Chinese Hamster Embryo Cells Research Articles

Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting a requirement for de novo protein synthesis.

Identifying inhibitors of queuine modification of tRNA in cultured cells

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2 cm 2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [ 3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thioguanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.

Carcinogen-mediated co-activation of two independent genes in Chinese hamster cells.

Following carcinogen treatment, elevated expression levels of dihydrofolate reductase (dhfr) were measured by labeling cells with fluorescent methotrexate which binds to this enzyme. Fractionation of carcinogen-treated, Simian virus 40 (SV40)-transformed Chinese hamster embryo cells (C060) into subpopulations differing in their levels of dhfr expression revealed co-expression, at enhanced levels, of dhfr and SV40 T antigen in the same cells. The increased expression of dhfr was amplification independent, while the T antigen coding sequences were amplified. The co-expression of dhfr and the bacterial chloramphenicol acetyltransferase gene linked to the early SV40 regulatory region was measured in CHO cells stably transformed by pSV2CAT-SVgpt (CC24). Both these sequences were expressed at higher levels in treated cells and the elevated expression levels were observed in the same subpopulation of cells, although no increase in their gene copy number was detected. The concomitant activation of enhanced expression of two independent genes in the same cells suggests that cellular factors governing gene expression are activated in the carcinogen-treated cells. The implications of these findings to cellular control mechanisms and to the carcinogenic process are discussed.

Alpha-radiation-induced amplification of integrated SV40 sequences is mediated by a trans-acting mechanism.

Treatment of Chinese hamster embryo cells with alpha irradiation (4 MeV, emitted by 241americium) induces a 15-fold amplification of integrated SV40 sequences. The extent of amplification depends on the dose of irradiation and on the presence of a functional T-antigen encoded by the SV40 A gene. The inducing signal can be transmitted to a non-irradiated nucleus following cell fusion. Amplification is therefore the result of some trans-acting process, and this could explain how SV40 amplification can occur after doses of alpha irradiation that are too low to cause direct DNA damage within the SV40 replicon.

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine.

Hypoxanthine induces the differentiation of certain transformed cells in vitro, so analyses were undertaken to determine whether this purine metabolite might influence the expression of transformed phenotypes induced in normal cells by chemical agents. Chinese hamster embryo cells and human skin fibroblasts in culture were treated with the promoting agent phorbol-12,13-didecanoate (PDD) with or without prior treatment with 3-methylcholanthrene (MCA), and various phenotypic effects were monitored. Hypoxanthine was found to inhibit significantly the formation of type III foci and the increase in saturation density observed for Chinese hamster cells treated with MCA plus the phorbol ester. Inosine and the hypoxanthine analogue allopurinol could also mediate the effect on saturation density, while xanthosine could not. An increase in the saturation density of human skin fibroblasts, which can be induced by the phorbol ester alone, was also inhibited by hypoxanthine. There was no significant effect on the growth rate or the intracellular nucleotide pools with hypoxanthine-treated cells. The results suggest that a normal purine metabolite, hypoxanthine, can modulate the expression of transformed phenotypes induced in vitro by the known tumor promotor PDD. These observations could help in elucidating the cellular basis for promotion of carcinogenesis.

A simple and reliable in vitro test system for the analysis of induced aneuploidy as well as other cytogenetic end-points using Chinese hamster cells.

Although aneuploidy is a serious human health problem, the experimental methodology devised until now to study the mechanisms involved in the induction of aneuploidy and for the screening of aneuploidy-inducing agents has not been so much employed to have the necessary validation. A procedure using primary cell cultures of Chinese hamster embryo cells grown on cover glasses is described. To avoid the excessive scattering and subsequent loss of chromosomes, a hypotonic treatment with a 0.17% sodium chloride solution, at room temperature, followed by in situ fixation has been standardized. This procedure improves the method through the reduction of the spontaneous frequency of aneuploid cells. Experiments carried out with cells treated with X-rays, X-rays plus caffeine, and the synthetic estrogen diethylstilbestrol (DES) demonstrated the accuracy of the system since the average chromosome number remained constant in spite of the induction of high frequencies of aneuploid cells. Moreover, the method allows for the analysis of other cytogenetic endpoints such as anaphase-telophase alterations, structural chromosome aberrations or sister chromatid exchanges.

Amplification of Oncogenes and Integrated SV40 Sequences in Mammalian Cells by the Decay of Incorporated Iodine-125

Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (“initiators”) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus—vaccinia virus—similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in C0631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

Selective Gene Amplification in Mammalian Cells after Exposure to 60 Co g Rays, 241 Am a Particles, or uv Light

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.

Induction of microchromosomes by chemical carcinogens correlates with SV40-DNA amplification in SV40-transformed Chinese hamster cells.

Radiations and chemical carcinogens induce the amplification of viral DNA inserts and of their cellular flanking sequences in SV40-transformed Chinese hamster embryo cells. In the cell line CO60, the phenomenon is easily measured by in situ hybridization using SV40-DNA as a probe. We found that the appearance of microchromosomes (MC) in CO60 metaphases correlated well with the induction of SV40-DNA amplification (SDA) mediated in the same cells by chemical carcinogens. SDA and MC formation had the same inducers and were essentially transient phenomena whose occurrence and disappearance were simultaneous. The banding properties of MC and the respective time courses of induction of chromosome aberrations and MC formation/disappearance indicated that MC were not chromosome or chromatid breaks but rather acentric double minute-like chromosomes. Double minute chromosomes (DM) have been shown to contain amplified genes. They specifically occur in established cell lines and in vivo tumor cells. Therefore, the reported correlations between SDA and MC formation and the homologies between MC and DM in CO60 cells further support the existence of specific relationships between mutagenesis, gene amplification, in vivo selective pressures and tumor cytogenetics.

Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells.

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.

Inhibition of initiator-induced SV40 gene amplification in SV40-transformed Chinese hamster cells by infection with a defective parvovirus.

Amplification of SV40 genes in SV40-transformed Chinese hamster embryo cells (CO631) by chemical carcinogens as well as by herpes simplex virus infection can be inhibited by infection with the defective parvovirus, AAV-5. This is shown by in situ hybridization with SV40 DNA of AAV-5-infected CO631 cells after treatment with herpes simplex virus type 1 or with chemical carcinogens: the initiator-induced selective amplification of SV40 sequences is prevented in the presence of the parvovirus. During HSV-infection and also in the presence of carcinogens, in CO631 cells parvovirus DNA is synthesized and AAV-5-specific antigens are expressed as revealed by hybridization with cloned AAV-5 DNA or by immunofluorescence with monoclonal antibodies, respectively. The inhibition of initiator-induced gene amplification could point to the mechanism of parvovirus-mediated inhibition of tumor development and may indicate an important role of selective gene amplification in oncogenesis.

Herpes simplex virus-induced amplification of SV40 sequences in transformed Chinese hamster embryo cells.

Infection with Herpes simplex viruses (HSV) induces amplification of SV40 sequences in SV40-transformed Chinese hamster embryo cells (CO631). This is shown by in situ hybridization of the infected cells with cloned 32P-labelled SV40 DNA. The HSV-mediated synthesis of SV40 DNA is more pronounced than after treatment with chemical carcinogens. This initiator-like effect of HSV may, in concert with the previously reported mutagenic activity of this virus (Schlehofer and zur Hausen, 1982), point to a possible mechanism of HSV infection in human genital cancer.

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells.

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.

On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture.

The present results demonstrate that Chinese hamster embryo cell populations in culture can be adapted to grow in the presence of chloramphenicol. It is shown that tryptose phosphate broth and uridine, one of its components, prevent the growth inhibitory effect of the drug. Study of some respiratory parameters (cytochrome c oxidase, cytochrome spectra, oxygen consumption) indicated that neither the broth nor uridine prevented the inhibitory effect of chloramphenicol on mitoribosomal protein synthesis. The cells grew with mitochondria devoid of a functional respiratory chain. Auxotrophy for pyrimidines appeared to result from the absence of dihydroorotate dehydrogenase, a respiratory chain-linked enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis. These and other results suggest that the synthesis of orotic acid may be considered as one of the main contributions of mitochondria to the growth of animal cells in culture.

Queuine hypomodification of tRNA induced by 7-methylguanine

Transfer RNA isolated from Chinese hamster cells transformed by 7-methylguanine is hypomodified for queuine. 7-Methylguanine rapidly induces queuine hypomodification of tRNA in normal Chinese hamster embryo cells under conditions leading to transformation, and the enzyme catalyzing the queuine modification reaction, tRNA: guanine transglycosylase, is inhibited by 7-methylguanine in vitro .

Carcinogen-mediated amplification of specific DNA sequences.

A model experimental system based on SV40-transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen-mediated amplification of SV40 DNA sequences is regulated by the viral "A" gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.

Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells.

Exposure of simian virus 40 (SV40)-transformed Chinese hamster embryo cells to various chemical and physical carcinogens induced SV40 DNA synthesis. Although the carcinogen-mediated amplification of SV40 DNA is regulated by the viral A gene, the induction of viral DNA synthesis does not result in the rescue of infectious virus or the formation of complete viral DNA molecules. Instead, a heterogeneous collection of DNA molecules containing SV40 sequences was generated by treatment with 7,12-dimethylbenz[a]anthracene. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant showed that not all sequences in the integrated SV40 inserts are present. The possibility that amplification of SV40 sequences is a reflection of a general-gene-amplification phenomenon mediated by carcinogens is discussed.