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J C Barrett

Publications and source records attributed to J C Barrett.

At least 271 records · Page 15Linked to original sources
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

Genetic and cellular basis of multistep carcinogenesis.

Experimental evidence indicates that cancer development is a multistep process, and that multiple genetic changes are required before a normal cell becomes fully neoplastic. These genetic changes involve oncogenes, tumor suppressor genes, and possibly senescence genes. From studies in vivo using several different animal models, the stages are broadly defined as initiation, progression, and clearly involve both genetic and epigenetic events. Studies in vitro using cell culture systems have allowed the multistep process to be dissected in greater detail at both the cellular and molecular genetic level. In the Syrian hamster embryo cell culture model, neoplastic progression requires four heritable changes, involving activation of two oncogenes and loss of two tumor suppressor genes. Like the experimental systems, a limited number of studies of human tumors suggest that the multistep paradigm is also applicable, and that similar genetic events are involved in the development of cancer in humans.

Animals↗
PubMed1990

Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts.

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.

Aneuploidy↗
PubMed1990

Sodium bisulfite induces morphological transformation of cultured Syrian hamster embryo cells but lacks the ability to induce detectable gene mutations, chromosome mutations or DNA damage.

The ability of sodium bisulfite to induce morphological transformation and mutagenesis of cultured Syrian hamster embryo cells was examined. Treatment of the cells at neutral pH for 15 min with 5-20 mM sodium bisulfite resulted in a dose-dependent increase in cell transformation but no induction of gene mutations measured at two genetic loci. Treatment of the cells for 24 h increased the level of transformation, but also failed to induce chromosome aberrations, aneuploidy or DNA strand breaks in the cells. The only positive response for a DNA alteration was an increase in sister chromatid exchanges, but this effect was observed only with the longer exposures and not with the 15 min exposure, which also transformed the cells. Possible mechanisms for bisulfite-induced cell transformation are discussed.

Animals↗
PubMed1990

Activation of cellular oncogenes by chemical carcinogens in Syrian hamster embryo fibroblasts.

Carcinogen-induced point mutations resulting in activation of ras oncogenes have been demonstrated in various experimental systems such as skin carcinogenesis, mammary, and liver carcinogenesis. In many cases, the data support the conclusion that these point mutations are critical changes in the initiation of these tumors. The Syrian hamster embryo (SHE) cell transformation model system has been widely used to study the multistep process of chemically induced neoplastic transformation. Recent data suggest that activation of the Ha-ras gene via point mutation is one of the crucial events in the transformation of these cells. We have now cloned the c-Ha-ras proto-oncogene from SHE cDNA-libraries, and we have performed polymerase chain reaction and direct sequencing to analyze tumor cell lines induced by different chemical carcinogens for the presence of point mutations. No changes were detectable at codons 12, 13, 59, 61, and 117 or adjacent regions in tumor cell lines induced by diethylstilbestrol, asbestos, benzo(a)pyrene, trenbolone, or aflatoxin B1. Thus, it is not known whether point mutations in the Ha-ras proto-oncogene are essential for the acquisition of the neoplastic phenotype of SHE cells. Activation of other oncogenes or inactivation of tumor suppressor genes may be responsible for the neoplastic progression of these cells. However, in SHE cells neoplastically transformed by diethylstilbestrol or trenbolone, a significant elevation of the c-Ha-ras expression was observed. Enhanced expression of c-myc was detected in SHE cells transformed by benzo(a)pyrene or trenbolone.

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

2,3,7,8-Tetrachlorodibenzo-p-dioxin enhancement of N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation of rat tracheal epithelial cells in culture.

The abilities of various dioxins to induce toxicity or transformation of rat tracheal epithelial cells in culture were examined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was not cytotoxic and did not induce transformation as measured by the induction of growth-altered, preneoplastic cells (termed enhanced growth variants). However, TCDD enhanced the transformation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated rat tracheal epithelial cells. Other dioxin congeners with 0 to 3 chloro substitutions were inactive in enhancing MNNG transformation. TCDD was most effective at a concentration of 0.3 nM and when treatment was administered immediately after MNNG exposure. The dose-response curves for enhancement of MNNG-induced transformation and induction of aryl hydrocarbon hydroxylase activity by TCDD were similar. These results are consistent with the hypothesis that the enhancement of cell transformation by TCDD is mediated through the TCDD receptor. TCDD also enhanced transformation when the cells were treated before MNNG treatment. The ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to enhance MNNG-induced rat tracheal epithelial transformation was also examined. In contrast to the findings with TCDD, the number of transformed colonies was increased only by pretreatment with TPA followed by MNNG. TPA-pretreatment enhanced equally the number of normal cells forming colonies and the total number of transformed colonies after selection; therefore, the transformation frequency (transformants per total surviving colonies) was unchanged by TPA. In contrast, TCDD treatment enhanced the transformation frequency in MNNG-exposed cultures since the number of transformed colonies increased while the number of total colonies remained constant. Thus, TCDD appears to act by a different mechanism than TPA. TCDD enhancement of MNNG-induced transformation may be attributed to a promotional effect, a comutagenic action, or a modulation of cell proliferation and/or differentiation mediated through the TCDD receptor.

Animals↗
PubMed1989

Mouse retroviral sequences acquired by cell lines after passaging through nude mice detected by hybridization of the fms probe pSM3.

The expression of a large RNA transcript, 8.5 to 9.5 kilobases, possibly related to the fms oncogene in mouse, rat, and human tumor cells, has been described in the literature. However, the pSM3 fms probe used to detect this gene transcript contains a significant amount of the pol gene of the Susan McDonough strain of feline sarcoma virus from which it was derived. Using a fms probe which does not contain any viral pol sequences, no such "fms-related" transcripts were detected in cell lines previously reported to express the large transcripts. These cell lines did express a large 9.5-kilobase transcript which hybridized to a probe for murine leukemia virus. Partial sequence analysis of the 9.5-kilobase transcript detected with the pSM3 probe in transformed rat cells indicated sequence homology with AKV murine leukemia virus. Thus, the presence of large RNA transcripts, interpreted by us and others as being related to the oncogene fms, appears to be due to the expression of mouse retroviral sequences which hybridize to the viral pol region contained in the pSM3 fms probe. In the case of rat and human cells, such sequences appear to be acquired after the cells have been passaged in nude mice. These results should serve as a reminder of the important biohazard and data interpretation implications for investigations in which cells transfected with retroviral vector constructs are injected into nude mice, because rescue of the recombinant sequences in these cells could occur following infection by endogenous murine retroviral particles.

Animals↗
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

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

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

Multiple mechanisms for the carcinogenic effects of asbestos and other mineral fibers.

Asbestos and other mineral fibers are carcinogenic to humans and animals but differ from many carcinogens in that they do not induce gene mutations. An understanding of these interesting human carcinogens, therefore, is an important problem in cancer research. Asbestos and other fibers induce predominantly two types of cancers: mesotheliomas and bronchogenic carcinomas. Fiber size is an important factor in the carcinogenic activity of these substances as has been shown for mesothelioma induction. For bronchogenic carcinomas, but not for mesotheliomas, a synergistic effect of asbestos exposure and cigarette smoke has been observed in humans. The mechanisms by which fibers alone versus fibers in concert with other carcinogens induce cancers are probably distinct. In addition to fiber dimensions, fiber durability and surface properties of fibers are important properties affecting carcinogenicity. Evidence exists that asbestos is a complete carcinogen, an initiator and a promoter. Multiple mechanisms must be operative to explain the diverse effects of mineral fibers. Although asbestos is inactive as a gene mutagen, there is now clear evidence that it induces chromosomal mutations (aneuploidy and aberrations) in a wide variety of mammalian cells including mesothelial cells. Asbestos also induces transformation of cells in culture including mesothelial cells and fibroblasts. A mechanism for cell transformation, which is dependent on fiber dimension, has been proposed. The fibers are phagocytized by the cells and accumulate in the perinuclear region of the cells. When the cell undergoes mitosis, the physical presence of the fibers interferes with chromosome segregation and results in anaphase abnormalities. The transformed cells show aneuploidy and other chromosome abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Induction of gene amplification by arsenic.

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Two arsenic salts (sodium arsenite and sodium arsenate) induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. The inability of arsenic to induce gene mutations may relate to the negative results of arsenic in long-term animal studies and suggests that these experiments may not detect some environmental agents that act late in the carcinogenic process in humans.

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↗