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     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Regulation of cell-cell communication in rat bone cells: the effect of phorbol esters.

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pH(i)) and cytosolic calcium ([Ca(+2)](i)) in this process. The results show that TPA (5 x 10(-)(8) M) caused a complete inhibition of intercellular communication within 40-60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pH(i). However, neither an increase of pH(i) alone nor exposure to TPA, under conditions preventing pH(i)-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pH(i)-shift itself rather than on the absolute value of pH(i). In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pH(i) and the increase of intracellular calcium are involved in repression of intercellular communication by TPA.     

Intercellular communication of transformed and non-transformed rat liver epithelial cells : Modulation by TPA

Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non-transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27 E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communication recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db-cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db-cAMP antagonized TPA effect [1] and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence [2], and suggest that TPA may not be an effective tumour promoter in rat liver.     

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     

Study of the biological effects of theophylline on V79 cells: Viability,membrane permeability,and metabolic cooperation

The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6-thioguanine-resistant (TG^r) and 6-thioguanine-sensitive (TG^s) V79 cells significantly increased the recovery of TG^r cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations <0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells.Abbreviations TG 6, thioguanine     

Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.     

Effect of cholesterol epoxides on the inhibition of intercellular communication and on mutation induction in Chinese hamster V79 cells

Cholesterol, cholesterol-5 alpha, 6 alpha-epoxide, cholesterol-5 beta, 6 beta-epoxide and cholestane-3 beta,5 alpha,6 beta-triol were tested for their ability to induce mutations at the Na+/K+-ATPase loci of the Chinese hamster V79 cells. None of these compounds induced ouabain-resistant mutations compared to the background mutation frequency in the control cells. These compounds were further tested for their ability to inhibit intercellular communication, using the Chinese hamster V79 cell metabolic cooperation assay. The diastereomeric epoxides and cholestane-triol, but not cholesterol, were found to be inhibitors of intercellular communication in a manner similar to other known tumor promoters.     

Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication

The naturally occurring stilbene/alexin trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is a promising agent for the prevention of cancer. We investigated the effect of resveratrol on gap-junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells because inhibition of GJIC is an important mechanism of tumor promotion. Seventeen to 50 microM resveratrol increased GJIC significantly by a factor of 1.3 compared with solvent vehicle controls, when the WB-F344 cells were exposed to resveratrol for 6 h. Most tumor promoters, including the phorbol ester TPA and the insecticide DDT, block GJIC. Resveratrol at 17-50 microM also significantly prevented down-regulation of GJIC by TPA and DDT, by a factor of 2.7 and 1.8, respectively. This recovery of GJIC from TPA inhibition was partly correlated with hindered hyperphosphorylation of Cx43. In conclusion, resveratrol was found to enhance GJIC and counteract the effects of tumor promoters on GJIC, and this is likely a mechanism that contributes to the antipromotional and anticarcinogenic properties of resveratrol.     

Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serum-treated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.     

Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP

The effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell-cell communication between mouse Balb/c 3T3 cells and between Chinese hamster V79 cells was inhibited almost completely by tumor-promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor-mediated, since specific binding of 3H-phorbol-12,13-dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.     

Effects of tumor promoters,genotoxic carcinogens and hepatocytotoxins on mouse hepatocyte intercellular communication

Intercellular communication via gap junctions may be an important mechanism of cellular growth control. Tumor promoters can inhibit intercellular communication between cultured cells, while genotoxic carcinogens apparently lack this capability. The inhibition of intercellular communication by tumor promoters may be an essential mechanism by which tumor promotion occurs in vivo. In this study, the liver tumor promoters phenobarbital, lindane (1,2,3,4,5,6-hexachlorocyclohexane, -isomer), DDT (1,1-Bis[4-chlorophenyl],-2,2,2-trichloroethane), Aroclor 1254 (a polychlorinated biphenyl mixture) and dieldrin inhibited intercellular communication between male B6C3F1 mouse hepatocytes in primary culture. Intercellular communication was detected as the passage of [5-³H]uridine nucleotides from pre-labelled donor hepatocytes to non-labelled recipient heptocytes. Mouse hepatocyte intercellular communication was also inhibited by the skin tumor promoter TPA (12-0-tetradecanoyl phorbol-13-acetate), but not by the bladder tumor promoter saccharin. The genotoxic hepatocarcinogens dimethylnitrosamine, diethylnitrosamine, benzo[a]pyrene and 2-acetylaminofluorene, and the hepatocytotoxins bromobenzene, acetaminophen, carbon tetrachloride, chloroform and methotrexate had no effect on mouse hepatocyte intercellular communication at non-cytotoxic levels. These results suggest that the ability to inhibit mouse hepatocyte intercellular communication is an effect specific to tumor promoters.Abbreviations DDT 1,1-Bis[4-chlorophenyl]-2,2,2-trichloroethane - FBS fetal bovine serum - LDH lactate dehydrogenase - TCA trichloroacetic acid - TPA 12-0-tetradecanoyl-phorbol-13-acetate     

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer of rat liver epithelial cells

We have studied the influence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the vitamin A derivative retinoic acid and the benzodiazepine diazepam on intercellular communication via established gap junctions in a monolayer of rat liver epithelial cells (RLB) at various times of incubation. Intercellular communication was measured as the transfer of [3H]hypoxanthine-derived nucleotides between RLB hypoxanthine guanine phosphoribosyl transferase+ (HPRT+) and RLB HPRT- cells. TPA only showed transient inhibition of metabolic cooperation: after 4 h of treatment, intercellular communication was reduced to about 40% of the control and longer treatments showed progressively less effect until 24 h of treatment, when no difference was seen between TPA-treated and control preparations. Retinoic acid was a more effective inhibitor: both 3 X 10(-6) M applied for 24 h and 10(-4) M applied for 6.5 h, caused a 50% inhibition of label transfer. The junctional communication could only be blocked at very high concentrations (5 X 10(-4) M) in short-exposure experiments, but this is possibly a consequence of non-specific effects on the cell membrane. When the incubation time was 24 h, a considerable portion of the gap junctions appeared to persist in the 'open' state. Diazepam showed no significant inhibitory effect in the experiments performed.     

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester.     

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.     

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.     

Inhibition of gap-junctional intercellular communication between Chinese hamster lung fibroblasts by di(2-ethylhexyl) phthalate (DEHP) and trisodium nitrilotriacetate monohydrate (NTA)

Di(2-ethylhexyl)phthalate and trisodium nitrilotriacetate monohydrate, two apparently nongenotoxic carcinogens, were tested for effects on gap-junctional communication between Chinese hamster V79 lung fibroblasts. Both compounds inhibited gap-junctional communication in a concentration-dependent manner. The inhibiting effects of these chemicals on gap-junctional communication in vitro correlate with their tumor-promoting activity. Such results further support the hypothesis that inhibition of gap-junctional communication is an in vitro biomarker for some tumor-promoting chemicals.Abbreviations CAS Chemical Abstracts Service - DEHP di(2-ethylhexyl)phthalate - GJIC gap-junctional intercellular communication - NTA trisodium nitrilotriacetate monohydrate     

ELF magnetic fields induce internalization of gap junction protein connexin 43 in Chinese hamster lung cells

We have previously demonstrated that exposure of Chinese hamster lung (CHL) cells to 50 Hz magnetic fields (MFs) and/or 12-O-tetradecanoylphorbol-3-acetate (TPA)-inhibited gap junctional intercellular communication (GJIC). To explore and compare the mechanisms of GJIC inhibition induced by extremely low frequency (ELF) MF and TPA, the number and localization of connexin 43 (C x 43) were studied. The localization of C x 43 was determined with indirect immunofluorescence histochemical analysis and detected by confocal microscopy after exposing CHL cells to 50 Hz sinusoidal magnetic field at 0.8 mT for 24 h without or with TPA (5 ng/ml) for the last 1 h. The C x 43 levels in nuclei and in cytoplasm were examined by Western blotting analysis. The results showed that the cells exposed to MF and/or TPA displayed individual plaques at regions of intercellular contact, which were fewer than the normal cells in number, while the number of C x 43 in cytoplasm increased and congregated near the nuclei. Western blot analysis further demonstrated the quantity of changes in location of Cx43. These results suggest that reduction of C x 43 at regions of intercellular contact may be one of the mechanisms of GJIC inhibition induced by ELF MF.     

The effects of several croton oil constituents on two types of DNA repair and cyclic nucleotide levels in mammalian cells in vitro.

The purpose of the present study was to determine the effects of two potent tumor-promoting agents on two DNA repair mechanisms and cyclic nucleotide levels in mammalian cells. Human amnion (AV3) cells were treated with low dose levels of either UV of N-acetoxy-acetylaminofluorene. Subsequently, DNA excision repair as measured by unscheduled DNA synthesis was followed in the absence or presence of non-toxic levels of either 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibenzoate (PDB), both potent tumor promoters, or phorbol, a non-promoter. Neither of these compounds inhibited DNA repair synthesis occurring in response to low doses of the carcinogenic agents. In addition, TPA did not inhibit "post-replication repair" in response to UV irradiation of growing Chinese hamster (V79-4) cells. However, both TPA and PDB did cause rapid dramatic increases in cyclic guanosine monophosphate levels in human amnion cells; phorbol had no effect. Neither of these compounds affected cyclic adenosine monophosphate levels. These results are discussed in the light of a possible mechanism of the action of tumor promoters involving "post-replication repair".