polymerase reaction inhibitors

We studied the cytotoxic effects of various DNA replication inhibitors on MMR-deficient and -proficient colon carcinoma cell lines. DNA polymerase (pol) inhibitors including aphidicolin and gemcitabine, and hydroxyurea were more toxic (1.7 to 2.8-fold) to hMLH1-deficient HCT116 than to hMLH1-proficient HCT116+ch3. Similarly, pol inhibitors were more toxic to hMSH2-deficient LoVo than to hMSH2-proficient LoVo+ch2. In contrast, DNA topoisomerase I inhibitors, such as CPT-11, SN-38, and topotecan, were more toxic to MMR-proficient cells. Our results suggest that MMR-deficient colon carcinoma cells are hypersensitive to inhibitors of the pol reaction.     

Analysis of microsatellite instability in acquired drug-resistance human tumor cell lines

Genomic instability characterized as microsatellite instability (MIN) is associated with loss of DNA mismatch repair (MMR) protein. Several studies have shown that loss of DNA MMR protein confers resistance to some interacting DNA chemotherapeutic drugs, but also that exposure of MMR-proficient cells to these drugs can result in loss of MMR protein accompanied by induction of MIN. Such associations were mainly reported for cisplatin, but scarce data are available for doxorubicin (a DNA interacting agent), and nothing is known about vinblastine (an antitubulin agent). Thus, in this study we have analyzed MIN frequency in different type of human tumor cell lines characterized by their MMR protein status and resistant to doxorubicin or to vinblastine. Relationship between MIN occurrence and drug resistance was firstly verified in cisplatin resistant cells, and showed a MIN enrichment (33%) only in the MMR-deficient cells. In order to determine whether treatment of MMR-proficient cells with doxorubicin might lead to induction of MIN, we analyzed two different MMR-proficient cell lines. Variations of MIN frequency were found with either high levels of MIN (66%) or no MIN at all (0%). Effect of vinblastine was analyzed according to the MMR status in two different MMR-proficient and -deficient cells. No major change in MIN frequency was found either in the MMR-proficient (0%) or -deficient (9%) cells. Our results demonstrate that MIN occurs only in tumor cells resistant to cisplatin or doxorubicin, thus supporting earlier findings reporting such associations only with drugs interacting with DNA. Moreover, the data show that MIN does not appear in all tumor cell lines, suggesting that induction of MIN in relation to MMR status is a complex phenomenon which does not only depend on the drug considered (interacting or not with DNA), but also on the tumor cell variant.     

MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells

In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.     

DNA mismatch repair genes hMLH1, hMSH2, and hMSH6 are not inactivated in bronchioloalveolar carcinomas of the lung.

BACKGROUND: Defective DNA mismatch repair (MMR) appears to be rare in nonsmall cell carcinomas of the lung. Defective DNA MMR results from genetic or epigenetic alterations that inactivate the DNA MMR genes hMLH1 or hMSH2, and rarely hMSH6. The loss of normal DNA MMR is thought to promote tumorigenesis by accelerating the accumulation of mutations in oncogenes and tumor suppressor genes. Inactivation of hMLH1, hMSH2, and hMSH6 is observed as a loss of expression of these proteins by immunohistochemistry. Bronchioloalveolar carcinoma is a subtype of adenocarcinoma with distinctive clinical and pathologic features. MATERIALS AND METHODS. An immunohistochemical study was performed on paraffin embedded sections of 33 bronchioloalveolar carcinomas (20 nonmucinous and 13 mucinous) for hmlh1, hmsh2, and hmsh6 proteins. RESULTS All the tumors showed normal expression of hmlh1, hmsh2, and hmsh6. CONCLUSIONS: These findings suggest that defective DNA MMR due to inactivation of hMLH1, hMSH2, or hMSH6 does not play a significant role in the pathogenesis of bronchioloalveolar carcinomas.     

Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.

PURPOSE: The relationships between mismatch repair (MMR) protein expression, microsatellite instability (MSI), family history, and germline MMR gene mutation status have not been studied on a population basis. METHODS: We studied 131 unselected patients with colorectal cancer diagnosed younger than age 45 years. For the 105 available tumors, MLH1, MSH2, MSH6, and PMS2 protein expression using immunohistochemistry (IHC) and MSI were measured. Germline DNA was screened for hMLH1, hMSH2, hMSH6, and hPMS2 mutations for the following patients: all from families fulfilling the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); all with tumors that were high MSI, low MSI, or that lacked expression of any MMR protein; and a random sample of 23 with MS-stable tumors expressing all MMR proteins. RESULTS: Germline mutations were found in 18 patients (nine hMLH1, four hMSH2, four hMSH6, and one hPMS2); all tumors exhibited loss of MMR protein expression, all but one were high MSI or low MSI, and nine were from a family fulfilling Amsterdam Criteria. Sensitivities of IHC testing, MSI (high or low), and Amsterdam Criteria for MMR gene mutation were 100%, 94%, and 50%, respectively. Corresponding positive predictive values were 69%, 50%, and 75%. CONCLUSIONS: Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.     

Mutated gene-specific phenotypes of dinucleotide repeat instability in human colorectal carcinoma cell lines deficient in DNA mismatch repair.

Mutations in DNA mismatch repair (MMR) genes in hereditary non-polyposis colon cancer (HNPCC) patients revealed the importance of MMR deficiency as a risk for carcinogenesis. Since diverse mutations occur in several MMR genes, the instability of repeat sequences dispersed in the genome, which are also governed by the MMR system, is a well used marker. However, the relationship between repeat sequence instability and MMR gene mutation in human cells has not been well defined mainly because precise systems to analyse repeat sequences have not been available. Using our newly developed system, we analysed alteration of dinucleotide repeats in human cell lines which harbour mutations in MMR genes. Among 24 subclones of DLD-1 cells (hMSH6-) only one had a dinucleotide repeat alteration in only one microsatellite locus, while LoVo cells (hMSH2-/hMSH6-) exhibited marked dinucleotide repeat instability (DRI). HCT116 cells, a hMLH1-mutant, showed an ultimate DRI phenotype. Interestingly, SW48 cells lacking hMLH1 expression also demonstrated DRI, albeit the extent of diversity being significantly lower than HCT116. These data suggest that the DRI phenotype in human cells is highly dependent on mutated MMR genes and on forms of mutation. The results of DRI analyses used to detect MMR-deficiency should be interpreted with caution.     

Mismatch repair and microsatellite instability in esophageal cancer cells

Using in vitro mismatch repair (MMR) assay, we have identified 3 of 22 esophageal cancer cell lines exhibiting reduced MMR activity. By means of gel-shift assay, decreased binding ability to GT mismatch and CA loop was observed in these 3 cell lines. However, we could not find any mutations in the hMSH2, hMSH3 and hMSH6 genes, the protein products of which exhibit mismatch binding activity in human cells. In addition, when using antibodies against 5 MMR-related proteins (hMSH2, hMSH3, hMSH6, hPMS2 and hMLH1), no aberrant expression was detected in any of them. When we examined 9 microsatellite loci in endogenous genomic DNA, these 3 esophageal cancer cell lines, deficient in MMR, did not exhibit microsatellite instability. However, when we examined the repetitious sequence on exogenous plasmid DNA which was introduced into these 3 esophageal cancer cells, the results suggested that MMR deficiency in esophageal cancer cells could result in moderate instability of the exogenous sequence.     

Immunohistochemical analysis of expression and allelotype of mismatch repair genes (hMLH1 and hMSH2) in bladder cancer

Mutation of human homologues of DNA mismatch repair (MMR) genes in tumours has been shown to be associated with the phenomenon of microsatellite instability (MSI). Several studies have reported the occurrence of MSI in bladder cancer, but evidence of involvement of MMR genes in the pathogenesis of this cancer is still unclear. We therefore utilized quantitative immunohistochemical (IHC) image analysis and PCR-based allelotype analysis to determine hMLH1 and hMSH2 genes alteration in a cohort of Egyptian bladder cancer samples. IHC analysis of 24 TCC and 12 SCC revealed marked- intra and intertumour heterogeneity in the levels of expression of the two MMR proteins. One TCC lost MLH1 expression and one lost MSH2, (1/24, 4%), and one SCC lost MSH2 (1/12, 8%). A large proportion of analysed tumours revealed a percentage positivity of less than 50% for MLH1 and MSH2 expression (44% and 69%, respectively). Complete loss of heterozygosity in three dinucleotide repeats lying within, or in close proximity to, hMLH1 and hMSH2 was rare (2/57, (4%) for MLH1; and 1/55, (2%) for MSH2), however allelic imbalance was detected in 11/57 (hMLH1) and 10/55 (hMSH2) at any of the informative microsatellite loci. These alterations in structure and expression of DNA MMR genes suggest their possible involvement in the tumorigenesis and/or progression of bladder cancer.     

Loss of expression of DNA repair enzymes MGMT,hMLH1, and hMSH2 during tumor progression in gastric cancer

BACKGROUND: Disorders of the DNA repair system that protects against alkylating mutagens are known to play an important role in carcinogenesis. METHODS: We investigated the expression of the DNA repair enzyme that protects against alkylating mutagens, O(6)-methylguanine DNA methyltransferase (MGMT), and the mismatch repair (MMR) enzymes, hMLH1 and hMSH2, in 135 gastric cancer specimens by immunohistochemical means. RESULTS: The immunoreactivity of MGMT and MMR proteins correlated significantly with several clinicopathologic factors. The survival curve in 116 patients showed that a loss of MGMT or hMLH1, but not of hMSH2, correlated with a poor prognosis. Combined evaluation of MGMT and hMLH1 revealed that the survival of patients with negative status for both MGMT and hMLH1 was shortest. However, this significant association between patient survival and MGMT or hMLH1 expression disappeared when early and advanced cancers were separately analyzed, indicating that synchronous losses of MGMT and hMLH1 increase during tumor progression and stage. Further evaluation according to histologic type revealed that loss of MGMT, hMLH1, and hMSH2 expression significantly differed between early and advanced cancer in differentiated-type cancers. In contrast, in undifferentiated-type cancer, loss of MGMT and MMR expression was frequently found even in intramucosal (m) cancer, and no significant difference was found in loss of hMLH1 and hMSH2 between early and advanced cancer. CONCLUSION: These findings demonstrate that the reduced expression of MGMT, hMLH1, and hMSH2 in differentiated-type cancer may play an important role during tumor progression between the early and advanced stage. On the other hand, in undifferentiated-type cancer, loss of MGMT and the MMR proteins appears to be an important event at carcinogenesis or at an earlier step of tumor progression.     

Apoptosis induced by overexpression of hMSH2 or hMLH1.

Mutations of the mismatch repair genes hMSH2 and hMLH1 have been found in a high proportion of individuals with hereditary nonpolyposis colon cancer (HNPCC), establishing the link between mismatch repair and cancer. Tumor cell lines that are deficient in mismatch repair develop a mutator phenotype that appears to drive the accumulation of mutations required for tumor development. However, mutations of other mismatch repair genes such as hPMS2 can lead to a mutator phenotype, although inherited mutations of these genes are rare in HNPCC families. Here, we show that overexpression of hMSH2 or hMLH1 but not of hMSH3, hMSH6, or hPMS2 induces apoptosis in either repair-proficient or -deficient cells. Furthermore, primary mouse embryo fibroblasts derived from Msh2-deficient mice lose their ability to undergo apoptosis after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These results suggest that the mismatch repair proteins hMSH2 and hMLH1 may be components of a pathway that influences apoptosis. We consider the possibility that loss of apoptosis as a result of hMSH2 or hMLH1 deficiency may be an additional factor in cancer predisposition in HNPCC.     

Differential radiosensitization in DNA mismatch repair-proficient and -deficient human colon cancer xenografts with 5-iodo-2-pyrimidinone-2'-deoxyribose.

PURPOSE: 5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR+) and -deficient (MMR-) human colon cancer xenografts. EXPERIMENTAL DESIGN: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR-, hMLH1-) and HCT116/3-6 (MMR+, hMLH1+). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine. RESULTS: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR-) tumor xenografts compared with HCT116/3-6 (MMR+) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11-14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR-) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR+) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups. CONCLUSIONS: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat "drug-resistant" MMR-deficient tumors as well as MMR-proficient tumors.     

Immunohistochemistry of proteins for DNA mismatch repair in correlation to prognostic factors of mammarian cancer

Mutations in genes of the DNA mismatch repair system (MMR) are strongly linked to the development of hereditary non-polyposis colorectal cancer and play a significant role in sporadic cancer too. Besides the repair of chromosomal mismatches produced during replication, the MMR is the linkage of DNA mismatches to cell cycle control. Proteins of the MMR are necessary for the induction of apoptosis in response to non-tolerable amounts of DNA damage. We correlated the immunoreactivity of the MMR proteins hMSH2, hMLH1 and PMS2 to the immunoreaction of p53, the proliferation marker Ki67 and clinical prognosis factors such as tumor grading and staging, steroid receptor expression and hemangiosis carcinomatosa or lymphangiosis carcinomatosa in 200 samples from patients with diagnosed breast cancer. No correlation could be detected among the expression of the three MMR-proteins hMSH2, hMLH1 and PMS2. The expression of hMSH2 correlated positively with the expression of p53, with the appearance of distant metastases, low differentiation and the appearance of hemangiosis carcinomatosa and lymphangiosis carcinomatosa, while it negatively correlated with the expression of the estrogen receptor. No correlation was detected between hMLH1 or PMS2 and any of the investigated factors. The expression of hMSH2 seems to be related with predictors of an unfavorable course of disease in breast cancer.     

Allelic imbalance at the DNA mismatch repair loci,hMSH2, hMLH1, hPMS1, hPMS2 and hMSH3, in squamous cell carcinoma of the head and neck

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. OBJECTIVE: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. MATERIALS AND METHODS: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. RESULTS AND CONCLUSIONS: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.     

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.