Jun-ichi Hamada

Somatic Mutations of the APC Gene in Primary Breast Cancers

APC gene mutations play an important role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) patients and non-FAP patients. Although the APC gene is expressed in most tissues, including the lung, liver, kidney, and mammary gland, its somatic mutations have rarely been found in primary tumors affecting these organs. We have developed a sensitive yeast-based assay for screening almost the entire coding region of the APC gene. By this method, we have been able to detect somatic mutations of the APC gene in 57% of colorectal cancers and none in non-small cell lung cancers. Interestingly, the assay detected somatic APC gene mutations in 18% of breast cancers, in which APC gene mutation was previously considered rare. In the breast cancers, most of the APC mutations were distributed outside the mutation cluster region that has been advocated for colorectal cancers. We also noted a difference in the mutation pattern of the APC between colorectal and breast cancers. In colorectal cancers, all base substitutions were observed at C residues (5 of 5), whereas in breast cancers the majority of them were found at G residues (4 of 5). Furthermore, APC mutations were observed at a significantly high frequency in advanced stages of primary breast cancers (TNM classification, P < 0.05; T category, P < 0.01). Our data suggest that the etiology of the APC mutations and their biological role in carcinogenesis may differ between colorectal and breast cancers.

Overexpression of homeobox gene HOXD3 induces coordinate expression of metastasis-related genes in human lung cancer cells

Homeobox‐containing genes are expressed in spatiotemporal fashion during embryogenesis and act as master transcription‐regulating factors which control the expression of a variety of genes involved in morphogenesis. They are also expressed in a tissue‐specific manner in normal adult tissues and appear to give cells spatial information in the maintenance of their architectural integrity. We transfected a HOXD3 class I homeobox‐containing gene into human lung cancer A549 cells and investigated alterations in gene expressions and phenotypes related to the maintenance of tissue architecture in HOXD3‐overexpressing A549 cells. In the HOXD3‐overexpressing cell lines, expression of E‐cadherin was lost and plakoglobin was strongly repressed, whereas integrin α3 and β3 were up‐regulated and N‐cadherin and integrin α4 were newly expressed. Compared with parental and control transfectant lines, the HOXD3‐overexpressing cell lines showed highly motile and invasive activity. Blocking experiments using anti‐integrin β1 and β3 suggested that the increased haptotaxis of the HOXD3‐overexpressing cells to vitronectin resulted from increased expression and activation of integrin αvβ3, and that overexpression of the HOXD3 gene converted the integrin β1‐dependent haptotaxis to fibronectin into both integrin β1‐ and β3‐dependent one. HOXD3 overexpression increased production of matrix‐degrative enzymes including matrix metalloproteinase‐2 and urokinase‐plasminogen activator. When the tumor cells were intravenously injected into the tail veins of nude mice, HOXD3 transfectants formed a significantly large number of metastatic foci in lungs compared with the control transfectants. These findings suggest that HOXD3 can act as a metastasis‐promoting gene in human lung cancer A549 cells.

Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression.

In this study, we investigated the role of E1AF, a member of ets family transcription factor, in the acquisition of metastatic capacity by non-metastatic mouse fibrosarcoma cell clone, QR-32. The QR-32 cell clone grows progressively after co-implantation with gelatin sponge in syngeneic C57BL/6 mice. The cell lines (QRsP) established from arising tumors after the co-implantation exhibited enhanced tumorigenicity and pulmonary metastasis in vivo as compared with parent QR-32 cells. The enhanced pulmonary metastasis of QRsP cells was correlated well with augmented production of matrix metalloproteinase-2 (MMP-2) and increased expression of membrane-type 1-MMP (MT1-MMP). The QRsP cells also acquired higher chemokinetic activities to fibronectin and higher invasive activities through a reconstituted basement membrane. Furthermore we observed the elevated mRNA expression of E1AF in QRsP cells compared to parent QR-32 cells. Therefore, we transfected QR-32 cells with E1AF cDNA. Overexpression of E1AF in the QR-32 cells resulted in the induction of MT1-MMP expression and converting an exogenously added precursor MMP-2 into active form. E1AF transfectants exhibited more motile and invasive activities, and moderately increased pulmonary metastatic activities than parental QR-32 cells in vivo, although their metastatic activities were lower than those of QRsP cells. These findings suggest that the increased expression of E1AF in fibrosarcoma contributes to invasive phenotypes including MT1-MMP expression and enhanced cell migration, but not sufficient for exhibiting highly metastatic activity in vivo.

A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors in rats

In order to analyze the mutational events and to understand the biological significance of the p53 gene in chemical carcinogenesis, we applied a new yeast‐based p53 functional assay to ovarian tumors induced by 7, 12‐dimethylbenz[a]anthracene (DMBA), as well as to transitional cell carcinomas of the urinary bladder induced by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) in rats. The assay demonstrated that 15 of 19 DMBA induced tumors harbored clonal p53 mutations, which is consistent with the expectations of the “clonal expansion” hypothesis. The majority of the mutations were purine (AG) to pyrimidine (CT) transversions (12/19) on the non‐transcribed (sense) strand (NTS), which is likely to be due to depurination created by DMBA adduct formation on the NTS. In contrast, we found no purine to pyrimidine transversion on the NTS. After cessation of BBN treatment, BBN‐induced multifocal lesions in the bladder contained heterogeneous p53 mutations at an early stage. In the later stage, however, clonal p53 mutations were identified in 4 out of 7 bladders analyzed, conforming with the concept of “field cancerization”. The observed base substitutions were G→A (1/6) or C →T transitions (2/6), and mutations at T (3/6) on the NTS in clonal mutations, together with non‐clonal mutations, showing a preference of C→T to G→A (17 vs. 0). Thus, preferential repair was found in the transcribed strand of the p53 gene, whether modified by DMBA or by BBN carcinogens. Very similar mutation patterns were observed between clonal and non‐clonal mutations in the DMBA‐ and BBN‐induced tumors, indicating that the rat yeast p53 functional assay can be a potential tool for the characterization of in vivo mutation patterns of p53, when modified by chemical carcinogens. Int. J. Cancer 83:700‐705, 1999.

The Mechanism of Actinomycin D-Mediated Increase of Borna Disease Virus (BDV) RNA in Cells Persistently Infected by BDV

The transcriptional mechanism of Borna disease virus (BDV) has been poorly understood. We have analyzed transcription of the virus upon various stimuli in Madin‐Darby canine kidney cells which were persistently infected by BDV (MDCK/BDV). Treatment with actinomycin D (ActD) increased the level of BDV RNA, shifting the size of RNA from 1.9 kb to 2.3 kb beginning 5 hr after the treatment. To understand the mechanism of this unique modulation of BDV RNA, we conducted several experiments. The RNA increase occurred at the stage in which synthesis of cellular intrinsic mRNA was intact, suggesting BDV does not compete with cellular transcriptional machinery for intrinsic RNA polymerase II. The BDV transcription was also enhanced by cycloheximide treatment, indicating that newly synthesized viral or cellular proteins are not necessary for viral transcription. However, a shift in the RNA size was not observed for cycloheximide‐induced BDV RNA. The increase in viral transcription persisted during the cellular apoptotic process consequent to p53 gene accumulation beginning 1 hr after ActD treatment. Caspase inhibitors Z‐VAD and DEVD‐CHO repressed the apoptotic process but failed to block the increase in BDV transcription. In addition, adenovirus‐mediated transduction of wild‐type p53 did not alter the BDV transcription, indicating that the increase in BDV transcription was independent of the p53‐mediated apoptotic process. Other various stimuli that evoke cellular signal transductions failed to alter BDV transcription. Agents inhibitory to topoisomerase except adriamycin failed to enhance BDV transcription, indicating that the increase in BDV transcription is not mediated by an inhibitory action to the topoisomerase II of ActD. Adriamycin showed an increase and size‐shift of BDV RNA similar to ActD. These results suggest that intercalation of the viral genome itself with ActD is related to the stabilization of viral RNA and alteration of RNA size rather than secondary host cell changes.

Inhibitory Effects of Malotilate on in vitro Invasion of Lung Endothelial Cell Monolayer by Human Oral Squamous Cell Carcinoma Cells

We have previously reported that malotilate (MT) inhibited the invasion and metastasis of rat mammary carcinoma cells through the modification of host endothelial cells. In this study, we examined the inhibitory effects of MT on invasion of human cancer, using five oral squamous cell carcinoma cells (SAS, Ca9-22 and HSC-2, -3 and -4). MT did not affect the growth of these tumor cells and the invasion of reconstituted basement membrane, Matrigel. In an in vitro invasion assay using rat lung endothelial (RLE) cells, invasion of tumor cells which had been treated with MT (10 ng/ml, 24 h) was not affected; however, when RLE cells had been treated with MT, invasion was significantly inhibited in three cell lines (SAS, Ca9-22 and HSC-4) and a tendency to inhibition was also observed in other cell lines. Electron-microscopical examination of the RLE monolayer treated with MT (MT-RLE) showed the development of gap and tight junction-like structures. Subsequently, junction-associated proteins, connexin 43, zonula occludin and desmoglein, were examined by Western blotting. Protein levels of connexin 43 and zonula occludin were elevated dose dependently, and connexin 43 was chronologically enhanced by MT whereas desmoglein was not. The enhanced gap junctional communication of MT-RLE cells was observed in the scrape-loading assay using lucifer yellow CH. These results suggest that MT promotes the development of cell-to-cell adhesion, e.g. gap junction and tight junction in endothelial cells, resulting in the inhibition of invasion by the tumor cells.