The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair.

Chinese hamster lung fibroblast V79 cells have been widely used in studies of DNA damage and DNA repair. Since the p53 gene is involved in normal responses to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79 cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was 76 and 75% homologous to human and mouse p53 respectively, and was 95% homologous to the Syrian hamster cells. The V79 p53 sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and nonfunctional. Our results indicate that caution should be used in interpreting studies of DNA damage, DNA repair and apoptosis in V79 cells.     

Inability of diethylstilbestrol to induce 6-thioguanine-resistant mutants and to inhibit metabolic cooperation of V79 Chinese hamster cells

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01–10 μg/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N′-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5–7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.     

Inhibition of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

Abstract. The tumour promotor 4 β -phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

Species-specific posttranscriptional regulation of interferon synthesis

Human fibroblast and Syrian hamster embryo cells were induced to synthesize interferon (IF) with rIn . rCn and rIn . rCn + DEAE-dextran, respectively. Following induction, these cells synthesized IF for only a short time before entering into a repressed state and shutting off the synthesis of IF. Homologous and heterologous whole cell translational systems were developed to investigate the molecular basis for the shut-off of IF synthesis. These systems allowedd for the introduction of exogenous hamster and human IF-mRNAs into intact normal and repressed hamster and human cells via an improved CaCl2 precipitation technique. Human IF-mRNA was translated in normal human and hamster cells and in repressed hamster cells but not in repressed human cells. In contrast, the hamster IF-mRNA was translated in normal human, normal hamster, and repressed human cells but not in repressed hamster cells. These results indicate that a species-specific mechanism inhibiting translation of IF-mRNA is directly responsible for the shut-off of IF synthesis in human fibroblasts and Syrian hamster embryo cells.     

The mutation of V79 cells by N-nitrosobis(2-oxopropyl)amine activated by pancreas acinar and duct tissue from Syrian hamsters and MRC-Wistar rats

The mutagenicity of N-nitrosobis(2-oxopropyl)amine was measured in the V79 assay using homogenates of acinar cells and duct tissue from the pancreases of Syrian hamsters and MRC-Wistar rats as the activating systems. Mutations at the sodium/potassium ATPase and hypoxanthine:guanine phosphoribosyltransferase loci were measured by resistance to ouabain and 6-thioguanine (TG). The order of effectiveness in generating mutagens from BOP was hamster duct, hamster acinar, rat duct, rat acinar. These data show extensive differences in BOP activation by hamster acinar and duct tissue.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.     

Suppression of synthesis of pro-alpha 1(I) and production of altered pro-alpha 2(I) procollagen subunits in 4-nitroquinoline-1-oxide-transformed fibroblasts

The collagen phenotype of a 4-nitroquinoline-1-oxide-transformed line of Syrian hamster embryo fibroblasts, NQT-SHE, was markedly altered from that of normal Syrian hamster embryo cells, which synthesized mainly type I procollagen [pro-alpha 1(I)]2 pro-alpha 2(I). Total collagen synthesis in the transformant was reduced to about 30% of the control level primarily because synthesis of the pro-alpha 1(I) subunit was completely suppressed. The major collagenous products synthesized consisted of two polypeptides, designated as N-33 and N-50, which could be completely separated by precipitation with ammonium sulfate at 33 and 50% saturation, respectively. N-33 migrated similarly to pro-alpha 2(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-50 migrated slightly more slowly. The collagenous regions of these chains were more sensitive to protease than the analogous region of procollagen I, but alpha-chains could be obtained by digestion for 2 h at 4 degrees C with high ratios of protein:pepsin. Staphylococcus V8 protease and cyanogen bromide peptide maps of N-33 alpha and N-50 alpha chains indicated that the chains were homologous with, but different than, alpha 2(I) chains and that they differed from each other. Considering their similarity to pro-alpha 2(I), it was surprising to find that the N-collagens were secreted to the same extent as was type I procollagen from Syrian hamster embryo cells and that there were no disulfide bonds between N-collagen chains. Intrachain disulfides were present. One possible explanation for the unusual collagen phenotype of NQT-SHE cells is that transformation induced one or more mutations in the pro-alpha 2(I) structural gene while suppression of synthesis of the pro-alpha 1(I) subunit may be due to a mutation in the regulatory region of its gene or in a general regulatory gene.     

Formation of 7,12-dimethylbenz[alpha] anthracene-DNA adducts in 7,8-benzoflavone-treated hamster embryo cells.

Pretreatment of secondary cultures of Syrian hamster embryo cells with 7,8-benzoflavone (7,8-BF) inhibited both the metabolism of 7,12-dimethylbenz[alpha] anthracene (DMBA) and the formation of DMBA-DNA adducts. The DMBA-deoxyribonucleoside adducts from 7,8-BF-treated cultures had the same elution profiles on Sephadex LH-20 columns as those from cultures exposed to DMBA alone, but 7,8-BF-treated cultures contained smaller amounts of DMBA-DNA adducts per mg DNA. As the concentration of 7,8-BF was increased, the decrease in the amount of DMBA-DNA adducts per mg DNA was logarithmic with respect to the decrease in the amount of DMBA metabolized. The results suggest that more than one metabolic step is required for the binding of DMBA to DNA in hamster embryo cells.     

Ribosomal protein differences between animal cells

Ribosomal proteins of human (HeLa), Syrian hamster, Chinese hamster, chick (embryo) and rat (Novikoff hepatoma) cells have been examined by two-dimensional polyacrylamide gel electrophoresis. The results show that although there are many similarities between the electrophoretic patterns, species-specific marker proteins can be identified for Syrian hamster, chick, rat and possibly HeLa cells, which could be used in genetic analysis. No specific protein marker has been identified for Chinese hamster. The similarity in electrophoretic mobility of the hamster, chick and rat marker proteins suggests an overall structural relationship between them.     

7,12-Dimethylbenz[a]anthracene-DNA adduct formation in Wistar rat and Syrian hamster embryo cell cultures

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA was examined in Syrian hamster and Wistar rat embryo cell cultures exposed to DMBA for 5, 24, 48 and 72 h. The level of binding of DMBA to DNA was about twice as great in the hamster embryo cells as in the rat embryo cells at all times. Analysis of the DMBA-deoxyribonucleoside adducts by immobilized boronate chromatography demonstrated that the ratio of adducts with no cis vicinal hydroxyl groups to those containing cis vicinal hydroxyl groups was much greater in the rat embryo cells (from 2.2:1 to 2.9:1) than in the hamster embryo cells (from 1.3:1 to 1.6:1). The hamster embryo cells contained three major DMBADE-DNA adducts: based upon their chromatographic behavior and comparison with the three major DMBA-DNA adducts described by Dipple et al. in mouse embryo cell cultures (Biochemistry, 24 (1985) 2291), two were tentatively identified as resulting from the reaction of anti-DMBADE (the isomer of 1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydro-DMBA with the epoxide and benzylic hydroxyl on the opposite faces of the molecule) with deoxyguanosine and deoxyadenosine and one adduct resulted from reaction of syn-DMBADE (epoxide and benzylic hydroxyl on the same face of the molecule) with deoxyadenosine. The anti-DMBADE-deoxyguanosine, syn-DMBADE-deoxyadenosine, and anti-DMBADE-deoxyadenosine adducts were present in hamster embryo cell DNA in a ratio of 1.2:2:1. The Wistar rat embryo cell DNA contained a much larger proportion of the syn-DMBADE-deoxyadenosine adduct. The relative proportions of the three major DMBA-DNA adducts in Syrian hamster embryo cells were similar at all times, but the proportion of syn-DMBADE-deoxyadenosine adduct decreased slightly with time in the rat embryo cells. These results indicate that there are species specific differences in the stereospecificity of activation of DMBA to DNA-binding diol epoxides which parallel those observed for benzo[a]pyrene (BaP). The high proportion of deoxyadenosine adducts suggests that they may have an important role in the induction of biological effects by DMBA.     

Susceptibility of cells with different stage of malignancy to cytostatic action of resident peritoneal macrophages of Syrian hamsters

Syrian hamster non-activated resident peritoneal cells (PC) and peritoneal macrophages (Mph) was demonstrated. The in vivo selection of highly tumourigenic and highly metastatic variants of this strain correlated with their resistance to CSA PC and Mph in four cell variants out of five examined. The highly tumourigenic Syrian hamster embryo cells in vitro transformed by Rous sarcoma virus were highly resistant to CSA PC without selection in vivo. The resistance of highly malignant cells to CSA PC appeared to be unrelated to their ability to produce immunosuppressing prostaglandins of E type.     

Induction by modified purines (2-aminopurine and 6-N-hydroxylaminopurine) of chromosome aberrations and aneuploidy in Syrian hamster embryo cells

The modified purines, 2-aminopurine and 6-N-hydroxylaminopurine, are known point mutagens in prokaryotic organisms. 2-Aminopurine is much less potent than 6-N-hydroxylaminopurine in inducing gene mutation in mammalian cells in culture and this corresponds to the relative activity of these two compounds in inducing tumors in rats and neoplastic transformation of Syrian hamster embryo cells in culture. We report here that these modified purines can induce chromosome aberrations, including chromatid gaps, breaks, and exchanges, as well as numerical chromosome changes in Syrian hamster embryo cells. These chromosome mutations occur over the concentration range of chemical needed to induced morphological transformation of the same cells. It is not known how nucleic base analogs induce chromosome mutations; however, this activity must be considered in attempting to understand the mechanism by which these agents induce neoplastic transformation of cells.     

An evaluation of three pesticides: Piritione,supercypermethrin and metolachlor in transformation bioassays of BHK21 and hamster embryo cells

In a study of potential carcinogenicity of pesticides, Piritione, metolachlor (in the form of Dual and VUCHT 524) and Supercypermethrin (in the form of Supercypermethrin EC and Supercypermethrin TP) were assayed for induction of anchorage independent growth of BHK21 cells and morphological transformation of Syrian hamster embryo cells. The activity of these substances in both transformation assays was compared to the activity of the direct-acting ultimate carcinogen N-methyl-N-nitrosourea. In comparison to the very strong transforming activity of N-methyl-N-nitrosourea all pesticides tested with or without S9 fraction manifested a very weak, weak, medium or strong effect. The ability to induce anchorage independent growth was graded as follows: Dual < Supercypermethrin EC < Supercypermethrin TP Piritione < VUCHT 524. Results of Syrian hamster embryo cell transformation assay were very similar to the BKH21 transformation assay. VUCHT 524 strongly induced transformation whereas Dual was inactive. Piritione and Supercypermethrin EC and Supercypermethrin TP elicited a slight but significant positive response.     

Application of alkaline unwinding assay for detection of mutagen-induced DNA strand breaks

The frequency of single-strand breaks in parental DNA and gaps in nascent DNA in various cells exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) was investigated by alkaline unwinding assay using two types of alkaline lysis conditions, 22°C lysis versus 0°C lysis. The DNA damage induced by MMS and MNU is considered to be characteristic of lesions produced in DNA by alkylating agents. The aim of our research project was to adjust this method to be able to detect the greatest number of DNA lesions induced by alkylating agents in parental DNA of different mammalian cells. In our experiments we used human cell lines EUE, GM637 and XP12, Chinese hamster V79 cells, and Syrian hamster embryo cells. The higher level of strand interruptions was detected under conditions of lysis of cells at 22°C. Probably the level of strand interruptions found after the lysis of cells at 22°C correlates with the increased number of disrupted alkali-labile sites of DNA. It is remarkable that the different lysis conditions did not influence the number of gaps detected in nascent DNA of alkylated cells. Comparing induction of breaks and gaps in radiolabelled strands of parental and daughter DNA under different lysis conditions, we succeeded in defining the optimum conditions for detection of alkali-labile sites of parental DNA.     

Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolize N-2-acetylaminofluorene to 2-aminofluorene and N-hydroxy-acetylaminofluorene, products that transform hamster embryo cells. Large amounts of N-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels of N-hydroxy-acetylaminofluorene were formed after incubation of N-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested with N-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system.     

The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

The detection of hamster connexins: a comparison of expression profiles with wild-type mouse and the cancer-prone Min mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.