Tumour-promoting phorbol esters rapidly inhibit bud formation in hydra

Summary The effects of tumour promoters and carcinogens on bud formation were investigated in an attempt to clarify the primary process of bud formation in hydra. Treatment with 1.0ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate (PDD) or mezerein added immediately after feeding rapidly and completely inhibited the formation of new buds in Hydra japonica. Treatment with TPA 3–6 h after feeding also suppressed bud formation 24 h later, but suppressed buds appeared 48 h later. Buds suppressed by TPA also formed in the presence of a diluted homogenate of hydra and during starvation. Carcinogens, such as benzo(a)pyrene and 20-methylcholanthrene, did not have an inhibitory effect on bud formation within 2 days. The tumour promoters and carcinogens used in this experiment did not inhibit the regeneration of tentacles. These results indicate that tumour-promoting phorbol esters, but not carcinogens, rapidly suppress the process by which the formation of buds is initiated by hydra, and the effects of these esters depend on the timing of treatment after feeding.     

Millimeter wave exposure reverses TPA suppression of gap junction intercellular communication in HaCaT human keratinocytes

The effect of 30.16 GHz millimeter wave (MMW) exposure at 1.0 and 3.5 mW/cm2 on gap junction intercellular communication (GJIC) was studied in cultured HaCaT keratinocytes, using the fluorescence recovery after photobleaching (FRAP) technique and laser confocal scanning microscopy to follow the intracellular movement of 5,6-carboxyfluorescein diacetate dye. While MMW exposure alone for 1 h at either 1.0 or 3.5 mW/cm2 did not affect GJIC, MMW exposure in combination with 5 ng/ml TPA treatment reversed TPA induced suppression of GJIC. Exposure at 1.0 mW/cm2 resulted in a partial reversal, and exposure at 3.5 mW/cm2 resulted in essentially full reversal of the TPA suppression.     

The role of nerve cell density in the regulation of bud production in hydra

Summary The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.     

Enhanced production of human interferon-gamma by the use of a combination of inducers--phytohemagglutinin, picibanil and tumor promoting agent

Phytohemagglutinin (PHA), picibanil (OK432) and tumor promoting agent (TPA) were tested in various combinations for optimal induction of human interferon-gamma (HuIFN-gamma). It was found that the use of a mixture of all 3 inducers resulted in IFN production 2-3 times higher than either PHA (10 micrograms/ml) in combination with TPA (5 ng/ml) or picibanil (10 micrograms/ml) alone. The IFN was produced by T lymphocytes and could be neutralized by specific IFN-gamma antiserum. It was pH 2.0 labile and species specific.     

Divergent effects of 1-oleoyl-2-acetylglycerol and tumor-promoting phorbol ester on rat granulosa cell steroidogenesis in vitro

The present study was undertaken to compare the effects of diacylglycerol (synthetic 1-oleoyl-2-acetylglycerol; DG) and TPA (12-O-tetradecanoylphorbol 13-acetate) on FSH- and dibutyryl cyclic AMP ((Bu)2cAMP)-stimulated granulosa cell pregnenolone (P5), progesterone (P), and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) secretion. Granulosa cells from immature rats pretreated with pregnant mare's serum gonadotropin (PMSG) were incubated for up to 24 h with DG (0-80 micrograms/ml) or TPA (0-80 ng/ml) in the presence or absence of FSH (150 ng/ml) or (Bu)2cAMP (1.5 mM). DG, when continually present in the culture medium (MEM), significantly stimulated basal P5 (in the presence of 25 microM cyanoketone to block further metabolism), P, and 20 alpha-OH-P secretion during 6 h and 24 h of incubation. Pretreatment with TPA for 1 h caused a substantial increase in the subsequent progestin (P + 20 alpha-OH-P) secretion. However, the phorbol ester had little or no effect on steroid secretion during 6 h of incubation, significantly inhibited the secretion of P5 and P, but stimulated 20 alpha-OH-P production in 24 h. DG and TPA exerted divergent effects on FSH- and (Bu)2cAMP-stimulated progestin secretion. Accumulation of P5 throughout the culture periods (1-24 h) was markedly increased by DG (20 micrograms/ml) but significantly inhibited in the presence of TPA (40 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 50 Hz magnetic fields on gap junctional intercellular communication.

To explore whether the extremely low frequency (ELF) electromagnetic fields (EMFs) may act as cancer promoters or be synergistic with 12-O-tetradecanoylphorbol-13-acetate (TPA) in cancer promotion, an experiment was conducted on the effects of 50 Hz magnetic fields (MFs) on gap junctional intercellular communication (GJIC) of Chinese hamster lung (CHL) cells. Lucifer dye was loaded into CHL cells by iontophoretic injection, and the number of dye-coupled cells (DCC) 5 min after the injection was adopted as the index of GJIC. The effects of TPA at different concentrations and magnetic fields at different intensities, combined with 5 ng/ml TPA, were studied. The results showed that the suppression of TPA on GJIC was dependent on TPA concentration; the threshold concentration of TPA for CHL cells was between 1 and 5 ng/ml. After exposure to 0.8 mT magnetic field for 24 h, the number of DCC decreased to 6.08 +/- 1.59, whereas the number of DCC in the control group was 9.84 +/- 2.27 (P < .05). When the cells were exposed at 0.2, 0.4, and 0.8 mT for 24 h, combined with 5 ng/ml TPA treatment during the last 1 h, the number of DCC decreased to 5.52 +/- 1.53, 5.00 +/- 1.22, and 4.00 +/- 1.29, respectively, which were significantly lower than the values for the group treated with 5 ng/ml TPA alone (6.38 +/- 1.39). It is suggested that certain intensities of 50 Hz magnetic field might act as cancer promoters, be additive with other promoters in cancer promotion, or both.     

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.     

Stimulation of prostaglandin production in bone by phorbol diesters and melittin

The production of prostaglandin E₂ (PGE₂) and bone resorption were studied in neonatal mouse calvaria in organ culture. Two tumor promoters 12- -tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12, 13-di-decanoate, but not the non-tumor promoters 4α-phorbol-12,13-didecanoate and phorbol, stimulated both PGE₂ synthesis in bone and bone resorption. The effect of TPA was maximum at about 25 ng/ml, and half-maximum stimulation occurred at about 8 ng/ml TPA. The effects of TPA on the production of PGE₂ and bone resorption were inhibited completely by indomethacin (5.6 × 10⁻⁸ to 5.6 × 10⁻⁷ M). The bee venom toxin, melittin, was also a potent stimulator of prostaglandin synthesis in bone and bone resorption. The effect of melittin was maximum at about 25 ng/ml, and the dose-response curve was biphasic. The effects of melittin on the production of PGE₂ and bone resorption were also inhibited by indomethacin. Indomethacin did not inhibit the bone resorption-stimulating activity of exogenously added PGE₂. We conclude that phorbol diesters, which have irritant and tumor-promoting activity in mouse skin, and the polypeptide melittin can act directly on bone to stimulate resorption by a mechanism involving the local production of PGE₂ or possibly other indomethacin-inhibited metabolites of arachidonic acid.     

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

AFP, CEA, CA 19-9 and TPA in hepatocellular carcinoma.

The present study is based on the assay of four markers (AFP, CEA, TPA, Ca 19-9) using IRMA methods in 36 normal subjects, 44 cirrhosis and 66 HCC patients. Parametric and non parametric tests were used to test differences and correlations. ROC curves and discriminant functions were also elaborated. Normal 95% "cut-off" was determined by the "boostrap" method yielding: CEA 3.4 ng/ml; Ca 19-9 55 U/ml; TPA 58U/l and AFP 5.2 ng/ml. In HCC patients the values of the four markers were, on average, significantly different from those of normal subjects. However, only AFP and TPA exhibited high diagnostic accuracy (90%) for detection of the tumor. Higher than normal mean values for all markers were, also observed in cirrhotic patients. Only AFP yielded effective discrimination between HCC and cirrhosis. The positive prediction for the presence of the tumor on cirrhotic ground was 95% for AFP values higher than 18.5 ng/ml, with a 78% negative predictive value with a 6 ng/ml threshold. Association of AFP with TPA showed only a marginal diagnostic improvement. Results were not improved at all by combining CEA and Ca 19-9 with AFP and/or TPA. In conclusion, AFP is and remains the best marker for HCC and the only one effective in discriminating of HCC from cirrhosis. TPA may be considered a valid alternative if cirrhosis is not present. CEA and Ca19-9 are of no use.     

Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL)

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.     

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.     

Characterization of the effects of phorbol esters on rat mast cell secretion

When applied to the skin, phorbol esters (PEs) elicit signs of acute inflammation, suggesting they may induce the release of mediators from mast cells. Therefore, we have studied the effects of PEs on purified rat peritoneal and thoracic mast cells both alone and in conjunction with the calcium ionophore, A23187, and various other secretagogues that interact with immunoglobulin E (e.g., anti-IgE and Con A) or other cell surface receptors, e.g., somatostatin and compd 48/80. PEs alone caused little or no release of histamine. However, the PE 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) tremendously potentiated release induced by the calcium ionophore A23187, reducing the EC50 for A23187 from 832 ng/ml to 56 ng/ml. In the presence of suboptimal A23187 (50 ng/ml), only active tumor promoting PEs elicited histamine release. The EC50 values of the various active PEs were: TPA 5 ng/ml; 4 beta-PDD, 83 ng/ml; and 4-O-methyl-TPA, 807 ng/ml, with maximal histamine release ranging from 54 to 80%. TPA synergistically enhanced stimulation of histamine release by anti-IgE and Con A over the entire concentration-response range. In contrast, this synergism was absent when cells were stimulated with somatostatin and compd 48/80. Phorbol esters may act by increasing the activity of a calcium/phospholipid-dependent protein kinase (Ca/PL-PK). Mast cells do have Ca/PL-PK activity, and TPA in the presence of suboptimal A23187 induces protein phosphorylation comparable with other secretagogues. These results suggest that in the purified mast cell, PE-induced mediator release increases the sensitivity of release mechanisms for calcium, acts syngergistically with secretagogues interacting with IgE, and as suggested from structure-activity relationships, occurs via a specific mechanism of action perhaps involving the Ca/PL-PK.     

Differential effects of non-genotoxic carcinogens and proliferating agents on cell growth, survival and apoptosis in hepatic cells in vitro.

Many nongenotoxic carcinogen's (ngc) produce hyperplastic lesions from which neoplastic foci may arise. Modulation of the rate of apoptosis by some ngc's within these lesions may be critical to their mechanism of tumour promotion but some may be cytotoxic. To establish if these compounds are apoptotic or necrotic in vitro, three ngc's (12-0-tetradecanoyl phorbol-13-acetate (TPA); nickel, and di(2-ethylhexyl-phthalate (DEHP), two noncarcinogenic hepatoproliferating agents (1,4-dichlorobenzene (DCB; HGF) and an in vitro genotoxic reference compound (7-hydroxy-2-acetylaminofluorene (70H2AAF) were used to induce mitogenic or growth responses in two liver cell-lines HepG2 and JTC-15. MTT and 3H-thymidine incorporation assays were used to measure cell growth and DNA replicative activity respectively. Rates of apoptosis were assayed using FITC-annexin V with propidium iodide staining and flow cytometry. Responses in HepG2 cells were HGF (proliferation at > or = 3 ng/ml), TPA (cell growth at > or = 8 ng/ml), DEHP (proliferation at > or = 0.05 microg/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.001 microg/ml and 100 ng/ml respectively. An equivocal result was obtained for DCB. Responses in JTC-15 cells were HGF (proliferation, 3 ng/ml), TPA (DNA replication, 10 ng/ml), and DEHP (cell mass, 2.5 microl/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.01 microg/ml and 110 mg/ml respectively. Equivocal results were obtained for DCB. In flow cytometry assays apoptotic and necrotic populations were not clearly separable. Approximate rates of apoptosis in HepG2 were: control 8.7%; DEHP, 10.19%. NiCl2, 12.67%; 70H2AAF, 16.56%; TPA, 19.72%; HGF, 23.73%; DCB, 24.59%; positive apoptotic control (taxol) 26.94%. These data show apoptosis was increased in chemically activated populations of HepG2. The ngc, DEHP, unexpectedtly produced proliferation in HepG2 and almost totally suppressed apoptosis in vitro in HepG2 relative to the non-carcinogenic hepatoproliferators. The rate of apoptosis induced by the ngc TPA was not considered to be sufficiently different to the rates of apoptosis induced by the noncarcinogenic hepatoproliferators. The results emphasize the importance of considering necrotic reactions from effects on apoptosis in detecting non-genotoxic carcinogens.     

Phorbol ester-stimulated murine myelopoiesis: role of colony-stimulating factors

Tumor promoting phorbol esters, such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulate colony formation in vitro by murine granulocyte-macrophage progenitors (GM-CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony-stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA-containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L-cell CSF inhibited colony formation stimulated by L-cell CSF and WEHI-3 CSF, but had no effect on colony formation induced by TPA. Cells from long-term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF-producing macrophages to less than 1.0%. TPA inhibited binding of radioiodinated L-cell CSF to marrow cells, especially if the cells were first exposed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM-CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites.     

Stimulation and inhibition of neoplastic cell growth by tumor promoter-treated macrophages

Macrophages treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent inflammatory and tumor-promoting agent, can have the diametrically opposed functions of contact-mediated tumor cytotoxicity and release of soluble clonal proliferation factor(s) for tumor cells. In vitro TPA treatment of macrophages at 1.0 ng/ml induced prostaglandin E₂ release and morphological changes analogous to cell activation. In addition, conditioned medium from macrophages pulsed with TPA enhanced M109 carcinoma colony formation in vitro. Although macrophages were not rendered tumoricidal by TPA in vitro, cytotoxic macrophages were recovered from mice following ip treatment with TPA at 1–100 μg/kg. This indicated an indirect pathway for the activation of macrophages by TPA. The very weak tumor promoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate lacked effects on macrophages at all doses tested. The possibility that macrophage secretions (e.g., prostaglandin E₂, angiogenesis-stimulating factor(s), and clonal proliferation factor(s) for carcinogen-triggered cells) may be involved in the tumor promotion process is discussed.     

Effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 in human hepatoma cell lines

The effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 (TGF-beta 1) in human hepatoma cell lines, Mahlavu and PLC/PRF/5, were investigated. TGF-beta 1 (2.5 to 10 pM) alone could not inhibit the growth of Mahlavu cells, whereas in the presence of 12-O-tetradecanoyl phorbol 13-acetate (TPA) at 1 ng/ml, TGF-beta 1 could suppress their growth in a dose-dependent manner. The growth of PLC/PRF/5 cells could be inhibited by addition of TGF-beta 1 (2.5 to 10 pM) alone in a dose-dependent manner, and this action was not affected by TPA (1 ng/ml). The TGF-beta 1 inhibition induced by TPA in Mahlavu cells could not be cancelled by addition of protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) (10 microM) or staurosporin (1 nM). Thus, TPA could induce TGF-beta 1 inhibition of cell growth in Mahlavu cells which did not respond to TGF-beta 1 alone, and activation of protein kinase C does not seem to be behind this TPA action.