Kiyotsugu Yoshikawa

The AID enzyme induces class switch recombination in fibroblasts.

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from Cμ to one of the other CH genes. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

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.

Activation‐Induced Cytidine Deaminase Links Class Switch Recombination and Somatic Hypermutation

Abstract: Activation‐induced cytidine deaminase (AID), a putative RNA‐editing cytidine deaiminase that is expressed strictly in activated B cells, is indispensable in three apparently distinct genetic alterations of immunoglobulin genes—namely, class switch recombination, somatic hypermutation, and gene conversion. Recent findings led us to propose a common DNA cleaving mechanism, in which the transient secondary structure of the S and V region DNA is recognized by a nicking enzyme regulated by the putative RNA‐editing activity of AID.

Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation

Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.

RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells

Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to induce somatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro.

Activation of fibroblast-derived matrix metalloproteinase-2 by colon-cancer cells in non-contact co-cultures

Stromal fibroblasts interact with invading cancer cells by secreting and activating matrix metalloproteinases (MMPs). To elucidate the mechanisms involved in the expression and activation patterns of MMPs, human colon‐cancer cell lines Caco‐2 and LoVo and colon‐fibroblast cell line CCD18‐Co were co‐cultivated in non‐contact and contact conditions which mimic in vivo interaction between cancer cells and fibroblasts before and after cancer invasion respectively. Gelatin zymography disclosed that MMP‐2 was secreted from the fibroblasts but not from the cancer cells. The quantity of fibroblast‐derived MMP‐2 in conditioned medium was not significantly changed in either the contact or the non‐contact co‐cultures when compared with that of individual cultures of CCD18‐Co fibroblasts. Cancer cells in non‐contact co‐cultures, however, enhanced the activation of fibroblast‐derived MMP‐2. Transcripts of membrane‐type matrix metalloproteinase‐1 (MT1‐MMP), which is thought to be present on the cell surface and to work as a candidate activator of MMP‐2, were detected in both cancer cell lines. Plasma membrane extracts of cancer cells also activated MMP‐2 in conditioned media in cell‐free conditions. This activation of MMP‐2 may be caused by MT1‐MMP of the cancer cells, since it was inhibited by a series of MMP inhibitors, including ethylenediaminetetraacetic acid (EDTA), the tissue inhibitor of metalloproteinase‐2 (TIMP‐2), and the MMP inhibitor CGS 27023A, but not by TIMP‐1. Our data demonstrate that in non‐contact co‐cultures colon‐cancer cells activate fibroblast‐derived MMP‐2 on their plasma membranes. These findings should help to elucidate the mechanism involved in the initial destruction of basement membrane by cancer cells. Int. J. Cancer 87:165–171, 2000.

Single-Agent Gemcitabine for Biliary Tract Cancers

Objective: The aim of this study was to investigate the outcomes of gemcitabine-treated patients with inoperable biliary tract cancers. Methods: We conducted a retrospective study of consecutively treated 22 inoperable biliary tract cancer patients with gemcitabine (500–1,000 mg/m2 on days 1, 8, 15 every 4 weeks) as first-line, and 17 patients as second- or third-line treatment. Results: The response rate of patients treated with gemcitabine as first-line and second- or third-line treatment was 5.3 and 28.5%, respectively. The median overall survival time in the first-, and second- or third-line treatment groups was 8.3 and 17.0 months, and the 1-year survival rate was 44.0 and 50.9%, respectively. The present study also suggests the possibility that the prognosis of patients with high levels of C-reactive protein and total bilirubin, or a low level of albumin might be worse. Conclusions: Our results indicate that the treatment of inoperable biliary tract cancers with gemcitabine is feasible. There was no difference in the response rate and overall survival between biliary tract cancer patients in the first- and second- or third-line treatment groups. We also present the systematic review of literature of the recent treatment results of biliary tract cancers treated with gemcitabine.

Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal‐like 4 (SALL4) as a dispersion factor in basal‐like breast cancer. Our shRNA‐mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal‐like breast cancer.

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze‐drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long‐term storage conditions and analyses of freeze‐dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)‐PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze‐dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT‐PCR analyses, respectively. Overexpression of c‐erbB‐2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze‐dried human breast cancer tissues. Although macromolecules in freeze‐dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Prevalence and incidence of anemia in Japanese cancer patients receiving outpatient chemotherapy.

Anemia in cancer patients has been under-recognized and little studied in Japan. To gain some insight into cancer-related anemia in Japanese patients undergoing outpatient chemotherapy, we performed a single-center retrospective study of the prevalence and incidence of anemia in 148 patients with solid tumors treated at the Kyoto University Hospital Outpatient Oncology Unit. We classified the cases of anemia in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0). Of 148 patients, 65 (44%) were anemic at the start of chemotherapy, 19 (13%) of whom had anemia of grade 2 or higher. Chemotherapy further increased the number of anemic patients, with 125 (84%) being anemic at some point during chemotherapy, and 61 (41 % ) of these having anemia of grade 2 or higher. Among the 83 patients without anemia at the start of chemotherapy, 60 (72%) developed anemia during chemotherapy, 15 (18%) of whom had anemia of grade 2 or higher. This is the first report showing a high prevalence and incidence of anemia in Japanese patients undergoing outpatient chemotherapy. Better recognition and management of cancer-related anemia are required in Japan. To this end, randomized controlled trials evaluating the effects of erythropoietic agents on patients’ survival and quality of life are necessary.