Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

Epigenetic mechanism of tumor promotion. Interrelationship between inflammation and ornithine decarboxylase activity induced in vitro by the carcinogenic 12-O-tetradecanoyl-phorbol-13-acetate

Topical application of 2 micrograms 12-O-tetradecanoyl-phorbol-13-acetate (TPA) regularly induced two early events in mouse skin: inflammatory reaction localized in dermal compartment and stimulation of ornithine decarboxylase activity in epidermal cells, in relation to polyamine synthesis and cell division. These reactions were followed, after 48 hrs. by an epidermal hyperplasia. At this stage, another TPA treatment induced a strong ODC activity concurrent with severe inflammation of the dermis. Inhibition of the synthesis of inflammatory factors may antagonize TPA-induced ODC, but the protective potencies differs according to the evolutive stages of the cell. After the first TPA treatment the anti-inflammatory compounds dexamethasone and indomethacin effectively inhibited ODC activity. In contrast during the proliferative stage epidermal cell function may be less dependent on inflammatory factors; thus only partial protection was observed with the inflammatory inhibitors.     

Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate

Since the enhancement of the activity of the natural glutathione (GSH)-dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we have compared the effectiveness of diverse intracellular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L-ornithine carboxylase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report at a 2-mM concentration, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine, and L-2-oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 microM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA-treated cells and is the most potent inhibitor of TPA-decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA-induced ODC activity, suggesting that the potential antitumor-promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation-promotion protocol.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

Hyperthermia and polyamine biosynthesis: decreased ornithine decarboxylase induction in skin and kidney after heat shock

The effect of hyperthermia treatments on ornithine decarboxylase (ODC) induction in mouse tissue was determined both in vitro and in vivo. In vitro, the addition of 12-0-tetradecanoylphorbol-13-acetate (TPA) to adult mouse skin pieces incubated at 37 degrees C in serum-free MEM led to a dramatic increase in epidermal ODC activity 5 hours following treatment. In contrast, incubation temperatures of 40 degrees C for the entire 5 hour incubation period rendered the skin pieces unresponsive to TPA for ODC induction. This inhibition of ODC induction was not the result of thermal skin kill, inactivation of TPA, or a general effect on epidermal protein synthesis. The inhibition of ODC induction could be reversed by switching the incubation temperature back to 37 degrees C. In vivo, raising the core body temperature in male mice to 41 degrees C for 1 hour resulted in a 78% decrease in kidney ODC activity. The kidney DNA synthesis and protein synthesis remained unaltered following the whole body hyperthermia treatments.     

Haemosiderin-like properties of free-radical-modified ferritin.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.     

Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G1-chalone, a tissue-specific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G1-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentration-independent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G1-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase alpha, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G1-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

Staurosporine, a potent protein kinase C inhibitor, fails to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells

Staurosporine, a most potent protein kinase C inhibitor, actually inhibited protein kinase C activity obtained either from cytosol or particulate fraction of mouse epidermis. Staurosporine at the concentrations which exert protein kinase C inhibition, however, failed to inhibit, but markedly augmented 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused ornithine decarboxylase (ODC) induction in isolated mouse epidermal cells. Staurosporine by itself induced ODC activity as TPA does. Mechanism of ODC induction seems different between these two compounds. Another protein kinase C inhibitor, H-7, inhibited both staurosporine- and TPA-caused ODC induction.     

Effects of garlic and onion oils on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione and ornithine decarboxylase induction in isolated mouse epidermal cells treated with tumor promoters

Garlic oil, onion oil and one of its constituents, dipropenyl sulfide, all increase, to diverse degrees, glutathione (GSH) peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity in isolated epidermal cells incubated in the presence or absence of the potent tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The stimulatory effects of these oils on epidermal GSH peroxidase activity are concentration-dependent and long-lasting, and thus, abolish totally the prolonged inhibitory effect of TPA on this enzyme. Moreover, garlic oil (5 micrograms/ml) inhibits by about 50% TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activity in the same epidermal cell system. This concentration of garlic oil also increases remarkably GSH peroxidase activity and inhibits ODC induction in the presence of various nonphorbol ester tumor promoters. Since the same oil treatments inhibit dramatically the sharp decline in the intracellular ratio of reduced (GSH)/oxidized (GSSG) glutathione caused by TPA, it is suggested that some of the inhibitory effects of garlic and onion oils on skin tumor promotion may result from their enhancement of the natural GSH-dependent antioxidant protective system of the epidermal cells.     

Cyclosporin A inhibits biological effects of tumor promoting phorbol esters

The antilymphocytic and antiphlogistic agent cyclosporin A (CsA) inhibits in vivo various effects induced by the tumor promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). These include the edema of the mouse ear, the alkaline phosphatase (AP) activity and the ornithine decarboxylase (ODC) activity in mouse epidermis as well as the generation of a specific arachidonic acid (AA) metabolite in mouse epidermis. AA metabolism in an epidermal cell-free system of mouse epidermis was not suppressed by CsA. According to thin layer chromatography the TPA-induced and as yet unidentified AA metabolite exhibits a polarity between that of 5-HETE and 12-/15-HETE. Studies with inhibitors indicate it to be a lipoxygenase product.     

Inhibitory effect of 18beta-glycyrrhetinic acid on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in mice

Glycyrrhetinic acid is an aglycone of glycyrrhizic acid, another major active component of licorice roots. Licorice root extract has been used for a long time as a medicine and a natural sweetening additive. In the present study, we found that glycyrrhetinic acid inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA) mediated oxidative stress and tumor promotion in murine skin. Topical application of TPA alone in mouse skin enhances ornithine decarboxylase activity and also increases [3H]-thymidine incorporation in DNA. Topical application of TPA also resulted in the depletion of glutathione, activities of glutathione metabolizing and antioxidant enzymes. Application of glycyrrhetinic acid prior to TPA treatment reduces this enhanced ODC activity, [3H]-thymidine incorporation in DNA and oxidative stress. Glycyrrhetinic acid was also found to inhibit DMBA/TPA-induced skin tumor formation at doses of 1.25 and 2.5 mg by reducing the number of tumors per mouse by 24% (P < 0.05) and 62% (P < 0.05), respectively. These results suggest that glycyrrhetinic acid, an antioxidant, is a potential chemopreventive agent that can inhibit DMBA/TPA-induced cutaneous oxidative stress and tumor promotion.     

Flavanones structure-related inhibition on TPA-induced tumor promotion through suppression of extracellular signal-regulated protein kinases: involvement of prostaglandin E2 in anti-promotive process

Biological functions of flavanones have been studied extensively, however, the structure-related activities of flavanones on 12-o-tetradecanoylphorbol 13-acetate (TPA)-induced promotive effects are still unclear. In this study, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the most significant dose-dependent inhibition on TPA-induced proliferative effects among eight tested flavanones in NIH3T3 cells. TPA-induced mitogen activated protein kinases (MAPK) phosphorylation, ornithine decarboxylase (ODC), c-Jun, and cyclooxygenase 2 (COX-2) protein expressions in a time-dependent manner, and the maximal inductive time point is at 1 h for MAPK phosphorylation and 6 h for others. Flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the dose-dependent inhibition on TPA-stimulated MAPK phosphorylation, COX-2, ODC, c-Jun protein expressions. Induction of, prostaglandin E(2) (PGE(2)) production was detected in TPA-treated NIH3T3 cells, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone inhibited significantly PGE(2) production induced by TPA. Addition of PGE(2) reverses the inhibitory activities of flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone on TPA-induced proliferation. And, PD98059, a specific inhibitor of ERKs, inhibited TPA-induced MAPK phosphorylation, accompanied by decreasing COX-2, c-Jun, and ODC protein expression, and showed dose-dependent inhibition on TPA-induced proliferation in cells. These results demonstrated that PGE(2) is an important mediator in TPA-induced proliferation, and MAPK phosphorylation was located at the upstream of COX-2, c-Jun, and ODC gene expressions in TPA-induced responses. Furthermore, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone (100 microM) suppressed TPA-induced colony formation associated with blocking MAPK phosphorylation, ODC, c-Jun, and COX-2 proteins expression. And, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone did not perform potent anti-radical activities among these eight tested compounds. In conclusion, this study provided molecular evidences to demonstrate that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone were potent inhibitors on TPA-induced responses without notable cytotoxicity through suppression of PGE(2) production; and anti-radical activity of flavanones was not correlated with preventing the occurrence of tumor promotion. We proposed that blocking TPA-induced intracellular signaling responses might be involved in the anti-promotive mechanism of flavanones.     

Purification and characterization of epidermal G2- and G1-chalones]

Highly purified epidermal G1- and G2-chalones from rat skin inhibit the entering of epidermocytes to S and M phases of cell cycle respectively. Their biological activity is characterized by tissue-specificity and not by species-specificity. Both of them are tissue-specific glycoproteins as for their antigenic properties. Molecular weight of G1-chlone is 21 000, G2-chalone--34 000, isoelectric point (pH) 5.55 and 5.85 respectively. G2-chalone is the fastest as compared to G1-chalone in 5% acrylamide gel electrophoresis, pH 8.3. When injected in rabbits, G2-chalone produced monospecific antibodies which have no cross-reactivity with G1-chalone. The amino acid composition of both chalones and immunofluorescent localization of G2-chalone in epidermal tisues are given.     

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: D,L-alpha-difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4-8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE2 or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis [( 3H]leucine incorporation), and the cell cycle progression from G2/M to G1, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G2/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.     

促癌物TPA诱导小鼠表皮鸟氨酸脱羧酶mRNA表达和维甲类化合物对该表达的影响

強皮肤促癌物十四烷酰佛波醋酸酯(TPA)局部应用时可触发一系列的生物化学改变,其中最明显的事件之一就是对ODC活性的短暂而急到的诱导,而这种诱导作用与其促癌作用密切相关。利用Northern印迹分析和条带(Slot)印迹分析证明,10nmol/L TPA一次局部处理小鼠背部皮肤可刺激ODC mRNA(2.0kb大小)表达,在4h左右最为明显,随后逐渐降低。10nmol/L TPA多次处理小鼠皮肤(每2天一次,共4次)也有类似的促进作用,但却在6h左右最为明显。在二甲基苯蒽和巴豆油诱发的二阶段小鼠皮肤乳头瘤和癌组织中也观察到了相同大小的ODC mRNA的高水平表达,尤以癌组织最高。新维甲类化合物R8605虽能明显抑制巴豆油诱导的ODC活性,但却未见对TPA诱导的ODC mRNA增加有明显抑制作用。     

Specificity of epidermal chalone extracts for human epidermoid tumour cells in vitro: a preliminary report.

In order to test the mitosis-inhibiting effect and the tissue specificity of the epidermal G2 chalone for tumour cells, extracts from hairless mouse epidermis were tested in short-term tissue cultures of cells from human respiratory tract epidermoid carcinomas and adenocarcinomas. The chalone inhibited strongly the mitotic activity in two cases of histologically proven epidermoid carcinoma, and had no effect in two cases of adenocarcinoma. In one case of a supposed epidermoid carcinoma, the chalone had no effect. Revision of the histology, and the result of autopsy 11 months later, showed that in this case the lesion in the lung had been a poorly differentiated metastasis from an adenocarcinoma of the ovary. Liver extracts produced in the same way as the epidermal extracts showed no mitotic inhibition in any of the cultures. These results indicate that epidermal G2 chalone produced from mouse skin is tissue specific for human epidermoid tumour cells, and also indicate that a chalone test might be used as a diagnostic tool for poorly differentiated carcinomas to see whether they are of epidermoid origin or not.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

Role of arachidonic acid release in the G2 delay induced by tumor promoter TPA in HeLa cells

The tumor promoter TPA2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells (V. Kinzel, J. Richards, and M. St?hr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (greater than 10(-4) M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetylglycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.