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M Oshimura

Publications and source records attributed to M Oshimura.

At least 145 records · Page 8Linked to original sources
PubMed1995

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.

Animals↗
PubMed1995

Complementation analysis of the murine scid cell line.

It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene.

Animals↗
PubMed1995

Studies on phenotypic complementation of ataxia-telangiectasia cells by chromosome transfer.

Cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) show an abnormal response to ionizing radiation-induced DNA damage, such as an increased cell killing and a diminished inhibition of DNA synthesis. The enhanced killing of A-T (group D) cells by X-rays can be corrected by multiple cDNAs, mapping to different chromosomes (6, 11, 17, and 18). In order to examine whether genes located on these chromosomes complement AT-D cells, normal neo-tagged chromosomes 6, 11, 17, and 18 were introduced into AT-D cells by microcell-mediated chromosome transfer. However, correction of the enhanced killing of AT-D cells by X-rays could only be achieved by chromosome 11 and by none of the other chromosomes tested. The enhanced killing of A-T (complementation group C) cells was also corrected by chromosome 11. Usually, but not in all microcell hybrid clones, chromosome 11 also corrected the radioresistant DNA synthesis (RDS) phenotype of AT-D and AT-C cells. These results (i) confirm findings by others suggesting assignment of the ATD and ATC genes to chromosome 11, (ii) demonstrate that several genes can modify the cellular radiation response when they are taken out of their normal genomic context and/or control, and (iii) indicate that the RDS phenotype and the enhanced cell killing in A-T are independent pleiotropic features resulting from the primary mutations in A-T. Also, our findings underscore that, in establishing cDNAs as candidate genes for A-T, microcell-mediated chromosome transfer studies are needed to exclude nonspecific correcting effects of these candidate cDNA genes.

Animals↗
PubMed1995

[Studies on tumor suppressor genes by gene/chromosome transfer].

The gene/chromosome transfer into cultured cancer cells has not only confirmed that genes isolated by the positional cloning method can actually suppress various transformed phenotypes of the cells, but also allowed to speculate cellular functions of cloned or uncloned tumor suppressor genes. One of the important functions of uncloned ones is to induce the cellular senescence program in immortal cancer cells, as revealed by the chromosome transfer. This method has also identified novel chromosomes (or chromosomal regions) carrying putative tumor suppressor genes, which have never been suggested by other approaches, e.g., LOH analyses. Thus, the studies on tumor suppressor genes using cultured cancer cells have different advantages from those of the molecular genetics/molecular biology studies.

Animals↗
PubMed1994

Evidence for multiple pathways to cellular senescence.

Normal cells in culture generally senesce whereas tumor-derived cells are often, but not without exception, immortal and grow indefinitely. For cells to escape the senescence program, normal genes must be lost or inactivated as shown by somatic cell genetic studies. For example, the introduction of specific chromosomes by microcell-mediated chromosome transfer has been shown to induce senescence of human and rodent tumor cell lines, and the mapping of over ten senescence genes has been achieved by this method. In this study, we observed that two different normal chromosomes induce senescence in the same human endometrial carcinoma cell line, which suggests that multiple pathways to senescence are inactivated in this cell line. This hypothesis has implications for the mechanisms of cellular senescence and its role in carcinogenesis. Furthermore, this hypothesis can explain why not all tumor-derived cells are immortal.

Animals↗
PubMed1994

Differential proliferative responses of Syrian hamster embryo fibroblasts to paraquat-generated superoxide radicals depending on tumor suppressor gene function.

Oxygen radicals have been widely implicated in neoplastic transformation; however, little is known regarding their mode of action. In an attempt to delineate potential mechanisms of action, an analysis of superoxide effects on cell growth was studied in normal and two nontumorigenic, immortal cell lines derived from normal Syrian hamster embryo (SHE) fibroblasts. The two immortal cell lines differed in their ability to suppress tumorigenicity of tumor cells in cell hybrids. One cell line suppressed tumorigenicity (sup+), while a second clone was unable to suppress tumorigenicity (sup-). Paraquat was used to generate superoxide through its capacity to be reduced by NAD(P)H and to generate superoxide radicals. The growth response of the various cell types was measured by colony-forming ability as well as by tritiated thymidine incorporation using autoradiography. At low paraquat concentrations (25 microM), primary SHE cells and two sup+ clones showed up to a 40% enhancement in colony formation, while two sup- clones showed no increase. Toxicity was observed at high doses, starting at approximately 100 microM paraquat. Since oxygen radicals are also mutagenic, primary SHE cells were examined for chromosomal aberrations. Chromatid gaps and breaks were induced at all concentrations of paraquat used. Thus, superoxide not only causes cellular toxicity at high doses but at low doses enhances cell growth of certain cells (primary SHE cells and sup+ cells) but not others (sup- cells). Therefore, differing responses of cells at different stages of neoplastic progression must be considered in understanding oxygen radical effects in growth control and carcinogenesis.

Animals↗
PubMed1994

Suppression of metastasis of rat prostatic cancer by introducing human chromosome 8.

In previous allelotype analyses of human prostatic cancer specimens, allelic loss on the short arm of chromosome 8 is frequently observed. However, it is still unclear whether this allelic loss is an initial event or a later one in development of prostatic cancer. Our previous studies demonstrate that introduction of human chromosome 11 into highly metastatic rat prostatic cancer cells results in suppression of metastatic ability without suppression of the in vivo growth rate or tumorigenicity of the hybrid cells (T. Ichikawa et al. Cancer Res., 52: 3486-3490, 1992). To clarify the role of human chromosome 8 in prostatic cancer, this chromosome was introduced into highly metastatic rat prostatic cancer cells using microcell-mediated chromosome transfer. Introduction of human chromosome 8 resulted in suppression of metastatic ability of the microcell hybrids, whereas no suppression of the in vivo growth rate or tumorigenicity was observed. These results demonstrate that human chromosome 8 contains metastasis suppressor gene(s) for prostatic cancer derived from a rat. These also suggest that human chromosome 8 has an important role in development of prostatic cancer.

Animals↗
PubMed1994

In vitro growth suppression and morphological change in a human renal cell carcinoma cell line by the introduction of normal chromosome 3 via microcell fusion.

Cytogenetic and molecular studies of human renal cell carcinoma (RCC) have suggested that the genetic and functional losses of one or more putative tumor suppressor genes on the short arm of chromosome 3 play a crucial role in the development of this disease. To examine whether the introduction of chromosome 3 has any effects on the biology of RCC cells, we introduced either chromosome 3, 7, or 11 from normal human fibroblasts into a newly established human RCC cell line with loss of heterozygosity for 3p, via microcell-mediated chromosome transfer. Microcell hybrids containing an introduced, intact chromosome 3 showed a significant reduction in in vitro growth rate and saturation density together with morphological alteration; these properties were not altered in microcell hybrids containing an introduced chromosome 7 or 11. During long-term cultivation, one of the clones that had lost the introduced chromosome 3 showed growth properties and morphology similar to those of the parental cell lines. Thus, our findings provide additional evidence for the presence of a putative tumor suppressor gene or genes on normal chromosome 3p and indicate that the gene is a dominant, negative growth regulator whose loss promotes progressive features of the neoplastic phenotype.

Carcinoma, Renal Cell↗
PubMed1994

Increased actin cable organization after single chromosome introduction: association with suppression of in vitro cell growth rather than tumorigenic suppression.

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms.

Actin Cytoskeleton↗
PubMed1994

A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1-->q11.1.

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with 60Co gamma-rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22-->qter or del(8)q23--> qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybrids, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1--> q11.1.

Animals↗
PubMed1994

Simple purification of human chromosomes to homogeneity using muntjac hybrid cells.

Chromosome sorting from hybrid cells offers enormous advantages for gene mapping and cloning, but purification of most chromosomes has been largely hindered by their similarity in size to other chromosomes. We have developed a novel cell line and strategy that allows simple, mass purification of mammalian chromosomes, permitting significant target genome enrichment. This strategy takes advantage of the small number of giant chromosomes (1,2,X) of the female Indian muntjac, a barking deer, avoiding the problem of size similarity. We introduced human chromosomes into a cell line derived from a muntjac and purified them to homogeneity using a relatively simple technique. This strategy should facilitate the isolation of chromosomes from species other than human for which hybrid cells are not available currently.

Animals↗
PubMed1994

Human arylhydrocarbon receptor: functional expression and chromosomal assignment to 7p21.

We isolated the human arylhydrocarbon receptor (AhR) cDNA from a human lung cDNA library, by using mouse AhR cDNA as a labeled probe. The nucleotide sequence of cloned human AhR cDNA is identical to the previously reported human AhR sequence [Dolwick et al. (1993), Mol. Pharmacol. 44, 911-917] from cell line HepG2. The overall amino acid identity with mouse AhR from cell line Hepa-1 is 72.5%. The human AhR expressed either in COS-7 cells or in a reticulocyte lysate in vitro translation system showed specific dioxin-binding activity and Arnt-dependent DNA-binding activity. Chromosomal localization of the AhR gene was determined to be chromosome 7p21 by fluorescent in situ hybridization and DNA blot hybridization using 23 human x mouse or Chinese hamster hybrid cell DNAs.

Amino Acid Sequence↗
PubMed1994

Plasmid-associated bacteriocin production by a Lactobacillus plantarum strain.

A Lactobacillus plantarum strain, LTF154, isolated from a fermented sausage, produces a bacteriocin, designated plantacin 154. Plantacin 154 was stable to heat treatment, and its activity was sensitive to proteolytic enzymes. The molecular mass, as indicated by activity detection after SDS-PAGE, was estimated to be 3.0 kDa or less. A plasmid-curing experiment and transformation analysis indicated that a 9.5-MDa plasmid, pLP1542, may be involved in the production of plantacin 154.

Bacteriocins↗
PubMed1994

Functional complementation studies with X-ray-sensitive mutants of Chinese hamster cells closely resembling ataxia-telangiectasia cells.

In order to isolate a human gene complementing the defect in A-T-like hamster cell mutants, the mutants were used as recipients for genomic DNA transfection, using either HeLa chromosomal DNA or DNA from a human cosmid library. Three primary transformants with an intermediate X-ray sensitivity and almost normal sensitivity to MMS, but retaining radioresistant DNA synthesis (RDS), were obtained. To identify the human chromosome that complements the defect in the A-T-like mutants, and to assess the degree of complementation for survival and RDS, microcell-mediated chromosome transfer was used. At least 20 independent hybrid clones between the mutant and each one of the human chromosomes 1, 2, 4, 5, 15, 17 or 18 were isolated. All hybrid clones remained X-ray sensitive, except one with chromosome 4, and another with chromosome 15, both showing an intermediate X-ray sensitivity. By using in situ hybridization we found that this partial correction was due to the presence of a mouse chromosome. In these two hybrids containing the mouse chromosome together with human chromosome 4 or 15, RDS was fully complemented only in the hybrid with chromosome 4 but not in the one containing chromosome 15, suggesting that RDS and X-ray sensitivity may be complemented independently.

Animals↗
PubMed1994

Inhibition of tumorigenicity of a murine squamous cell carcinoma (SCC) cell line by a putative tumor suppressor gene on human chromosome 7.

Alterations in oncogenes and tumor suppressor genes (TSG) are considered to be critical steps in oncogenesis. However information on putative TSG involved in the development of squamous cell carcinomas (SCC) is very limited. In this study we confirmed the existence of a tumor suppressor gene (TSG) on human chromosome 7 (hchr 7) that suppresses the tumorigenicity of squamous cell carcinomas (SCCs). We injected seven clones of CH72 cells (a murine SCC-derived cell line) bearing a hchr 7 (CH72/hchr 7) introduced by microcell fusion, two clones bearing human chromosome 12 (CH72/hchr 12) and parental CH72 cells into athymic Balb/c nude mice. The sizes of the tumors were determined twice a week until tumors reached 12 mm diameter. In situ hybridization for centromeric repetitive sequences of the transferred chromosomes were performed on the cell lines injected and the tumors arising after the injection. Southern blots and polymerase chain reaction (PCR) amplifications of near terminal sequences and (CA) microsatellite repeats were done to test the integrity of the introduced chromosomes. Five out of seven CH72/hchr 7 clones had a twofold and threefold longer latency periods than CH72 cells. The remaining CH72/hchr 7 clones (MF 6 and 13 no. 4) had latency periods similar to that of parental CH72; MF 6 had a deletion in the introduced chromosome 7 involving q31.3-q31.3, whereas the other hybrid (MF 13 no. 4) seemed to have an intact hchr 7. Tumor-derived cells from CH72/hchr 7 hybrids with a delayed latency had lost centromeric and telomeric sequences of Chr 7. In contrast, tumors derived from the MF 6 and MF 13 no. 4 as well as the CH72/hchr 12 clones retained the introduced human chromosome as shown by chromosome 7 or 12 centromeric and telomeric sequences. These results indicate that the tumorigenicity of CH72 murine SCC cells was suppressed by hchr 7 and that the CH72/hchr 7 regain the tumorigenic phenotype after loss of the introduced chromosome, suggesting the presence of a TSG on hchr 7.

Animals↗
PubMed1993

Human chromosome 11 complements ataxia-telangiectasia cells but does not complement the defect in AT-like Chinese hamster cell mutants.

It has been shown that the X-ray-sensitive Chinese hamster V79 mutants (V-E5, V-C4 and V-G8) are similar to ataxia-telangiectasia (A-T) cells. To determine whether the AT-like rodent cell mutants are defective in the gene homologous to A-T (group A, C or D), human chromosome 11 was introduced to the V-E5 and V-G8 mutant cells by microcell-mediated chromosome transfer. Forty independent hybrid clones were obtained in which the presence of chromosome 11 was determined by in situ hybridization. The presence of the region of chromosome 11q22-23 was shown by molecular analysis using polymorphic DNA markers specific for the ATA, ATC and ATD loci. Seventeen of the obtained monochromosomal Chinese hamster hybrids contained a cytogenetically normal human chromosome 11, but only twelve hybrid cell lines were shown to contain an intact 11q22-23 region. Despite the complementation of the X-ray sensitivity by a normal chromosome 11 introduced to A-T cells (complementation group D), these twelve Chinese hamster hybrid clones showed lack of complementation of X-ray and streptonigrin hypersensitivity. The observed lack of complementation does not seem to be attributable to hypermethylation of the human chromosome 11 in the rodent cell background, since 5-azacytidine treatment had no effect on the streptonigrin hypersensitivity of the hybrid cell lines. These results indicate that the gene defective in the AT-like rodent cell mutants is not homologous to the ATA, ATC or ATD genes and that the human gene complementing the defect in the AT-like mutants seems not to be located on human chromosome 11.

Animals↗
PubMed1993

Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer.

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell-mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3-1.8 x 10(-6). Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100-123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.

Adenocarcinoma↗