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M Koi

Publications and source records attributed to M Koi.

34 records · Page 2Linked to original sources
PubMed1990

Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts.

Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.

Chromosome Mapping↗
PubMed1990

Induction of cellular senescence in immortalized cells by human chromosome 1.

The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.

Animals↗
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↗
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

Role of a tumor-suppressor gene in the negative control of anchorage-independent growth of Syrian hamster cells.

Tumor-suppressor genes control the neoplastic phenotype of tumor cells, but the function of these genes in normal cells is unknown. In this report we show that the loss of a tumor-suppressor gene function releases negative controls on the growth of cells in agar. This conclusion is based on observations of cell hybrids and studies of cell variants that have retained or lost a tumor-suppressor gene function. Nontumorigenic cell hybrids between normal Syrian hamster embryo cells and a benzo[a]pyrene-transformed tumor-cell line (BP6T) continued to secrete autocrine and/or paracrine growth factors produced by the tumor cells but failed to respond to these factors by growing in agar. Normal diploid cells also failed to grow in agar in response to the growth factors produced by the tumor cells. Clonal variants of nontumorigenic, immortal Syrian hamster cell lines were isolated that either retained (termed supB+) or had lost (termed supB-) the ability to suppress tumorigenicity of BP6T tumor cells after cell hybridization. Neither supB+ nor supB- variants grew in agar under conditions that allowed efficient growth of the tumor cells. However, supB- cells were reversibly induced to grow in agar with high colony-forming efficiencies in the presence of tumor cell-conditioned medium or by supplementation of the medium with a combination of growth factors. Under the same conditions, the supB+ cells failed to grow in agar. This enhanced growth-factor responsiveness in agar was used to select for supB- variants existing at a low frequency in the supB+ population. These two phenotypes, loss of tumor-suppressor function and enhanced growth-factor responsiveness in agar, were seen to cosegregate. These results indicate the tumor-suppressor gene function in these cells negatively regulates the growth response of cells in agar to mitogenic stimuli. This growth regulation may depend on cell shape or adhesion because supB+ and supB- cells grown attached to plastic responded similarly to growth factors.

Animals↗
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↗
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↗
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↗
PubMed1986

Loss of tumor-suppressive function during chemically induced neoplastic progression of Syrian hamster embryo cells.

Cell hybrids between normal, early-passage Syrian hamster embryo cells and a highly tumorigenic, chemically transformed hamster cell line, BP6T, were formed, selected, and analyzed. Tumorigenicity and anchorage-independent growth were suppressed in the hybrid cells compared to the tumorigenic BP6T cells. These two phenotypes segregated coordinately in these cells. To determine at what stage in the neoplastic process this tumor-suppressive function was lost, two chemically induced immortal cell lines were examined at different passages for the ability to suppress the tumorigenic phenotype of BP6T cells following hybridization. Hybrids of BP6T cells with the immortal, nontumorigenic cell lines at early passages were suppressed for tumorigenicity and anchorage-independent growth. This tumor-suppressive ability was reduced in the same cells at later passages and in some cases nearly completely lost, prior to the neoplastic transformation of the immortal cell lines. Subclones of the cell lines were heterogeneous in their ability to suppress tumorigenicity in cell hybrids; some clones retained the tumor-suppressive ability and others lost this function. The susceptibility to neoplastic transformation of these cells following DNA transfection with the viral ras oncogene or BP6T DNA inversely correlated with the tumor-suppressive ability of the cells. These results suggest that chemically induced neoplastic progression of Syrian hamster embryo cells involves at least three steps: induction of immortality, activation of a transforming oncogene, and loss of a tumor-suppressive function.

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↗
PubMed1983

In vitro production of immune interferon (IFN gamma) by murine spleen cells when different sensitizing antigens are used in vivo and in vitro.

OK-432, a killed preparation of Streptococcus pyogenes, as well as bacillus Calmette-Guérin (BCG) and Corynebacterium parvum are all known to induce immune interferon (IFN gamma) in mice. To examine the mechanisms of IFN gamma induction by OK-432, DDI mice were sensitized with various doses of OK-432, either by a single injection of a 1-mg dose or repeated injections of 0.1-mg doses given intraperitoneally. Spleen cells removed from the mice 7-9 days after the last injection produced high-titered IFN gamma (600-800 IU/ml) in vitro in the presence of 5 micrograms/ml of OK-432. In the absence of OK-432, however, in vitro IFN gamma production of sensitized spleen cells was quite limited. Moreover, when inducers of different antigenic entities such as serologically unrelated Streptococcus faecalis, Listeria monocytogenes, or Con A were added in vitro, instead of OK-432, to the OK-432-sensitized spleen cells, high-titered IFN gamma production also occurred. This may indicate that the signal required by T cells to produce IFN gamma in vitro need not necessarily be the same as that required to sensitize mouse macrophage in vivo. Once sensitized with OK-432, mice spleen cells continued to produce high-titered IFN gamma for more than 3 but less than 5 weeks.

Animals↗
PubMed1981

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice.

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.

Animals↗
PubMed1977

Two-step chromosomal control of tumorigenicity of Chinese hamster cells in nude mice.

A simple method for microinjecting isolated chromosomes into a single living cell under an inverted microscope has been developed. Of the 368 injected cells, 85 were able to form a colony and could be cloned. Clones of Chinese hamster V79 cells microinjected with chromosomes isolated from murine D56 cells [V79 (D56) cells] were tested for tumorigenicity in immunodeficient nude mice and for colony-forming ability in soft agar. Untreated recipient V79 cells were highly tumorigenic and had a high colony-forming ability in soft agar. In contrast, two out of the 21 microinjected clones tested were non-tumorigenic in nude mice and had only weak colony-forming ability in soft agar. The chromosome banding pattern was analyzed in microinjected clones and tumors derived from cells of these clones. In cells of the two non-tumorigenic clones, a telocentric chhromosome 1 (t1) was specifically involved in translocations with other chromosomes or chromosome fragments. In all tumor cells obtained from nude mice, a supernumerary piece or a whole biarmed chromosome 14 (b14) was specifically found. The results suggest that the t1 chromosome bears the gene which controls in vitro transformation and that the additional genetic change, i.e. the extra piece of b14 chromosome, was required for tumor formation in vivo.

Animals↗