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Biomedical subjects

M Oshimura

Publications and source records attributed to M Oshimura.

At least 199 records · Page 11Linked to original sources
PubMed1990

Nonrandom chromosome alterations that correlate with progression to immortality in rat tracheal epithelial cells transformed with N-methyl-N'-nitro-N-nitrosoguanidine.

Primary rat tracheal epithelial cells can be transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine. The earliest recognizable morphological transformant is the enhanced growth variant (EGV), characterized by enhanced proliferative capacity. Transformed EGV colonies can progress to give rise to immortal cell lines. The purpose of this study was to determine if specific chromosome changes occur which correlate with immortalization. A total of 34 EGV colonies were isolated, of which five were able to progress in culture to become immortal (greater than or equal to 100 population doublings). Early passages of all five immortalized cultures exhibited additional copies of chromosomes 4, 7, and 11 as a common or recurrent abnormality. These numerical alterations were rarely observed in the primary EGV colonies from which the cell lines were derived, suggesting that these alterations occurred during progression. Structural alterations involving chromosome 1 (resulting in a net gain of 1q) and chromosome 3(3q) also occurred in four out of five immortalized cultures. In all cases, structural alterations involving 1q and/or 3q were detected in the primary EGV colonies from which the immortal cell lines arose. Comparison of the frequency of the structural and numerical alterations observed in the immortalized cultures with their frequency in the 29 EGV colonies which did not become immortal indicated that these changes correlated (P less than or equal to 0.005) with the ability to become immortal. These results suggest that structural alterations occur in primary EGV colonies which predispose cells to immortalization and that subsequent numerical changes occur during progression that correlate with acquisition of the immortal phenotype.

Aneuploidy↗
PubMed1990

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines.

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.

Animals↗
PubMed1990

Restoration of radiation resistance in ataxia telangiectasia cells by the introduction of normal human chromosome 11.

In order to identify the human chromosome which carries a mutated gene in cells from a patient with the hereditary disorder ataxia telangiectasia belonging to complementation group D (AT-D), we performed chromosome transfer experiments via microcell fusion. A single, pSV2neo-tagged chromosome, either 11 or 12, derived from normal human fibroblasts was introduced into AT-D cells by microcell fusion, and clones which were resistant to the antibiotic G418 were isolated. All 3 hybrid clones containing an additional copy number of chromosome 11 showed a restoration of the resistance of wild-type cells to killing by X-irradiation, whereas all 3 hybrid clones containing an additional copy number of chromosome 12 remained hyper-radiosensitive, like the parental AT cells. The results indicate that a defective gene of AT-D cells is also located on chromosome 11, since a genetic linkage analysis has previously suggested that a defective gene of its complementation group A is located on this chromosome.

Ataxia Telangiectasia↗
PubMed1990

Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells.

A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9, 7 of 11 recipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts of UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A.

Blotting, Southern↗
PubMed1990

Establishment of a novel immortalized cell line from ataxia telangiectasia fibroblasts and its use for the chromosomal assignment of radiosensitivity gene.

An immortalized cell line was established from a female ataxia telangiectasia (AT) patient by the transfection of primary skin fibroblasts with origin-defective SV40 DNA. The cell line was characterized by a hypodiploid chromosome constitution and radiation hypersensitivity. The established cell line was used as a recipient for microcell-mediated chromosome transfer. Among seven G418-resistant clones obtained by the fusion with microcells from mouse A9 cells carrying a pSV2neo-tagged normal human chromosome 11, three clones showed restoration of radiation resistance with concomitant gain of an extra intact chromosome 11, while the others contained no recognizable or deleted chromosome 11. The association of the presence of 11q14----qter region with the radioresistance suggests the presence of AT gene in this chromosomal region.

Ataxia Telangiectasia↗
PubMed1990

Normal human chromosome 1 carries suppressor activity for various phenotypes of a Kirsten murine sarcoma virus-transformed NIH/3T3 cell line.

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2-neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.

Animals↗
PubMed1990

Introduction of normal chromosome 3p modulates the tumorigenicity of a human renal cell carcinoma cell line YCR.

It has been suggested that loss and/or mutational inactivation of a gene or genes on the short arm of chromosome 3 (3p) may play a crucial role in the development of human renal cell carcinoma (RCC). If it is correct, the normal allele may carry suppressor activity for a tumor-associated phenotype(s). In order to test the hypothesis, we introduced a single chromosome containing 3p into a human renal cell carcinoma cell line YCR via microcell fusion, and examined tumorigenicity in nude mice and in vitro growth-properties. The following chromosomes derived from normal human fibroblasts were transferred to YCR or 6-thioguanine-resistant YCR cells: t(X;3) consisting of Xpter greater than Xq26::3p12 greater than 3pter, X, pSV2neo-tagged chromosome 11, and 3/t consisting of pSV2neo-tagged 3p and unknown segments. The introduction of t(X;3) or 3/t resulted in suppression of tumorigenicity or modulation of tumor-growth rate, whereas transfer of other chromosomes, i.e., X and 11, had no effect on tumorigenicity or tumor-growth rate of the cells. In vitro growth properties, i.e., cell-growth in medium containing 1% or 10% serum, growth in soft-agar and saturation density, were not correlated with the tumor-growth. In addition, the tumor-growth rate of 6-thioguanine-resistant segregants which have lost the t(X;3) became similar to that of the parental YCR cells. Thus, the introduction of 3p modulated at least the tumor-growth, indicating the presence on the 3p of a putative tumor-suppressor gene(s) for human RCC.

Animals↗
PubMed1990

Suggestive evidence for functionally distinct, tumor-suppressor genes on chromosomes 1 and 11 for a human fibrosarcoma cell line, HT1080.

One approach for identifying chromosomes which carry putative tumor-suppressor genes is the introduction of specific chromosomes into the tumor cells of interest. We examined the ability of human chromosomes derived from normal fibroblasts to suppress or modulate tumorigenicity in nude mice and the in vitro properties of HT1080, a human fibrosarcoma cell line. We first isolated mouse A9 cells containing a single human chromosome (1, 2, 7, 11, or 12) integrated with pSV2neo plasmid DNA. Following fusion of microcells from these A9 cells with the HT1080 cells, clones that were resistant to G418 were isolated and karyotypically analysed. Three of 4 microcell-hybrids with an introduced chromosome 1 were non-tumorigenic (#1-7, -8 and -13), whereas the parental HT1080 cells were highly tumorigenic. The other microcell-hybrid clone (#1-1) formed tumors, the cells of which had lost one copy of chromosome 1. Two clones from the #1-1 cells were isolated; one contained an extra copy of chromosome 1, and the other one did not. The former was non-tumorigenic and the latter was tumorigenic. The introduction of chromosome 11 also suppressed the tumorigenicity of HT1080 cells, while the introduction of other chromosomes, i.e., 2, 7, or 12, had minimal or no effect on the tumorigenicity of these cells. Cells from tumors formed by microcell-hybrids with the introduction of chromosome 2, 7, or 12 still contained the introduced chromosome. Interestingly, only the microcell-hybrids with an introduced chromosome 1 had an alteration in cellular morphology and modulation of in vitro transformed properties, i.e., cell-growth and saturation density in a medium containing 10% calf serum and cell-growth in soft-agar. Thus, the results indicate the presence of putative tumor-suppressor genes for HT1080 cells on chromosomes 1 and 11, and further suggest that the genes on these chromosomes control different neoplastic phenotypes.

Animals↗
PubMed1990

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer.

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.

Cell Fusion↗
PubMed1989

Concordant deletions of chromosome 3p and loss of heterozygosity for chromosomes 13 and 17 in small cell lung carcinoma.

Common regions of loss of heterozygosity on chromosomes 3, 13, and 17 were determined by restriction fragment length polymorphism analysis in 34 tumors and nine cell lines from 27 patients with small cell lung carcinoma. The common regions of loss of heterozygosity on chromosomes 3, 13, and 17 reside between D3S2 (3p14-p21) and ERBA beta (3p22-p24.1), between D13S1 (13q12) and D13S2 (13q22), and distal to MYH2 (17p13.1), respectively. Allele loss in each of these regions has been previously shown in several human tumors. Thus, the present findings indicate the pleiotropy of recessive genetic lesions in these genomic areas. Cytogenetic analysis was performed on three small cell lung carcinoma cell lines which had allele loss on all three chromosomes, and although chromosome 3p deletions were observed in two of three cell lines, no obvious structural abnormalities involving chromosomes 13 and 17 were detected. Mitotic recombination or mitotic nondisjunction rather than deletion may thus be the frequent chromosomal mechanism for attaining homozygosity of chromosomes 13 and 17 in small cell lung carcinoma.

Alleles↗
PubMed1989

Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa.

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfection, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418-resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo-tagged human chromosome 11 in all of the five SiHa-microcell hybrids. Two SiHa-microcell hybrids that contained a single copy of neo-tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa-11) were suppressed; four of the five SiHa-11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa-11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor-suppressor gene (or genes) for human cervical tumors.

Cell Line, Transformed↗
PubMed1989

Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma.

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gamma-glutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.

Animals↗
PubMed1989

Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol.

Treatment of Syrian hamster embryo cells with diethylstilbestrol (DES) resulted in the induction of immortal cell lines that progressed and formed tumors in nude mice. Four independently treated cell lines were analyzed cytogenetically at several passages during neoplastic progression. The immortal cell lines at the early passages had no structural abnormalities but did have numerical changes. For example, gain of chromosome 11 was found in all immortal cell lines, and gain of chromosome 19 was found in two of four cell lines. Tumorigenic cells showed not only a variety of numerical abnormalities but also structural abnormalities. Loss of a sex chromosome and gain of chromosome 19 were found in six of seven tumors. Gain of chromosome 11, which was found in all immortal cell lines, disappeared in five of seven tumors. Structural abnormalities involving chromosomes 2 and 3 were found in three of seven tumors. Many marker chromosomes were also found in the tumors. These results support our hypothesis that DES-induced nondisjunction is important in its ability to induce cell transformation and suggests that gain of chromosome 11 and/or 19 may play a role in DES-induced neoplastic progression. Furthermore, these results indicate that for the acquisition of tumorigenicity, additional numerical or structural changes are needed, suggesting that multiple genetic events are required in the multistep process of carcinogenesis.

Animals↗
PubMed1989

Longterm survival of normal, diploid Syrian hamster-cells in tumors induced by transfection with v-Ha-ras and v-myc oncogenes.

Tumors were induced following transfection of normal, diploid hamster embryo cells with plasmids containing the viral Harvey ras and viral myc oncogenes. Direct cytogenetic studies of the tumors performed at 3-7 weeks after injection of the transfected hamster cells into nude mice revealed that 100% of the hamster cells were aneuploid and no detectable diploid cells in mitosis were observed. However, when tumor explants were cultured in vitro, diploid Syrian hamster cells were frequently detected at early passages. The percentage of diploid hamster cells in the cultures varied from 2 to 94% at the first passage. After several passages in vitro, only aneuploid hamster cells were observed. The diploid hamster cells in culture had a flat morphology and senesced. The aneuploid cells in the cultures were readily cloned and formed tumors after reinjection in nude mice. Reconstruction experiments consisting of injecting cloned, aneuploid tumor cells mixed with 6 X 10(6) normal Syrian hamster embryo cells were performed. Cell cultures derived from these mixed tumors contained both normal and aneuploid hamster cells indicating that normal cells survived in vivo during the growth of the tumor cells for up to 4 weeks. During this period, some normal cells transplanted in vivo did not die or terminally differentiate. Since normal cells can persist in vivo, tumor-derived cell cultures are mixed populations and caution should be exercised in interpreting quantitative molecular or cellular studies of these uncloned populations.

Aneuploidy↗
PubMed1989

Construction of mouse A9 clones containing a single human chromosome (X/autosome translocation) via micro-cell fusion.

Cell hybrids between hypoxanthine guanine phosphoribosyl transferase (HGPRT)-deficient mouse cell lines (A9 or RAG) and each of 12 different human fibroblasts (GM cells) containing various X/autosome translocations were formed, selected and isolated. Several human chromosomes including an X/autosome translocation carrying HGPRT locus were found in these hybrid cells. To construct A9 cell clones that contain a single X/autosome translocation, micro-cell fusion was undertaken to transfer these chromosomes from the hybrids to A9 cells. Karyotype analysis revealed that most of the resulting micro-cell hybrids contain, in a background of mouse chromosomes, only the human X/autosome translocations which were present in the GM cells used for cell hybridization. Sublines of A9 cells were established containing the following autosomal segments: 1q23----1qter; 1q12----1pter; 3p12----3pter; 3q21----3qter; 11q13----11qter; 11q13----11pter; 11p11----11qter; 11q23----11pter; 12q24----12pter; 16q24----16pter; 17q11----17pter.

Animals↗