Hiromichi Hemmi

Abnormal expression of BRCA1 and BRCA1‐interactive DNA‐repair proteins in breast carcinomas

Breast cancer is one of the most common malignancies among women. The molecular mechanisms involved in breast carcinogenesis, however, remain to be elucidated. Although somatic mutation of BRCA1 is rare, BRCA1 protein expression is reduced in about 30% of sporadic breast carcinomas (Yoshikawa et al., Clin. Cancer Res., 5:1249–1261, 1999), indicating its possible involvement even in sporadic breast carcinogenesis. Among the BRCA1‐interactive proteins are hRAD51 (a human homologue of Escherichia coli rec A protein), BARD1 (BRCA1‐associated RING domain 1) and p53, all of which are involved in DNA repair. We have analyzed the expression patterns of the hRAD51, BARD1 and p53 proteins in five breast cancer cell lines, including a BRCA1‐deficient cell line, and in 179 breast cancer tissue samples from Japanese women, including 113 sporadic, 47 hereditary (i.e., BRCA1 status unknown), and 19 BRCA1‐associated cases. Of the 179 breast carcinomas, fifty‐four (30%) exhibited reduced hRAD51 expression, and sixty‐two (35%) exhibited p53 overexpression. On the other hand, reduced expression level of BARD1, and of hMSH2 and hMLH1, which are components of DNA mismatch‐repair pathway and are involved in colorectal carcinogenesis, was observed respectively in only 10 (6%), 8 (5%) and 3 (2%) cases. The overall frequency of sporadic breast carcinomas with abnormal expression of either BRCA1 or the BRCA1‐interactive proteins was 67% (76/113). These results indicate that there may be an important role for the BRCA1‐associated DNA‐repair pathway, not only in BRCA1‐associated breast carcinomas, but also in sporadic breast carcinomas. Int. J. Cancer 88:28–36, 2000.

A change in microsatellite instability caused by cisplatin-based chemotherapy of ovarian cancer

To clarify the mechanism of acquired CDDP resistance in ovarian cancer, we compared the microsatellite instability (MSI) by the amplification of 10 microsatellite loci and immunohistochemical detection of hMSH2 and hMLH1 expression between the primary resected tumours and the secondary resected residual tumours after 5 or 6 courses of CDDP-based chemotherapy in the 24 cases of ovarian cancer. Of the 24 primary resected tumours, 9 (37.5%) showed MSI (7 cases of MSI-L, 2 cases of MSI-H), while 15 (72.5%) were microsatellite stable tumours (MSS). The primary tumours also had MSI in the residual tumours after CDDP-based chemotherapy. However, all of the cases with MSS in the primary resected tumours exhibited MSI (2 cases were MSI-L, and 13 cases were MSI-H) in the residual tumours after CDDP-based chemotherapy (P < 0.001). Furthermore, 11 (73.3%) of these cases which changed from MSS to MSI also had a change in the expression of hMLH1 from positive to undetectable (P < 0.001). Our data suggest that tumour MSI changes during CDDP-based chemotherapy, and that the loss of hMLH1 expression is one of the factors that has the greatest effect on this transformation.

Molecular analysis and diagnosis in Japanese patients with Wilson's disease.

Abstract Background: Wilson’s disease is characterized by the toxic accumulation of copper in the liver, brain, cornea and other organs. It is caused by both impaired excretion via the bile and impaired incorporation of copper into ceruloplasmin in the liver. The Wilson’s disease gene (ATP7B) has been cloned as a putative copper‐transporting P‐type ATPase gene. We therefore analysed mutations of ATP7B in Japanese patients with Wilson’s disease.

Complementation of an hMSH2 defect in human colorectal carcinoma cells by human chromosome 2 transfer

The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination–proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild‐type hMSH2. Complete correlation was found between the presence of the wild‐type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2‐positive LoVo hybrids also showed an increased sensitivity to N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. This enhanced toxicity is associated with G2 cell‐cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug‐resistant phenotypes in chromosome 2‐transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin‐resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2. Mol. Carcinog. 29:37–49, 2000. Published 2000 Wiley‐Liss, Inc.

Microsatellite instability of papillary subtype of human gastric adenocarcinoma and hMLH1 promoter hypermethylation in the surrounding mucosa

Gastric cancer has striking heterogeneity in histological pattern, cellular phenotype, genotype, biomarkers, and biological behavior. We focused on the specific morphological papillary phenotype of gastric adenocarcinoma and attempted to identify its distinct molecular characteristics. In our comparative study, early stage papillary (papillary‐dominant) gastric cancer showed a significantly higher and more widespread high‐frequency microsatellite instability (MSI‐H) than other morphological types. Analysis of mutations in a panel of five putative microsatellite instability (MSI)‐associated genes in the MSI‐H cases revealed that papillary or papillary‐dominant cancer displays a unique profile of mutations compared to profiles previously reported in gastric cancer. Immunohistochemical staining and methylation analysis revealed that silencing of hMLH1 by methylation in its promoter region was responsible for the failure of mismatch repair in papillary‐type gastric cancer, whereas aberrant promoter methylation of hMLH1 was not found in any cases without the unique mutator phenotype. Promoter hypermethylation of the hMLH1 genes was found to a lesser degree in the adjacent non‐tumor mucosa in four of the 10 cases with tumor having the mutator phenotype. Microsatellite instability itself could not be detected in the adjacent non‐tumor mucosa. Inactivation of hMLH1 expression by promoter hypermethylation may be an early event in carcinogenesis of this type of gastric cancer, preceding the development of the clear MSI phenotype of papillary carcinoma.

Various AGC repeat numbers in the coding region of the human transcription factor gene E2F-4.

The E2F family of transcription factors regulates the expression of genes required for DNA synthesis and cell cycle control. The AGC triplet repeat in the coding region of the E2F‐4 gene, a member of the family, has been reported to be mutated in colorectal cancers with a microsatellite instability (MSI) phenotype. We found a wider range variation of the repeat number in DNAs from tumors, the corresponding normal mucosa, and healthy individuals. A total of 5 repeat variants, ranging from 8 to 17 AGC repeats, was detected in 6 (9.7%) of the 62 healthy individuals and 8 (8.9%) of the 90 normal DNAs of the patients. The wild‐type 13 repeat was present in all of these individuals. The variation of the AGC repeat number may be a polymorphism. Further, loss of heterozygosity (LOH) at the E2F‐4 locus in the tumor tissues of 2 (25%) of the 8 informative cases was detected. The variation may be a useful marker for detection of LOH in primary tumors. Hum Mutat 15:296–297, 2000.

Growth inhibition due to complementation of transforming growth factor-β receptor type II-defect by human chromosome 3 transfer in human colorectal carcinoma cells

The transforming growth‐β receptor type II (TGF‐βRII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF‐βRII gene, both located on the short arm of chromosome 3. Introduction of the wild‐type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR‐deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF‐βRII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF‐β‐TGF‐βRII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild‐type as well as mutated TGF‐βRII mRNA. In addition, phosphorylation of TGF‐βRI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF‐β. The amount of TGF‐β1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF‐β produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti‐TGF‐β antibody. Taken together, these results strongly suggest that the TGF‐βRII gene defect in HCT116 is complemented by a wild‐type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF‐β. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.