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Biomedical subjects

M Oshimura

Publications and source records attributed to M Oshimura.

At least 217 records · Page 12Linked to original sources
PubMed1989

Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion.

Normal human fibroblasts (MRC-5 or NTI-4) were transfected with pSV2-neo plasmid DNA. Fifty G418-resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabain. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10, 11, 12, 15, 18, 19 or 20 were established. Isozyme analyses conformed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2-neo plasmid DNA.

Animals↗
PubMed1989

Multiple chromosomes carrying tumor suppressor activity, via microcell-mediated chromosome transfer, for various tumor cell lines.

The ability of normal human fibroblast-derived chromosomes to suppress tumorigenicity in nude mice and in vitro growth properties of various tumor cell lines was examined. Normal human chromosomes tagged with pSV2neo gene by DNA transfection were transferred to the following human tumor cell lines by microcell-fusion: SiHa (uterine cervical carcinoma), A204 (rhabdomyosarcoma), SK-NEP-1 (Wilms' tumor), HHUA (uterine endometrial carcinoma), SK-N-MC (neuroblastoma), YCR (renal cell carcinoma), HT1080 (fibrosarcoma), and CC1 (chorionic carcinoma). The results indicate the presence of a putative tumor-suppressor gene(s) in multiple chromosomes, and suggest that multiple genes may normally be involved in suppressing the transformed phenotypes at different stages in some tumors. Thus, the microcell transfer of chromosomes to specific tumor cell lines is a useful technique to demonstrate the presence of tumor-suppressor genes on individual chromosomes, and may also be useful in cloning of tumor-suppressor genes as well as elucidating their function in cell-growth and differentiation.

Animals↗
PubMed1988

Role of chromosome loss in ras/myc-induced Syrian hamster tumors.

It has been shown previously that normal Syrian hamster embryo cells are neoplastically transformed by transfection with two cooperating oncogenes, v-myc plus v-Ha-ras. Karyotypic analyses of the cells from the tumors revealed a nonrandom chromosome change, monosomy of chromosome 15. In order to clarify the role of chromosome loss in these tumor cells with defined oncogene alterations, molecular and cytogenetic studies were performed on hybrids between normal Syrian hamster embryo cells and ras/myc tumor cells. Following fusion of the tumor cells with the normal cells which are not immortal, the majority of the cell hybrids senesced after less than or equal to 20 population doublings indicating that immortality was recessive. Some of the hybrids escaped senescence and grew indefinitely. These immortal hybrid cells retained the expected numbers of chromosome 15 indicating that escape from senescence did not involve loss of this chromosome. The tumorigenicity and anchorage-independent growth of the nonsenescent hybrids were still suppressed significantly. In these suppressed hybrid cells, RNAs complementary to the v-Ha-ras and v-myc oncogenes were expressed. Furthermore, radioimmune precipitation with a monoclonal antibody to p21ras of [35S]methionine-labeled cell extracts followed by polyacrylamide gel electrophoresis/sodium dodecyl sulfate electrophoresis showed that the suppressed hybrid cells contained high levels of the mutated ras protein. These results indicate that tumorigenicity is suppressed in the hybrids even though the oncogenes are expressed. When the hybrid cells were passaged, anchorage-independent variants appeared in the cultures. At this time, morphological changes occurred in the cultures and the cells were tumorigenic. Karyotypic analyses of the transformed segregants versus the parental hybrid cells revealed a nonrandom loss of one copy of chromosome 15 in the transformed segregants. No other nonrandom chromosome change was observed. These results suggest that the loss of chromosome 15 results in the loss of a cellular tumor suppressor gene which effects a phenotypic change necessary for expression of neoplastic transformation. In addition, the cellular factors responsible for the senescence of the hybrids may provide another mechanism involved in suppressing tumorigenicity.

Animals↗
PubMed1988

Cytogenetic changes in rat tracheal epithelial cells during early stages of carcinogen-induced neoplastic progression.

The cytogenetic changes in enhanced growth (EG) variants of rat tracheal epithelial cells in culture were examined. These variants which are detectable at 35 days after carcinogen exposure are the first phenotypic alteration in the multistep neoplastic process studied in this model system. Karyotypic analysis of N-methyl-N'-nitro-N-nitrosoguanidine-induced EG variants at Day 35 was made possible by the development of an in situ method of cytogenetic analysis on intact colonies containing too few cells for conventional chromosome preparation methods. Of the transformed EG variant colonies in both control and N-methyl-N'-nitro-N-nitrosoguanidine-treated groups, 62-78% had abnormal karyotypes which included numerical and structural changes. There were no specific chromosome changes, although aberrations of chromosomes 3 and 4 were recurrently observed. However, some colonies of even the most morphologically transformed EG variants were composed of only diploid cells. To confirm this finding 10 EG variant colonies were bisected and half of the clone was prepared for chromosome analysis and the other half was subcultured to measure the clonogenicity and karyotypes of the cells. Cells from 3 colonies plated very poorly on 3T3 feeders and therefore no karyotypic analysis of the colony-forming cells was possible; the cells of the 3 parental colonies were diploid. Three other parental colonies were predominantly diploid (80-90%) but upon replating the resultant daughter colonies had progressively smaller fractions of diploid cells indicating a selection for cells with abnormal karyotypes. When more selective conditions were used (i.e., growth after removal of the feeder cells), the percentage of abnormal cells increased even further. In one case the parental cells had a karyotypic alteration in the long arm of chromosome 4 and this karyotypic alteration was accentuated in the daughter colonies. Thus, selection of cells with increased growth ability upon subculturing or growth in the absence of feeder cells (properties associated with the acquisition of immortality) resulted in concomitant selection for cells with abnormal karyotypes. Since some of the carcinogen-induced rat tracheal epithelial cells expressing the EG variant phenotype were diploid, it is possible that the first step in this transformation process is an epigenetic change. However, most of the diploid cells became terminal. The aneuploid subpopulations present in these colonies have a selective growth advantage and comprise the cell compartment that expresses continued growth, immortality, and ultimately tumorigenicity.

Aneuploidy↗
PubMed1988

Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse.

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.

Animals↗
PubMed1987

Correlation of V-src gene amplification with the tumorigenic phenotype in a Syrian hamster embryo cell line.

A preneoplastic cell line (10W) isolated after treatment of Syrian hamster embryo cells with asbestos was cotransfected with pSV2-neo DNA and Rous sarcoma virus DNA. Six of these colonies contained v-src DNA; however, none of the six initially expressed v-src RNA. Five of the clones failed to grow in soft agar (frequency, less than 10(-6)). One clone (61) grew in soft agar, but with a low frequency. Three of the clones (41, 61, and 62) were tumorigenic in nude mice and three were nontumorigenic. Cells cloned from soft agar or established from tumor explants expressed the v-src gene. The gene copy number of v-src, which was three to 10 in the original neoR clones, was increased approximately 10-fold in the soft agar-derived cell clones and tumor-derived cell lines. Cytogenetic analyses indicated that cells with amplified v-src contained double minute chromosomes. The results suggest that gene amplification influences the expression of the transfected oncogene and is a mechanism which can overcome the initial suppression of transcription of the v-src oncogene in the 10W cell line.

Animals↗
PubMed1987

Chromosome aberrations in mouse bone marrow cells following in vivo exposure to 1,3-butadiene.

Chronic exposure to 1,3-butadiene (BD) results in a marked increase in the incidence of thymic lymphoma in male B6C3F1 relative to NIH Swiss mice whereas no demonstrable differences in bone marrow (target organ) toxicity exist. Repeated exposure to BD is known to produce a macrocytic anemia and an increase in the frequency of micronuclei in circulating erythrocytes in both strains. The present study was undertaken to determine if chromosomal breakage, aneuploidy or both reflect differences in BD leukemogenicity observed between B6C3F1 and NIH Swiss mice. Mice were exposed to a single concentration of BD (1250 p.p.m.) for 6 h. Bone marrow cell preparations were made at 24, 48, 72 and 96 h after cessation of exposure. In both strains comparable increases in the frequency of chromosomal aberrations (of the chromatid type) were observed following exposure to BD. Significant differences in the number of chromosomes were not observed, although a pattern of chromosomal loss in cells from treated animals was observed. These results indicate that BD-treatment in vivo produces significant increases in chromatid aberrations but not aneuploidy in both strains. Therefore it is concluded that bone marrow toxicity, including cytogenetic abnormalities, is not predictive of leukemogenicity in these mice.

Animals↗
PubMed1986

Chemically induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer.

A growing body of evidence from human and animal cancer cytogenetics indicates that aneuploidy is an important chromosome change in carcinogenesis. Aneuploidy may be associated with a primary event of carcinogenesis in some cancers and a later change in other tumors. Evidence from in vitro cell transformation studies supports the idea that aneuploidy has a direct effect on the conversion of a normal cell to a preneoplastic or malignant cell. Induction of an aneuploid state in a preneoplastic or neoplastic cell could have any of the following four biological effects: a change in gene dosage, a change in gene balance, expression of a recessive mutation, or a change in genetic instability (which could secondarily lead to neoplasia). To understand the role of aneuploidy in carcinogenesis, cellular and molecular studies coupled with the cytogenetic studies will be required. There are a number of possible mechanisms by which chemicals might induce aneuploidy, including effects on microtubules, damage to essential elements for chromosome function (ie, centromeres, origins of replication, and telomeres), reduction in chromosome condensation or pairing, induction of chromosome interchanges, unresolved recombination structures, increased chromosome stickiness, damage to centrioles, impairment of chromosome alignment, ionic alterations during mitosis, damage to the nuclear membrane, and a physical disruption of chromosome segregation. Therefore, a number of different targets exist for chemically induced aneuploidy. Because the ability of certain chemicals to induce aneuploidy differs between mammalian cells and lower eukaryotic cells, it is important to study the mechanisms of aneuploidy induction in mammalian cells and to use mammalian cells in assays for potential aneuploidogens (chemicals that induce aneuploidy). Despite the wide use of mammalian cells for studying chemically induced mutagenesis and chromosome breakage, aneuploidy studies with mammalian cells are limited. The lack of a genetic assay with mammalian cells for aneuploidy is a serious limitation in these studies.

Aneuploidy↗
PubMed1986

An early, nonrandom karyotypic change in immortal Syrian hamster cell lines transformed by asbestos: trisomy of chromosome 11.

Cytogenetic studies were performed on eight early passage Syrian hamster embryo cell lines independently derived following asbestos exposure. The modal chromosome number of all the immortal cell lines was near-diploid. At the earliest passage examined, six of eight cell lines had only numerical chromosome changes. Cells in each of these six cell lines had an extra chromosome #11, either as a sole karyotypic change or with other numerical changes. The remaining two cell lines displayed both numerical and structural chromosome changes, but without involvement of chromosome #11. Common abnormalities were -X or -Y, +3, and 8p- in one cell line, and -13 and t(13;21) in the other cell line. A nonrandom gain of chromosome #8 was also found in four cell lines. In three of the four cell lines, trisomy of chromosome #8 seems to have occurred during karyotypic progression. The observation that nonrandom changes in chromosome number are an early karyotypic change after carcinogen treatment supports our hypothesis that induction of aneuploidy by asbestos is mechanistically important in the transformation of Syrian hamster embryo cells in culture and, further, suggests that trisomy 11 plays a major role in the early steps of immortalization and neoplastic progression.

Animals↗
PubMed1986

Role of oncogenes and tumor suppressor genes in a multistep model of carcinogenesis.

We demonstrated previously that carcinogen-induced neoplastic transformation of Syrian hamster embryo (SHE) cells requires multiple steps. Normal, diploid SHE cells and carcinogen-induced preneoplastic cells were transfected with different oncogenes. The normal, early-passage cells were not transformed by the v-Ha-ras or v-myc oncogenes alone, but the two oncogenes combined caused tumors in nude mice and syngeneic hamsters. Cytogenetic analysis of the ras-plus-myc-induced tumors showed a nonrandom chromosome loss (monosomy of chromosome 15) in the ras/myc tumor cells. Tumorigenicity of the ras/myc tumor cells was suppressed following hybridization with normal SHE cells; reexpression of tumorigenicity at later passages correlated with loss of chromosome 15. The hybrid cells in which tumorigenicity was suppressed still expressed the ras and myc oncogenes. An early change in carcinogen-induced neoplastic progression of SHE cells is induction of immortality. At early passages, immortal cells retain the ability to suppress tumorigenicity in cell hybrids. This ability decreases with passaging of immortal cell lines. The susceptibility of immortal cell lines to neoplastic transformation by DNA transfection with the v-Ha-ras oncogene or tumor DNA inversely correlated with the tumor-suppressive ability of the cells in cell hybrids. These observations indicate that neoplastic transformation of SHE cells involves at least three steps: (1) induction of immortality, (2) activation of a transforming gene or oncogene, and (3) loss of or inactivation of a tumor-suppressor gene.

Animals↗
PubMed1986

Role of phagocytosis in Syrian hamster cell transformation and cytogenetic effects induced by asbestos and short and long glass fibers.

We have shown previously that asbestos and other mineral dusts, including glass fibers, induce cell transformation and chromosomal mutations in Syrian hamster embryo cells in culture. In the present study, we observed that both asbestos and glass fibers were phagocytized by these cells and accumulated in the perinuclear region of the cytoplasm. In order to understand the mechanism of fiber length-dependent cellular effects, we examined the phagocytosis and intracellular distribution of glass fibers of differing lengths in cells at various times after treatment. Glass fiber length was decreased by milling with a mortar and pestle. Cells treated with an equal dose of milled glass fibers (on a weight per surface area basis) were exposed to 7-fold more fibers since milling of glass fibers resulted in a 7-fold decrease in length with little change in diameter. However, cells exposed to milled glass fibers phagocytized a similar number of fibers as cells exposed to an equal mass of unmilled glass fibers, indicating that milled fibers were less readily phagocytized. In cells treated with either unmilled or milled glass fibers, the length of the intracellular fibers was more than 2-fold greater than the length of the fibers on the surface, suggesting that cells selectively internalized longer fibers. Fiber length, however, did not appear to affect the migration of intracellular fibers to the perinuclear region of the cytoplasm. Even though cells treated with milled glass fibers contained a similar number of fibers as those treated with unmilled glass fibers, the resulting cytotoxicity, transformation frequency, and frequency of micronuclei were greatly reduced in the cultures treated with milled glass fibers. Thus, fiber length appears to affect the phagocytosis of fibers as well as the ability of intracellular fibers to induce cytogenetic damage and the resultant transformation.

Animals↗
PubMed1985

Characterization of extramedullary tumors in a case of Ph-positive chronic myelogenous leukemia: possible involvement of immature T lymphocytes.

A 42-year-old male with chronic myelogenous leukemia (CML) developed acute transformation associated with subcutaneous tumors. Histopathologic examinations of the tumors were done on two occasions; the first study revealed reticulum cell sarcoma-like features, and the second suggested a blastoma. Chromosomal analysis showed that the cells of the tumors originated from the CML clone. The cells had a negative reaction for myeloperoxidase by electron microscopy. Furthermore, biochemical and surface marker studies revealed that the tumor cells contained a significant terminal transferase activity. However, they did not express E- or EAC-rosette receptors, Ia-like antigens, or common ALL antigens.

Adult↗
PubMed1985

Cytogenetic and ultrastructural studies on ten patients with acute promyelocytic leukemia, including one case with a complex translocation.

Chromosomal banding analyses and ultrastructural studies were performed on ten cases of acute promyelocytic leukemia (APL-M3). A reciprocal translocation, t(15q + ;17q-), was found in six of them, and the possible breakpoints of these chromosomes were assigned at bands 15q22 and 17q12. In addition, trisomy 8, trisomy 8 and 21, and an isochromosome of the long arm of the translocated #17, i(17q-), were observed in addition to the 15;17 translocation in three cases, respectively. Furthermore, one patient was found to have a complex translocation in the marrow cells, i.e., 47,XX,+X,t(1p+;5q-;15q+;17q-). Ultrastructural studies demonstrated that the leukemic cells obtained from six of the seven patients with the chromosomal changes involving 17q12 and from two of the three with normal karyotypes contained stellate rough surface endoplasmic reticulum (stellate rER) complexes and/or inclusion bodies in part of the dilated rER.

Adult↗
PubMed1985

Double nondisjunction during karyotypic progression of chemically induced Syrian hamster cell lines.

The karyotypic evolution of three chemically induced cell lines of Syrian hamster embryo in culture are described. The only karyotypic alteration of one clone was a trisomy of chromosome #11, which presumably arose by nondisjunction after carcinogen treatment. A pure population of cells with the trisomy was observed repeatedly upon karyotyping of cells at the first three passages after cloning. However, at a late passage, apparently normal diploid cells appeared in the culture, which we propose resulted from a second nondisjunction of one chromosome #11, reverting the cells from trisomy 11 to disomy 11. The karyotypic evolution of two other cell lines also involved double nondisjunction, which resulted in duplication of a translocated chromosome and concurrent loss of the normal nonrearranged chromosome. Taken together with the reported findings of others, the results indicate that double nondisjunction is a mechanism in karyotypic progression during neoplastic development.

Animals↗
PubMed1985

Comparison of arsenic-induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture.

Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. A linear dose-dependence with a slope of approximately 1 was observed with both compounds when the data were plotted on a log-log graph. The trivalent sodium arsenite was greater than 10-fold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at non-toxic as well as toxic doses. At low doses, enhanced colony-forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges were induced by the arsenicals with similar dose-responses. These results support a possible role for chromosomal changes in arsenic-induced transformation.

Animals↗
PubMed1985

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.

Animals↗