Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes

To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.     

Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer

The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC). On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref. 3). Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23. We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family.     

Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability

Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colon cancer and in a subset of patients with sporadic colorectal cancer (CRC). In sporadic CRC, three tumor phenotypes have been defined: microsatellite stable (MSS), low-frequency MSI, and high-frequency MSI (MSI-H). Although defective mismatch repair, consisting primarily of alterations in hMSH2 and hMLH1, is believed to be responsible for the MSI phenotype in the majority of patients with hereditary nonpolyposis colon cancer, the genetic defect responsible for this phenotype in sporadic CRC has yet to be clearly delineated. Somatic or germ-line alterations in these two genes have been identified in only a minority of these cases. Analysis of the protein expression patterns of hMSH2 and hMLH1 in unselected CRC, however, suggests that alterations in hMLH1 may account for a majority of the MSI-H cases. In an effort to explore the underlying molecular basis for these findings, we have examined the methylation status of the presumptive hMLHI promoter region in 31 tumors that vary in regard to their MSI status (MSI-H or MSS), their hMLH1 protein expression (MLH- or MLH+), and their gene mutation (Mut+ or Mut-) status. Hypermethylation of the hMLH1 promoter occurred in all 13 MSI-H/ MLH- tumors that did not have a detectable mutation within the hMLH1 gene. Of those MSI-H tumors containing germ-line or somatic alterations in hMLH1 (n = 7, including 3 frameshift, 1 nonsense, 2 missense mutations, and 1 tumor containing multiple mutations: missense, splice-site alteration, and a frameshift), four had a normal methylation pattern, whereas three others demonstrated hypermethylation of the hMLH1 promoter region. Two of these cases had a missense alteration, the other a frameshift alteration. The single MSI-H/Mut+ tumor that had normal hMLH1 and hMSH2 expression, as well as 9 of the 10 MSS cases, lacked methylation of the hMLH1 promoter. Hypermethylation of the hMSH2 promoter was not observed for any of the cases. These results suggest that hypermethylation of the hMLH1 promoter may be the principal mechanism of gene inactivation in sporadic CRC characterized by widespread MSI.     

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.     

Expression of the mismatch repair gene hMSH2 in sporadic colorectal cancer

At least four genes involved in DNA mismatch repair (MMR), hMSH2, hMLH1, hPMS1 and hPMS2, have been cloned and characterized. These genes have been demonstrated to be altered in the germline of patients with hereditary non-polyposis colorectal cancer (HNPCC). HNPCC is an autosomal dominant disease characterized by a preponderance of proximal colon, young age of onset, increased multiplicity, and improved stage-specific survival. In this study, we examined the expression of hMSH2 protein in sporadic colorectal cancer (CRC). As a result, the frequency of right-sided CRC and multiple CRCs were significantly higher in the patients with hMSH2-negative CRC than in those with hMSH2-positive CRC. The rate of p53 positivity was significantly lower in the hMSH2-negative tumours than that in the hMSH2-positive tumours. The disease-free survival rate tended to be higher in the patients with hMSH2-negative CRC than in the patients with hMSH2-positive CRC. Our findings suggest that both the clinicopathological and biological features of hMSH2-negative sporadic CRC seemed to be similar to those of HNPCC. To clarify the mechanism of carcinogenesis in HNPCC and sporadic CRC, further investigations of genetic alterations caused by MMR genes will be needed.     

Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.     

Mutation of hMSH3 and hMSH6 mismatch repair genes in genetically unstable human colorectal and gastric carcinomas

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.     

Mutations of the transforming growth factor-beta type II receptor gene are strongly related to sporadic proximal colon carcinomas with microsatellite instability

BACKGROUND: Mutations of the transforming growth factor-beta type II receptor gene (TGF-beta RII) have been found in several replication error-positive sporadic colorectal carcinomas and hereditary nonpolyposis colorectal carcinoma cell lines. The aim of this study was to clarify the role of TGF-beta RII in sporadic colorectal carcinogenesis. METHODS: The authors screened for mutations at simple repeated sequences in the TGF-beta RII gene by polymerase chain reaction-single strand conformation polymorphism. They also examined genomic instability, using five microsatellite DNA markers in 69 sporadic colorectal carcinomas. When the carcinomas exhibited the TGF-beta RII mutations, the authors screened further for mutations in two DNA mismatch repair genes, hMSH2 and hMLH1. RESULTS: Seven of the 69 cancers (10%) showed one or two A deletions in TGF-beta RII and resultant frameshift mutations in nucleotide positions 709-718 containing a (A) 10 repeated sequence; but none of these appeared in the corresponding normal DNA, indicating a somatic mutation. All of the seven cancers were located in the proximal colon; there were none in the distal colon (P < 0.01). On the other hand, 22 of the 69 carcinomas (32%) showed the replication error-positive phenotype. The frequency of replication errors in proximal colon carcinomas was higher than that in distal colon carcinomas (P < 0.05). All 7 cancers with TGF-beta RII mutations showed replication errors. One of them revealed a nonsense mutation at codon 413, and 1 revealed a loss of heterozygosity in hMSH2. CONCLUSIONS: These data indicate that mutations of TGF-beta RII are strongly related to proximal colon carcinomas with microsatellite instability and that the mechanism of carcinogenesis in some proximal colon carcinomas is similar to that in hereditary nonpolyposis colorectal carcinoma.     

Complex genetic predisposition to cancer in an extended HNPCC family with an ancestral hMLH1 mutation

Hereditary non-polyposis colorectal cancer (HNPCC) is characterised by a genetic predisposition to develop colorectal cancer at an early age and, to a lesser degree, cancer of the endometrium, ovaries, urinary tract, and organs of the gastrointestinal tract other than the colon. In the majority of families the disease is linked to mutations in one of the two mismatch repair genes, hMSH2 or hMLH1. We have found a novel hMLH1 nonsense mutation in a Swiss family with Lynch syndrome, which has been transmitted through at least nine generations. A different tumour spectrum of neoplasms of the skin, soft palate, breast, duodenum, and pancreas was observed in three branches of this family, where there was a virtual absence of colonic tumours. The hMLH1 mutation could not be detected in members of these branches suggesting that at least a second genetic defect predisposing to cancer is segregating in part of the kindred.     

Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome involving a predisposition to cancers of the colon, endometrium and several other extra-colonic sites, accounting for approximately 1-5% of all colorectal cancer cases. It is not easily recognized because of a lack of distinctive clinical markers, making diagnosis and management of this disease problematic. To provide a basis for uniformity in diagnosis of HNPCC, the Amsterdam criteria were proposed and are currently in use. More recently, the discovery of four human mismatch repair genes (hMSH2, hMLH1, hPMS1 and hPMS2) has provided novel insight into the genetic basis of this disease, and raised the possibility of genetic diagnosis for management of HNPCC patients and their family members. This report summarizes the clinicopathologic aspects of HNPCC, reviews the recent genetic findings and surveillance strategies, and suggests a novel designation of certain patients as suspected HNPCC.     

Reduced COX-2 protein in colorectal cancer with defective mismatch repair

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.     

Combining stimulus fading, reinforcement, and extinction to treat food refusal

The expression of mismatch repair proteins hMSH2 and hMLH1 was investigated in human ovarian cancer cell lines and in biopsies of ovarian carcinomas obtained from 20 patients undergoing surgical operation. By Western blotting analysis hMSH2 protein was detected in all the tumor samples analyzed and in eight out of nine human ovarian cancer cell lines, while hMLH1 was undetectable in four out of 20 ovarian tumors and in five out of nine human ovarian cancer cell lines analyzed. The possible presence of frameshift mutations in the BAX gene, which contains a sequence of eight contiguous guanines in its third exon, was tested in all the samples. All the cell lines presented the normal alleles for the BAX gene while only in one of the tumor samples a heterozygous frameshift mutation was found. The frameshift mutation was associated to a low, almost undetectable, level of BAX protein which was instead present at much higher levels in all the other samples investigated. The results indicate that frameshift mutations in the BAX gene, possibly arising as a consequence of microsatellite instability (detectable in these tumors), is detectable in human ovarian cancer although quantitatively it does not appear to be a major determinant of the low apoptotic response to chemotherapy observed in ovarian cancer cells.     

Correction of hypermutability, N-methyl-N'-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6

The human DNA mismatch repair genes hMSH2 and hMSH6 encode the proteins that, together, bind to mismatches to initiate repair of replication errors. Human tumor cells containing mutations in these genes have strongly elevated mutation rates in selectable genes and at microsatellite loci, although mutations in these genes cause somewhat different mutator phenotypes. These cells are also resistant to killing by certain drugs and are defective in mismatch repair. Because the elevated mutation rates in these cells may lead to mutations in additional genes that are causally related to the other defects, here we attempt to establish a cause-effect relationship between the hMSH2 and hMSH6 gene mutations and the observed phenotypes. The endometrial tumor cell line HEC59 contains mutations in both alleles of hMSH2. The colon tumor cell line HCT15 contains mutations in hMSH6 and also has a sequence change in a conserved region of the coding sequence for DNA polymerase delta, a replicative DNA polymerase. We introduced human chromosome 2 containing the wild-type hMSH2 and hMSH6 genes into HEC59 and HCT15 cells. Introduction of chromosome 2 to HEC59 cells restored microsatellite stability, sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment, and mismatch repair activity. Transfer of chromosome 2 to HCT15 cells also reduced the mutation rate at the HPRT locus and restored sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment and mismatch repair activity. The results demonstrate that the observed defects are causally related to mutations in genes on chromosome 2, probably hMSH2 or hMSH6, but are not related to sequence changes in other genes, including the gene encoding DNA polymerase delta.     

Neurofibromatosis and early onset of cancers in hMLH1-deficient children

Hereditary nonpolyposis colon cancer is a common hereditary disorder caused by the germ-line mutations of DNA mismatch repair (MMR) genes, especially hMLH1 and hMSH2. We report here the first identification of human compounds with a homozygous inactivation of a MMR gene. In a typical hereditary nonpolyposis colon cancer family, MMR-deficient children conceived from matings between heterozygotes for a hMLH1 deleterious mutation exhibited clinical features of de novo neurofibromatosis type I and early onset of extracolonic cancers. This observation demonstrates that MMR deficiency is compatible with human development but may lead to mutations during embryogenesis. On the basis of clinical symptoms observed in MMR-deficient children, we speculate that the neurofibromatosis type 1 gene is a preferential target for such alterations.     

Instability of micro-satellite sequences of DNA associated with genetic alterations in head and neck neoplasms. Review of the literature and preliminary results of a research plan

BACKGROUND: It is known that tumoral progression towards a metastatic stage is identifiable with a genomic instability. This instability leads to both primary and secondary genetic alterations. It can give some selective proliferative advantages. This study aims to review the literature on instability of the microsatellite sequences of DNA associated with genetic alterations in tumors of the head and neck. In particular, the studies on relations between instability of micro-satellite sequences and expression of MLH1, MSH2, PMS1, PMS2 genes have been reviewed. Further aim of this study is to present preliminary results of a research project into distribution of hMLH1 and hMLH2 proteins in oral tissues. METHODS: Nine formalin fixed and paraffin embedded samples (8M/1F; mean age 58 years) of oral squamous cell carcinomas tissues have been analysed for the presence of hMSH2 and hMLH1 by using streptavidin-biotin immunoperoxidase technique. Samples have been analysed by optical microscope by two observers evaluating the positive cell percentage for three representative fields. RESULTS: Immunohistochemistry demonstrated that hMLH1 and hMSH2 are widely expressed nuclear proteins in oral tissues. CONCLUSIONS: The localization of hMLH1 and hMSH2 in oral epithelium are consistent with the biochemical function of these proteins in DNA mismatch repair.     

Interactions of human hMSH2 with hMSH3 and hMSH2 with hMSH6: examination of mutations found in hereditary nonpolyposis colorectal cancer

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.     

Optimisation of sample presentation for the near-infrared spectra of pharmaceutical excipients

The two most common forms of hereditary ovarian cancer are: the breast ovarian cancer syndrome, and ovarian cancer associated with HNPCC (hereditary nonpolyposis colorectal cancer) syndrome. Studies have shown that these diseases may be associated with mutations in a number of tumor suppressor genes, mainly BRCA1 and BRCA2. Malfunction of the protein products of these genes have also been found to be involved in sporadic ovarian cancer, which makes up the majority of ovarian cancer cases. HNPCC-ovarian cancer associated families reveal frequent mutations in at least four genes (hMSH2, hMLH1, hPMS1, and hPMS2) involved in the repair of mismatched DNA. With ovarian cancer being such an important health issue, the push is on to design reliable screening tests to detect defective inherited or somatic alleles in individual carriers. So far, most progress has been demonstrated in those patients with family histories of the disease who are at increased risk. The ramifications of such research may impact a variety of scientific, clinical, legal, ethical, and psychosocial issues. In addition to current treatment modalities, positive results of these tests may indicate the need for increased clinical surveillance, prophylactic treatment, and genetic counseling of patients on an individual basis. It remains to be seen whether the technology can be made reliable enough to not only benefit high-risk individuals but also the general population.     

Mutation of a mutL homolog in hereditary colon cancer

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.     

A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype

Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a "two-hit" inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.