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.     

Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

A mutagenesis-enhancing activity of 12-O-tetradecanoylphorbol 13-acetate detected in Chinese hamster ovary cells

Tumour-promoting agents may bring about the completion of multi-step carcinogenesis by acting as enhancers of mutagenesis, recombinogens or clastogens. We report here that the classical mouse skin tumour promoter TPA, although non-mutagenic per se, can enhance the induction of OuaR CHO-K1 cell mutants by MNNG approximately 2-fold. This observation was made at a concentration approaching the compounds aqueous solubility limit which was non-cytotoxic. Mutagenesis enhancement was dependent on TPA being present throughout mutation expression and mutant selection. It was not accompanied by any modification of cell sensitivity to mutagen killing. In the same treatment protocol TPA did not enhance either EMS- or UV-induced mutagenesis. TPA exposure over 2 rounds of cell replication failed to produce an increase in the frequency of SCE in control or mutagen-treated CHO-K1 cultures. Likewise TPA exposure over 1 round of cell replication failed to produce an increase in the frequency of chromosomal aberrations. Apparently TPA is not a recombinogen or clastogen but in the right exposure regime is capable of acting to enhance mutagenesis by certain genotoxic agents, an action which may contribute to tumour promotion.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.     

Nature of the iron requirement for Chinese hamster V79 cells in tissue culture medium

The Chinese hamster V79 cell line can be grown in medium containing iron instead of lactalbumin hydrolysate and containing defined low molecular weight components instead of peptone. A rather large amount of inorganic iron must be supplied for optimum growth. Dose-response curves done with commercially available transferrins from various species show that this Chinese hamster cell line grows well with human and rabbit transferrins but poorly with porcine, bovine, and chicken egg white (conalbumin) transferrins. An assay of Chinese hamster serum in the presence and absence of iron shows that hamster serum is better at providing the V79 cells with iron than human or rabbit transferrin. Thus, the nature of the iron requirement of V79 cells lies in the requirement for a specific transferrin.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

Modulatory action of 12-O-tetradecanoylphorbol-13-acetate on bud production inHydra

Summary A low concentration of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.0 ng/ml) induced a transient inhibition of bud production in hydra which were fed daily. However, when hydra were starved following TPA-treatment, they produced further buds. Phorbol (1.0 ng/ml) and dimethyl sulfoxide (0.001%) did not influence bud production under either feeding or starvation conditions. These results indicate that TPA modulates asexual reproduction in hydra.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA).

TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.