The effect of a tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister-chromatid exchange formation in cultured Chinese hamster cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) does not increase the sister-chromatid exchange (SCE) frequency in either Chinese hamster ovary (CHO) or lung (V 79) cells which are cultured in the presence of 5-bromodeoxyuridine. Moreover, TPA does not alter the induction of SCEs in CHO cells by mitomycin C during the first 3 cycles following addition of the alkylating agent. These SCE induction data do not by themselves support the hypothesis that tumor promotion by TPA depends on the enhancement of mitotic recombination.     

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: Implication of a role for protein kinase C in the regulation of gap junctional intercellular communication

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6t-hioguanine-resistant (6-TG ^R ) and 6-thioguanine-sensitive (6-TG ^S ) V79 cells significantly increased the recovery of 6-TG ^R cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TG ^R cells was significantly greater than the expected (calculated) additive 6-TG ^R cell recovery. No synergistic increases in 6-TG ^R cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.Abbreviations DDT dichlorodiphenyltrichlorethane - MC metabolic cooperation defective - 6-TG 6thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

Effect of biological toxins on gap-junctionai intercellular communication in Chinese hamster V79 cells

Since chemical modulation of gap junctional intercellular communication has been implicated in several toxicological endpoints, a study to examine the ability of several biological toxins to inhibit this process was undertaken. Eight biological toxins were tested for their ability to inhibit metabolic cooperation, a measure of gap-junctional intercellular communication, in the Chinese V79 cell system. Aplysiatoxin, anhydrodebromoaplysiatoxin and debromoaplysiatoxin showed the strongest ability to inhibit metabolic cooperation while T2-toxin and vomitoxin inhibited metabolic cooperation to a lesser degree. Afatoxin B₁, afatoxin B₂ and palytoxin were inactive in the Chinese V79 system. Palytoxin, which was extremely cytotoxic, might act as a tumor promoter if it induces compensatory hyperplasia in vivo.Abbreviations 6-TG 6-thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

Effects of tetradecanoyl phorbol acetate,pyrethroids and DDT in the V79

The effects of the pyrethroids fucythrinate, cyfluthrin, bioallethrin and resmethrin on metabolic cooperation between V79 cells were investigated. Addition offucythrinate to cocultures of 6-thioguanine-resistant and 6-thioguanine-sensitive V79 cells significantly increased the mutant cell recovery, indicating inhibition of intercellular communication. No such effect was observed by the other pyrethroids tested. To compare the modes of action of TPA-, DDT-, and pyrethroid-induced inhibition of intercellular communication, co-exposure experiments were undertaken. Addition of TPA, together with increasing doses of fenvalerate or fucythrinate, produced a synergistic response. Various combinations of fenvalerate-, fucythrinate- and DDT-exposure gave results in accordance with an additive response. The result suggest different pathways of action for TPA and the insecticides investigated in this study.Abbreviations DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane - DMSO dimethyl sulfoxide - 6-TG 6thioguanine - TPA 12-0-tetradecanoyl phorbol-13-acetate     

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.     

Biochemical genetics of the mammalian oxidative phosphorylation system: analysis of the difference in the sensitivity of various Chinese hamster cell lines to inhibitors of the mitochondrial ATP synthase complex

Seven different Chinese hamster cell lines were found to vary greatly in their sensitivity to inhibitors of the mitochondrial ATPase. In plating-efficiency experiments, Chinese hamster lung V79 and bone marrow M3-1 cells were approximately 10,000-fold more resistant to oligomycin, 100-fold more resistant to efrapeptin, and 10-fold more resistant to ossamycin and leucinostatin than were ovary CHO or peritoneal B14 cells. In vitro experiments indicated that the increased resistance of V79 versus CHO cells to these inhibitors was due to an increased resistance of the mitochondrial ATPase. Heat-inactivation experiments indicated that there was a difference in the structure of the mitochondrial ATPase of V79 and CHO cells. Genetic experiments indicated that the difference in the sensitivity of V79 and CHO cells to inhibitors of the ATPase and the difference in the structure of the mitochondrial ATPase of V79 and CHO cells was due to a difference in both a nuclear and a cytoplasmic gene.     

Enhancement of aggregation of Chinese hamster V79 cells in rotation culture by 12-O-tetradecanoylphorbol-13-acetate

A potent mouse skin tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhanced the increase in the size of aggregates of Chinese hamster V79 C-2 cells cultured in rotation flasks for 24 h. The effective concentrations of TPA were 1-100 ng/ml. Phorbol used as the negative control did not enhance aggregate formation of V79 C-2 cells. When aggregates that had formed in culture with TPA for 24 h were transferred to normal medium and cultured for another 24 h in rotation culture, aggregate size was not markedly enhanced as compared with that in the control culture. These results suggest that some changes produced in the cell surface by TPA remain irreversible on further culture in normal medium. No such difference in aggregate-forming activity was found in aggregates formed with phorbol. Dimethyl sulfoxide (DMSO) as the solvent had no effect at the concentrations used in these experiments.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate

Previous studies have shown that metabolic cooperation between Chinese hamster V-79 cells is inhibited by the tumor-promoting phorbol esters, but not by those phorbol esters inactive in tumor promotion. Metabolic cooperation is believed to be mediated by gap junctions. Therefore, in the present study we have used freeze-fracture and quantitative morphological techniques to examine the effect of the most potent tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of the inactive analog, 4α-phorbol-12,13-didecanoate (4α-PDD) on the frequency of gap-junctional contacts between V-79 cells. In both control and in 4α-PDD treated cultures the junctions were frequent, though rather small in size. In contrast, in the TPA-treated cultures, gap junctions were few, and the area of membrane occupied by gap junctions was reduced more than 20-fold from that found for controls. These results suggest that the TPA-induced inhibition of metabolic cooperation between V-79 cells is a consequence of a decrease in the number of gap junctions.     

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells (K_d = 20.9 nM at 4 °C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.     

Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplomycin susceptibility

The Chinese hamster lung (V79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) cell. This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell. The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonal antibody specific for the enzyme. Labelling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 cells, and that 48 kDa subunits comprising bleomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105,000 X g pellet (microsomes) fraction obtained after subcellular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105,000 X g supernatant cytosol fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degraded forms of the enzyme that have lost enzymatic activity are associated.     

Evaluation of magnolia bark extract in chromosomal aberration assays

Magnolia bark extract (MBE) has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. The genotoxic potential of MBE was studied in two in vitro chromosomal aberration assays. In Chinese hamster ovary (CHO) cells, exposure for 3 h to MBE at concentrations of 0-30 microg/ml in the absence of a metabolic activation system (S9) and 0-7 microg/ml with S9 did not induce chromosomal aberrations, whereas higher concentrations were cytotoxic and did not allow for analysis of aberrations. Extended exposure for 18 h without metabolic activation at concentrations up to 15 microg/ml also resulted in a negative response. In V79 cells derived from Chinese hamster lung tissue, treatment for 6h with concentrations up to 52 and 59 microg/ml in the absence and presence of S9, respectively, did not increase the incidence of chromosomal aberrations compared to negative controls. Furthermore, MBE exposure for 24 h without metabolic activation did not induce aberrations. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro chromosomal aberration assays in CHO and V79 cells, and support the safety of MBE.     

Effects of a tumor promoter and an anti-promoter on spontaneous and UV-induced 6-thioguanine-resistant mutations and sister-chromatid exchanges in V79 Chinese hamster cells

The effects of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or an anti-promotor antipain (protease inhibitor) on spontaneous and ultraviolet-induced sister-chromatid exchanges (SCEs) and 6-thioguanine- resistant (6TG^r) recessive mutations were examined in V79 Chinese hamster cells in culture. TPA and/or antipain neither significantly altered base-line and UV-induced immediate SCE frequencies, nor decreased the level of delayed SCEs which persisted 6–7 days after irradiation. TPA and/or antipain appeared to enhance the recovery of UV-induced 6TG^r colonies at the plateau expression phase despite non-mutagenicity by themselves and unaltered metabolic co- operation. Thus, the results conceivably imply that the 6TG^r-recessive mutation expression, but not fixation, can be modulated at the cell level by the TPA and/or antipain. Our results, together with the recent results of Loveday and Latt, may argue against the notion that TPA enhances the antipain-suppressible SCEs as an index of mitotic recombination in relevance with a tumor-promotion mechanism.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines.

Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.     

Tyrosyltubulin ligase and colchicine binding activity in synchronized Chinese hamster cells

Tyrosyltubulin ligase (TTL) was found to be present in CHO and V79 Chinese hamster cells grown in tissue culture. The enzyme is soluble and requires potassium, magnesium, and ATP for maximum activity and requires tubulin as a substrate. TTL was analyzed through the cell cycle of V79 and CHO Chinese hamster cells. The enzyme showed two peaks of activity in V79 cells at 4 h and 7 h after mitotic selection, corresponding to the early S and mid to late S phases of the cell cycle. In CHO cells the enzyme displayed a major peak of activity at mid S and a minor peak or plateau during early S. Tubulin, as measured by (3H)colchicine binding, was shown to increase through S phase and reach a maximum late in the cycle during G2 approx. 3 h after maximum TTL activity.     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.     

Induction of the nuclear proto-oncogene c-fos by the phorbol ester TPA and v-H-Ras

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.     

Antipain and 12-O-tetradecanoyl-phorbol-13-acetate: Phenomenology of effects on UV mutagenesis in V79 Chinese hamster cells

Antipain (AP) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were tested in V79 Chinese hamster cells for cytotoxicity and effects on survival and 6-thioguanine-resistant (6TG^r) mutation after UV-irradiation. AP and/or TPA were relatively non-cytotoxic and had no significant effects on UV survival. Despite their non-mutagenicity, the recovery of UV-induced 6TG^r colonies was significantly enhanced by the pretreatment with either AP (0.5–2 mM) or TPA (0.1–1 μg/ml) only during the expression period before the 6TG selection at a low density of cells in the absence of AP or TPA. Such enhancing effects were maximal when AP or TPA was present during the late expression period after the mutation fixation and extensive dilution of DNA lesions. Reconstruction experiments revealed the antagonistic actions that TPA and AP tended to eliminate and increase, respectively, the metabolic co-operation. In the TPA-plus-AP treatment, AP abolished the TPA-enhanced recovery of induced mutants. Thus, it seems that TPA increases the mutant recovery largely through decreased metabolic co-operation and AP could modulate the mutation expression. Further, an error-prone inducible repair may not exist or, if it exists, AP may not inhibit it in V79 Chinese hamster cells.