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Frequent frameshift mutations of simple nucleotide repeats in the protein-encoding regions, as well as replication errors (RERs) at microsatellite loci, have recently been demonstrated in gastrointestinal tumors. These genetic instabilities have been considered indicative of an increased risk of accumulating mutations in cancer-associated genes and of developing multiple cancers. We studied frameshift (or insertion/deletion) mutations of simple nucleotide repeats in five genes (TGFbeta type II receptor [TGFbetaRII], E2F4, MSH2, MSH3, and MSH6) in 23 tumors from 12 patients who had synchronous cancers of the esophagus and other organs. Genetic instability at four microsatellite loci, as well as mutations in the TP53, APC, and KRAS2 genes, were also studied. No frameshift mutations were observed in the TGFbetaRII, MSH3, and MSH6 genes. RER and a deletion mutation of BAT26 in MSH2 were present in one (1/23; 4%) gastric cancer. This tumor also carried a deletion mutation in the serine (AGC) repeat of the E2F4 gene. Mutation screening of the TP53, APC, and KRAS2 genes revealed that the synchronous cancers did not carry the same mutations. Our results suggested that genetic instability, such as insertion/deletion mutations in simple nucleotide repeats, is not significantly associated with the development of multiple primary cancers of the esophagus and other organs, and that these synchronous cancers developed independently according to their different environmental factors.
PURPOSE: To clarify the cellular origin of extranodal marginal-zone B-cell lymphoma (EZML) of the mucosa-associated lymphoid tissue (MALT) type in ocular adnexa, the somatic mutation was analyzed in the immunoglobulin heavy-chain variable region (VH) gene. METHODS: Eight cases of EZML in the orbit and four in the conjunctiva were studied. The VH genes were amplified by a seminested PCR and sequenced directly. These were compared with the closest published VH germline segments to determine the somatic mutation frequency. Intraclonal microheterogeneity, which was termed the ongoing mutation frequency in the current study, was estimated by counting the number of single nucleotide substitutions in individual clones and dividing by the total number of nucleotides analyzed. Nine cases of gastrointestinal EMZL were also examined for comparison. RESULTS: The somatic mutation frequency varied between 2.0% and 12.7%, with a mean value of 7.9%. Ten cases with intraclonal microheterogeneity showed between one and six further substitutions. The average of ongoing mutation frequency was 0.11%, with a range of 0% to 0.25%. In the gastrointestinal EMZLs, the average of somatic mutation frequency was 8.5% (1.5%-14.2%) and of ongoing mutation frequency was 0.51% (0.25%-0.75%). CONCLUSIONS: The average of ongoing mutation frequency in ocular adnexal EMZL was lower than that in gastrointestinal EMZL. Both ocular adnexal and gastrointestinal EMZLs are derived from postgerminal center memory B cells, but the low ongoing mutation frequencies of ocular adnexal EMZL may result from less antigen stimulation and follicular colonization in the orbit relative to gastrointestinal EMZL.
BACKGROUND/AIMS: The genetic pathways of gallbladder cancer are not yet well defined, since the contribution of genetic abnormalities, clarified in other organs, remains questionable. Our aim was to evaluate the role of microsatellite instability in this organs carcinogenesis. METHODOLOGY: We investigated a group of 20 gallbladder carcinomas from Greek patients with regard to alterations in length of the BAT-26 mononucleotide marker--as an indicator of microsatellite instability. The findings were correlated with the presence of p53 and ras mutations, alterations of the bax and TGF-beta RII genes and tumors' clinicopathological features. Polymerase chain reaction and electrophoretic analysis in a non-denaturating 25% polyacrylamide gel was performed for the detection of BAT-26 size variations. RESULTS: None of our specimens showed microsatellite instability at the BAT-26 marker. CONCLUSIONS: BAT-26, an indicator of high-level microsatellite instability, may not be sufficient, when used alone, for determining the microsatellite instability status of gallbladder carcinomas, since these may be characterized by low-level instability. Bax and TGF-beta RII genes may not also be targets of instability in this type of tumors.
Nervous system-specific transcription factors that bind to the octameric deoxyribonucleic acid sequence motif ATGCAAAT (or ATTTGCAT) are known as N-Oct proteins. Neurons and glia contain the ubiquitous Oct-1 protein and four polypeptide complexes termed N-Oct-2, N-Oct-3, N-Oct-4, and N-Oct-5. Previously, we showed that N-Oct proteins are differentially expressed by human neuroblastoma and glioblastoma cell lines in vitro. We have now extended this work to freshly isolated human primary and metastatic brain tumors. Contrary to brain tumor cell lines, of the five astrocytomas and three glioblastomas analyzed, all but two tumors displayed the complete N-Oct protein profile, irrespective of histopathological tumor grade. Two astrocytomas were negative for N-Oct-4. Ten of 13 ependymomas exhibited N-Oct-2, N-Oct-3, and N-Oct-4 but lacked the N-Oct-5 complex. In contrast, brain metastases of two patients with extracerebral carcinomas contained only Oct-1, and cerebral metastases from two cases of B cell lymphomas showed Oct-1 and Oct-2 complexes, the characteristic Oct protein pattern of B lymphocytes. Thus, metastatic carcinoma and lymphoma expressed a non-nervous system phenotype of Oct proteins.
The genetic study of two cases of tubulovillous adenoma associated with poorly differentiated endocrine carcinoma (PDEC) is reported. Aim of this work was to assess whether the exocrine and endocrine growths share a common genotype. The analysis entailed the search for allelic loss (LOH) or imbalances of polymorphic microsatellite markers at the corresponding chromosomal loci of the genes MEN-1 (11q13), p53 (17p13). Deleted in Colorectal Carcinoma (DCC) (18q21) and hMSH-2 (BAT26) (2p21-22). Additionally, the exons 5-8 of the p53 gene were sequenced in the two PDECs only. One of the two cases investigated showed LOH for 18q DCC markers in the tubulo-villous adenoma while a point mutation of the p53 gene was observed in the PDEC component. No genetic abnormality was observed in both adenoma and PDEC components of the other case. In the two cases p53 protein accumulation was observed in both PDEC and adenoma cells. These data indicate that only the p53 gene abnormality is shared by both colon cancer and PDEC in the two cases reported. The lack of other common genetic defect may suggest a different histogenesis for the two tumor types. The development of colon PDEC implies the defect of p53 gene.
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Two tightly linked recombination activating genes, RAG-1 and RAG-2, are involved in VDJ recombination of the immune system. Although these genes were originally thought to be expressed exclusively in precursor B and T cells, RAG-1 transcripts were recently found in the murine central nervous system (CNS) [Chun et al., Cell 6, 189, (1991)]. We found that the RAG-2 gene was expressed in CNS tumor cell lines, melanoma (B-16) and skin fibroblast (A9). RAG-1 expression was not found in any non-lymphoid cell lines examined including CNS tumor cell lines. Another VDJ recombination-related gene RBP-Jk was expressed in all tumor cell lines examined. It remains to be seen whether expression of RAG-1 and RAG-2 in CNS is abortive or functionally important.
The deficiencies of nucleotide excision repair (NER) factors are genetic diseases, xeroderma pigmentosum (XP) increasing risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis for the XP, XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 62.1 % of ovarian tumors (18/29), 16.7% of colon (2/12) and 22.2% lung (2/9) carcinomas. Furthermore, 13.8% of ovarian, 8.3% of colon and 22% of lung carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCAI, BRCA2 and DCC. Although both microsatellite instability and LOH of NER factors were observed in some cases, there was no strong association between them in the present study. These observations raise the possibility that alterations of NER factors may be frequent in human sporadic carcinomas. Further study should be needed to find the direct evidence of NER gene abnormalities in human sporadic carcinoma tissues.
BACKGROUND: The expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and is expressed at high levels in malignant tumors. It is of interest whether expression of hTERT is regulated by methylation of the CpG island in the promoter of the hTERT gene. We examined hTERT expression and methylation status of the hTERT and other genes including p16. METHODS: We analyzed methylation status by bisulfite treatment and polymerase chain reaction with single-strand conformation polymorphism analysis (PCR-SSCP) and expression of the hTERT by RT-PCR, in 13 cancer cell lines, eight white blood cell samples and 24 colorectal cancer tissues. RESULTS: In the cancer cell lines, hTERT was expressed and the CpG island of the hTERT promoter was methylated. Most colorectal cancer tissues showed similar results. The promoter of hTERT was methylated in six cases, partially methylated in 17 cases and unmethylated in one case. All cases with methylation of hMLH1 or p16 also showed methylation of hTERT; however, some of the cases lacking p16 methylation also had hTERT methylation. CONCLUSION: Increased expression of hTERT is related to hypermethylation of hTERT in colorectal cancerous tissues as well as some cancer cell lines and disconcordant with hypermethylation of p16.
We report here a cancer drug therapy use of a gene involved in Down's syndrome. Using bioinformatics approaches, we recently predicted Single Minded 2 gene (SIM2) from Down's syndrome critical region to be specific to certain solid tumors. Involvement of SIM2 in solid tumors has not previously been reported. Intrigued by a possible association between a Down's syndrome gene and solid tumors, we monitored SIM2 expression in solid tumors. Isoform-specific expression of SIM2 short-form (SIM2-s) was seen selectively in colon, prostate, and pancreatic carcinomas but not in breast, lung, or ovarian carcinomas nor in most normal tissues. In colon tumors, SIM2-s expression was seen in early stages. Antisense inhibition of SIM2-s expression in a colon cancer cell line caused inhibition of gene expression, growth inhibition, and apoptosis. The administration of the antisense, but not the control, oligonucleotides caused a pronounced inhibition of tumor growth in nude mice with no major toxicity. Our findings provide a strong rationale for the genes-to-drugs paradigm, establish SIM2-s as a molecular target for cancer therapeutics, and may further understanding of the cancer risk of Down's syndrome patients.
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Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.
Defects in the mismatch repair (MMR) genes hMLH1 and hMSH2 have been found in 10% to 20% of sporadic colorectal carcinomas and also many cases of hereditary nonpolyposis colorectal cancer syndrome. Patients with these tumors have an improved prognosis and may show greater sensitivity to chemotherapy. We examined 458 resected colorectal carcinomas from 430 consecutive patients and used immunohistochemistry to determine which tumors lacked expression of these genes (MMR-d). We correlated the status of MMR-d or "intact" expression with stage, site, and histology. Eighty-nine of 458 tumors (19.4%) were MMR-d, including 80 hMLH1 and 9 hMSH2 tumors. A total of 6% of patients had synchronous tumors, and 37.7% of these were MMR-d (P=0.0008). A high proportion of patients with previous breast cancer (4 of 6 patients) had hMLH1-defective colorectal carcinomas. MMR-d tumors presented at an earlier stage than intact tumors, and the node-positive MMR-d tumors were less likely than intact tumors to have pericolonic extranodal tumor deposits (18.2% vs. 44%). The proportion of tumors at each site that were MMR-d increased progressively from cecum (32%) to ascending (35%) to transverse colon, where 41% of all tumors were defective. The proportions then rapidly decreased, reaching the lowest rate (4.7%) in the rectum. Both types of MMR-d tumors more often had expansive borders, intraepithelial lymphocytosis, peritumoral lymphoid, and Crohn's-like lymphoid responses than the intact tumors; the frequencies of these features diminished with advancing stage. Tumor budding was less common in stage II and III MMR-d tumors than in intact tumors. Keloid and myxoid type stromas correlated with stage and vascular invasion and were not related to mismatch repair status. Significant differences existed between the hMLH1 and hMSH2 tumors. The reported right-sided preponderance of MMR-d tumors is due to most hMLH1, but not hMSH2, tumors being found there (87.5% vs. 44.4%). hMSH2 tumors were most common in the rectum (55.6%). Mucinous tumors were common in hMLH1 tumors (36.3%) but not in hMSH2 tumors (11.1%). hMLH1 tumors were most likely to be poorly differentiated (70%), which was uncommon with hMSH2 tumors (22.2%). hMSH2 tumors were more likely to be confined to the wall (66.7%) than hMLH1 (20%) or intact tumors (23%). We conclude that hMLH1 and hMSH2-defective tumors have distinctly differing histologic features from each other.
Loss of heterozygosity (LOH) on the short arm of chromosome 8 occurs at high frequencies in many tumor types, including colorectal carcinoma. We have previously used microcell-mediated chromosome transfer (MMCT) to map an approximately 7.7 Mb colorectal cancer suppressor region (CRCSR) at 8p22-23.1. We have now taken a candidate gene approach to identify the putative tumor suppressor gene located within the CRCSR. CNOT7 encodes a subunit of the CCR4-Not transcription complex and is located at 8p22. We showed that CNOT7 is expressed in normal colonic mucosa and in colonic crypt cells, as well as in colorectal cell lines and primary tumors. We assembled a panel of 88 primary colorectal tumors comprising 20 MSI-high (high microsatellite instability), 19 MSI-low and 49 MSS (microsatellite stable) tumors for mutation analysis of the CNOT7 gene. Denaturing high-performance liquid chromatography (DHPLC) analysis of the entire coding region of the CNOT7 gene revealed only one somatic missense mutation in an MSS tumor. The rarity of somatic mutations in CNOT7, and its expression in primary colorectal tumors and cell lines, suggests that CNOT7 is not the target tumor suppressor gene in the 8p22-23.1 CRCSR.
In order to determine the effects of mismatch repair (MMR) deficiencies in sporadic colorectal carcinomas, 45 such cancers were examined using a sensitive method called crypt isolation technique. Loss of heterozygosity (LOH) in the MSH2 or MLH1 gene was more frequently observed in replication error (RER) (+) carcinomas than in RER (-) carcinomas, which implied that loss of one normal allele could partly affect repair capacity. MSH2 gene defects at both alleles were observed in two carcinomas, which showed severe repair deficiencies. Interestingly, unlike the situation observed in the p53 gene, the MSH2 and MLH1 genes did not show complete LOH. Novel crypt isolation-based subpopulation (CISP) analysis demonstrated that at least two distinct carcinoma subpopulations existed in most carcinomas that showed incomplete LOH; one with and one without LOH. In one carcinoma that had germline mutation and somatic incomplete LOH of the MSH2 gene, the mutator phenotype was only observed in populations affected in both alleles. Thus, the MSH2 gene appears to possess the two hits mechanism of tumor suppressor genes. However, unlike the tumor suppressor genes, MMR gene defects lead to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.
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.
Chromosome 18q is lost a high proportion of colorectal and pancreatic cancers. Three candidate tumor suppressor genes, DCC, Smad4 and Smad2 have been identified in this chromosome region. DCC and Smad4 aberrations have been previously identified in pancreatic and colorectal tumors. The aim of this study was to compare the presence of concurrent genetic aberrations in DCC and neighboring Smad4 and Smad2 genes during colorectal and pancreatic distal dissemination. We have used a panel of orthotopically implanted colorectal and pancreatic xenografts and corresponding metastases. We have shown that while LOH at DCC locus occurred at a similar frequency in both tumors, diminished DCC protein expression was exclusively present in colorectal tumors harboring intragenic DCC LOH. In contrast, in pancreatic xenografts loss of DCC protein and mRNA expression was restricted to metastases. Smad4 gene aberrations were detected at a similar frequency in both tumors and were selected for during distal dissemination. Acquisition of alterations in both genes occurred independently. Our results suggest that both DCC and Smad4 contribute to pancreatic and colorectal distal dissemination. However, the role of DCC may differ between both tumor types.