Somatic mutation of the APC gene in gastric cancer: frequent mutations in very well differentiated adenocarcinoma and signet-ring cell carcinoma.

We searched for somatic mutations of the adenomatous polyposis coli (APC) gene in DNA samples isolated from 57 sporadic gastric cancers, by means of a ribonuclease (RNase) protection analysis coupled with DNA amplification by the polymerase chain reaction (PCR). Examining 30% of the APC coding region, including a region where somatic mutations in colorectal tumors are known to be clustered, we detected somatic mutations in 12 tumors; seven in 17 very well differentiated adenocarcinomas, two in 19 well or moderately differentiated adenocarcinomas, and three in ten signet-ring cell carcinomas. So far, no somatic mutations have been identified in 11 poorly differentiated adenocarcinomas. Eight of the 17 somatic mutations found in 12 tumors caused truncation of the gene product due to a nonsense mutation and a 1-, 2- or 5-bp deletion; nine others were point mutations that altered amino acids. Our results suggest that inactivation of APC plays a role in development of some gastric cancers, particularly very well differentiated adenocarcinomas and signet-ring cell carcinomas.     

Somatic mutations of the beta-TrCP gene in gastric cancer

Beta-TrCP is a component of the ubiquitin ligase complex targeting beta-catenin for proteasomal degradation, and is a negative regulator of Wnt/beta-catenin signaling. To determine whether genetic alterations of the beta-TrCP gene are involved in the development or progression of gastric cancer, we analyzed its somatic mutations in 95 gastric cancers by single-strand conformational polymorphism and sequencing. We found five missense mutations (5.3%): A99V, H342Y, H425Y, C206Y, and G260E. Tissue carrying mutations showed moderate to strong cytoplasmic and/or nuclear staining of beta-catenin by immunohistochemistry. Thus, somatic mutations of the beta-TrCP gene may contribute to the development of gastric cancer through beta-catenin stabilization.     

Somatic mutation of the Caspase-5 gene in human lung cancer

Using cDNA array-based gene expression profiling, we previously found reduced expression of the Caspase-5 gene in highly metastatic subpopulations of a lung cancer cell line. The Caspase-5 gene contained poly(A) repeats in its coding region, an area that has been reported to be mutated in both endometrial and gastrointestinal tumors displaying evidence of microsatellite instability. In order to determine the contribution of Caspase-5 gene inactivation to lung cancer development and progression, the mutational status of the Caspase-5 poly(A) tract in 30 primary lung cancers with distant metastasis and 30 lung cancer cell lines was determined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Three somatic mutations of the Caspase-5 gene were found in two out of 30 lung cancer tissues, although no mutations were found in other genes that also contain small nucleotide repeats, such as hMSH3, hMSH6 and BAX. The results of the present study, combined with our prior cDNA array-based gene expression profiling data, suggest that Caspase-5 might be a suppressor gene of highly metastatic potential in lung cancer.     

Somatic mutation of the hBUB1 mitotic checkpoint gene in primary lung cancer

Mutations in mitotic checkpoint genes have been detected in several human cancers, and these cancers exhibit chromosomal instability. Aneuploid stem cells seem to result from chromosomal instability and have been reported in many lung cancers. To determine whether alteration of mitotic checkpoint regulators is involved in carcinogenesis and tumor progression in primary lung cancer, we screened the genomic DNA sequence of 30 human lung cancer cell lines and 30 primary lung cancer tumors for a mutation in the hBUB1 mitotic checkpoint gene. First, we designed 26 sets of intron-based primers to amplify each of the 25 exons of the hBUB1 gene to examine the entire coding region of the hBUB1 gene. Using these primers, we performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis as well as direct sequencing in the mutation analysis of the hBUB1 gene. Three different nucleotide substitutions were detected in the coding region of the hBUB1 gene in some of the cancer cell lines and primary tumors as follows. The hBUB1 gene of one adenocarcinoma tumor contained a somatic missense mutation, a cytosine-to-guanine substitution in codon 51 of exon 5 that resulted in a histidine-to-aspartic acid amino acid substitution. The hBUB1 gene of three lung cancer cell lines contained a thymine-to-cytosine substitution in codon 430 of exon 12, which did not result in an amino-acid substitution. We were unable to determine whether the nucleotide substitution in exon 12 was a polymorphism or a silent mutation because matched normal tissue was not available. A polymorphism in codon 93 of exon 4, a guanine-to-thymine substitution, in hBUB1 was found in one lung cancer cell line and one primary lung tumor. This is the first report of a somatic missense mutation of a gene involved in a mitotic checkpoint in primary lung cancer. The presence of a point mutation in the hBUB1 gene is consistent with the hypothesis that alteration of mitotic checkpoint genes is involved in the development of primary lung cancers. Because the frequency of hBUB1 gene mutations was low, future studies should focus on other mechanisms of inactivation of the hBUB1 gene as well as mutation analysis of other mitotic checkpoint genes in lung cancers.     

Somatic mutation induced by heliotrine in Drosophila

The pyrrolizidine alkaloid heliotrine has been shown to be a powerful mutagen in Drosophila. This report has evaluated the teratogenicity of heliotrine in this organism. The alkaloid was fed to larvae and its teratogenic effects measured in various developmental stages of the insect. The pupal stage is predominantly affected. The main consequences of treatment were failed eclosions at higher alkaloid concentrations (10(-4) M), while lower concentrations (10(-5) M) permitted the eclosion of adults, but these showed abdominal abnormalities ranging from severe distortions to reduced numbers of tergite bristles. mei-9 strains of Drosophila were more sensitive to the production of somatic chromosomal changes as well as the teratogenic effects of the alkaloid. These strains also showed reduced numbers of cells in histoblast nests of 6-hour-old prepupae. It is suggested that reduced numbers of histoblast cells in prepupae may be a consequence of genetic damage and this in turn leads to the abdominal distortions and reduced bristle numbers observed.     

Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer

Gastric carcinoma is one of the most common types of cancer in Taiwan. Somatic mitochondrial DNA (mtDNA) alteration in gastric carcinoma and its association with clinicopathologic features remain unclear. When we used polymerase chain reaction (PCR) and direct sequencing, 15 of the 31 (48%) gastric carcinomas displayed somatic mutations in the D-loop region, a hot spot for mutations in mtDNA of human cancers. Ten (67%) cancers with the somatic mutations in the D-loop had insertion or deletion mutations in nucleotide position (np) 303-309 in the mononucleotide repeat region. One carcinoma carried tandem duplication and triplication flanked by mononucleotide repeats starting at np 311 and 568, respectively, in the D-loop. We also detected the common 4,977-bp deletion in 17 (55%) of the noncancerous tissue samples, but only in three (9%) carcinomas. Moreover, we quantified the mtDNA content using a competitive PCR technique and found that mtDNA depletion occurred in 17 (55%) of the gastric carcinomas. Although no significant association was found between clinicopathologic features and the mtDNA mutations in the D-loop, mtDNA depletion was observed significantly in the ulcerated, infiltrating (Borrmann's type III) and diffusely thick (Borrmann's type IV) types of gastric carcinomas (P = 0.018). Our results suggest that somatic mtDNA mutations and mtDNA depletion occur in gastric cancer and that mtDNA depletion is involved in carcinogenesis and/or cancer progression of gastric carcinoma.     

Inactivating mutation of the pro-apoptotic gene BID in gastric cancer

There is evidence that deregulation of apoptosis is mechanistically involved in cancer development and somatic mutations of apoptosis-related genes have been reported in human cancers. BID, a pro-apoptotic member of the Bcl-2 family, interconnects the extrinsic apoptosis pathway initiated by death receptors to the intrinsic apoptosis pathway. To explore the possibility that genetic alterations of BID might be involved in the development of human cancers, this study analysed the entire coding region and all splice sites in the human BID gene in 67 advanced gastric carcinomas. Overall, four BID mutations (6.0%) were detected that consisted of one frameshift and three missense mutations. The tumour-derived BID mutants were expressed in 293T cells and it was found that, compared with wild-type BID, the frequency of apoptosis was significantly reduced in cells expressing the gene containing the frameshift mutation. Furthermore, expression of the inactivating frameshift mutant interfered with cell death by overexpression of death receptors, indicating that this mutant inhibits the extrinsic apoptosis pathway in a dominant-negative fashion. Also, the frameshift mutation rendered cancer cells resistant to apoptosis induced by the anti-cancer drug 5-fluorouracil (5-FU). This is the first report of BID gene mutation in human malignancy. The data suggest that such mutations occur rarely in gastric cancers and that only a small fraction of BID mutations may lead to the loss of its apoptotic function.     

ERBB2 kinase domain mutation in a gastric cancer metastasis

ERBB2 is a member of the epidermal growth factor receptor (EGFR) family. Recent studies revealed that the kinase domain of the ERBB2 gene was mutated in human cancers, including gastric cancer. Despite the importance of cancer metastasis in the pathogenesis of cancers, data on the ERBB2 kinase domain mutation in cancer metastasis are lacking. In this study, to explore the possibility that ERBB2 mutation is involved in the metastasis mechanism, we analyzed the kinase domain of ERBB2 for the detection of somatic mutations in 58 gastric adenocarcinomas with lymph node metastasis. We found one ERBB2 mutation, which was detected in the lymph node metastasis, but not in the primary tumor of the same patient. The ERBB2 mutation was a missense mutation which substituted an amino acid in exon 21 (V832I). We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA and BRAF genes in the sample with the ERBB2 mutation, and found that this metastatic carcinoma did not harbor any of the mutations. Our data suggest that ERBB2 kinase domain mutation occasionally occurs in metastatic gastric carcinoma and might play a role in the metastatic process of some gastric carcinomas.     

大鼠乳腺肿瘤线粒体基因突变研究

目的:了解大鼠乳腺癌组织、癌旁组织和正常组织的线粒体细胞色素b基因变异,探讨线粒体基因突变与肿瘤发生的关系。方法:提取细胞总DNA,用PCR法扩增细胞色素b基因,产物用DNA自动测序法进行序列分析。结果:癌组织发生了nt14931C^→G,nt15004C^→G和nt15435T^→C3处点突变。癌旁组织发生了nt15436A^→C突变。正常组织没有突变。结论:mtDNA突变可能是诱导核基因突变的内源性因素之一,对肿瘤的发生可能有促进作用。癌旁组织在DNA水平属非正常组织。     

Somatic mutation and recombination test in Drosophila melanogaster

A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans-heterozygous for the mutations multiple wing hairs (mwh) and flare (flr) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well-known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B1), DNA breaking agents (bleomycin), intercalating agents (5-aminoacridine, ICR-170), spindle poisons (vinblastine), and antimetabolites (methotrexate). In addition, the test detects mutagens unstable in aqueous solution (beta-propiolactone), gaseous mutagens (1,2-dibromoethane), as well as promutagens needing various pathways of metabolic activation (aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short-term test.     

Pin1 gene mutation is a rare event in gastric cancer

The peptidyl-prolyl isomerase Pin1 is strikingly overexpressed in human cancers and is a novel regulator of beta-catenin. To determine whether somatic mutation of the Pin1 gene is involved in the development and/or progression of gastric cancer, we searched for mutations of the Pin1 gene in 95 gastric cancer specimens. The effect of Pin1 on beta-catenin expression was further examined in wild- and mutant-type Pin1-transfected HEK 293T cells. We found only one missense mutation that led to the substitution of alanine by aspartic acid at codon 118 of the Pin1 gene. On transfection study, the mutant Pin1 showed an increased expression of beta-catenin. However, the mutation had no effect on expression of the Pin1 protein in the case with Pin1 mutation. These results suggest that Pin1 may not play a role in the development or progression of gastric cancer.     

Somatic mutation processes at a human minisatellite

Germline instability at human minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at minisatellites. An electrophoretic size-enrichment strategy was therefore developed at minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quantitated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over.      

Somatic mitochondrial DNA mutations in single neurons and glia

Somatic mitochondrial DNA (mtDNA) point mutations reach high levels in the brain. However, the cell types that accumulate mutations and the patterns of mutations within individual cells are not known. We have quantified somatic mtDNA mutations in 28 single neurons and in 18 single glia from post-mortem human substantia nigra of six control subjects. Both neurons and glia contain mtDNA with somatic mutations. Single neurons harbor a geometric mean (95% CI) of 200.3 (152.9–262.4) somatic mtDNA point mutations per million base pairs, compared to 133.8 (97.5–184.9) for single glia (p = 0.0251). If mutations detected multiple times in the same cell are counted only once, the mean mutation level per million base pairs remains elevated in single neurons (146.9; 124.0–174.2) compared to single glia (100.5; 81.5–126.5; p = 0.009). Multiple distinct somatic point mutations are present in different cells from the same subject. Most of these mutations are individually present at low levels (less than 10–20% of mtDNA molecules), but with high aggregate mutation levels, particularly in neurons. These mutations may contribute to changes in brain function during normal aging and neurodegenerative disorders.     

Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma

Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive the pathogenesis and progression of human malignancies. Previous investigations have revealed a high frequency of somatic mutations in the D‐loop control region of mtDNA in osteosarcoma. However, little is known with regard to whether or not somatic mutations also occur in the coding regions of mtDNA in osteosarcoma. To test this possibility, in the present study we screened somatic mutations over the full‐length mitochondrial genome of 31 osteosarcoma tumour tissue samples, and corresponding peripheral blood samples from the same cohort of patients. We detected a sum of 11 somatic mutations in the mtDNA coding regions in our series. Nine of them were missense or frameshift mutations that have the potential to hamper mitochondrial respiratory function. In combination with our earlier observations on the D‐loop fragment, 71.0% (22/31) of patients with osteosarcoma carried at least one somatic mtDNA mutation, and a total of 40 somatic mutations were identified. Amongst them, 29 (72.5%) were located in the D‐loop region, two (5%) were in the sequences of the tRNA genes, two (5%) were in the mitochondrial ATP synthase subunit 6 gene and seven (17.5%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, somatic mtDNA mutation was not closely associated with the clinicopathological characteristics of osteosarcoma. Together, these findings suggest that somatic mutations are highly prevalent events in both coding and non‐coding regions of mtDNA in osteosarcoma. Some missense and frameshift mutations are putatively harmful to proper mitochondrial activity and might play vital roles in osteosarcoma carcinogenesis.     

A germline E-cadherin mutation in a family with gastric and colon cancer

Inactivating mutations have been found in the cell-cell adhesion molecule E-cadherin (CDH1), which acts as a tumor suppressor gene in different kinds of cancers, e.g. primarily diffuse gastric cancer and lobular breast cancer. In this study, we screened for germline alterations in familial gastric and colon cancer cases. In total, 20 gastric and 18 colon cancer patients with both familial gastric and colon cancer were tested for germline E-cadherin alterations by using PCR/SSCP, specific restriction digestion test and sequencing. No pathogenic mutations were identified in the gastric cancer patients. In two colon cancer patients, a missense mutation in exon 12, codon 592 (Ala592Thr) was found. This alteration segregated with diffuse gastric cancer and colon cancer in one of the families. The prevalence of this alteration in the general population and colon cancer cases was almost the same. However, the fact that this alteration (Ala592Thr) segregated with colon cancer and diffuse gastric cancer in one big family, suggests that this E-cadherin missense alteration, beside predisposing to diffuse gastric cancer, also may play a role in colorectal carcinogenesis.