The Effects Of 12-O-tetradecanoylphorbol-13-acetate Research Articles

Protein kinase C activation enhances K+-stimulated endogenous dopamine release from rat striatal synaptosomes in the absence of an increase in cytosolic Ca2+.

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on axonal elongation and fasciculation.

Ultrastructural examination of neurons treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) confirmed our previous finding that TPA promoted neurite differentiation. At the low concentration of 16 nM TPA, the outgrowth of long neurites was correlated with the increased appearance of membrane-filled varicosities and filopodial extensions along the axons. In contrast, treatment with high concentrations of TPA (160 nM) produced dense outgrowths which were shorter in length and organized as thick fascicles. Increased neurite fasciculation appeared to result from the enhanced side-to-side interactions of neighboring neurites by a neural cell adhesion molecule. Axons within these fascicles were retracted and appeared congested with cytoskeletal and membranous components. Treatment with the antibody to the neural cell adhesion molecule defasciculated the thick outgrowths and permitted further axonal elongation.

Actions of a phorbol ester on factors regulating contraction in rabbit mesenteric artery.

The effects of 12-o-tetradecanoylphorbol-13-acetate (TPA) on isometric tension, unloaded shortening velocity determined using the slack test (Vmax), and phosphorylation of myosin light chain (MLC20) were investigated in intact and skinned muscle tissues of the rabbit mesenteric artery. In intact muscles exposed to 128 mM K+, the Vmax reached a maximum before the peak of the phasic component of contraction and reduced proportionally with changes in the amplitude of the subsequent tonic contraction. TPA (0.1 microM) enhanced to a greater extent the amplitude of the tonic than the phasic contraction. During the tonic phase, TPA enhanced the Vmax and amplitude of the contraction more than the phosphorylation of MLC20. In skinned muscles, Ca2+ (0.3-1.0 microM) increased the force, Vmax and MLC20 phosphorylation, in a dose dependent manner. The relation between any two of the above three parameters was always linear. TPA (0.1 microM) with phosphatidylserine (50 micrograms/ml) time-dependently increased the force and Vmax observed with 0.3-0.5 microM Ca2+. The relation between the force and Vmax shifted in the direction of that observed in the presence of high Ca2+, but in 0.5 microM Ca2+, TPA with phosphatidylserine showed little change in the phosphorylation of MLC20. As estimated from the actions of TPA, protein kinase C may contribute to activation of contractile proteins through MLC20 phosphorylation dependent and other regulatory processes.

Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue.

Second messengers have been implicated in the control of communication between cells of various tissues and of a number of cell lines. To assess whether protein kinase C (PKC) is involved in the regulation of gap junctions between primary differentiated cells, we studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on PKC translocation and junctional conductance of rat pancreatic exocrine cells. Our results show that although TPA induced the translocation of PKC from a "cytosolic" to a "microsomal" fraction within minutes, it failed to block the junctional conductance of acinar cell pairs up to 30 min after application. By contrast, analogous experiments on a liver-derived cell line (WB cells) showed that TPA-induced PKC translocation was paralleled by a marked and irreversible inhibition of intercellular coupling. These results indicate that, in contrast to the effects on transformed or dedifferentiated permanent cell lines, PKC is not involved in gating gap junctional channels between primary differentiated secretory cells of the pancreas.

Minimal effects of phorbol esters on glucose transport and insulin sensitivity of rat skeletal muscle.

Protein kinase C (PKC) has been suggested as a mediator of insulin's effect on glucose transport, and PKC-mediated modulation of tyrosine kinase activity in the insulin receptor has been implicated in regulating the insulin sensitivity of tissues. Because skeletal muscle is a major target of insulin action, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-oleoyl-2-acetyl-rac-glycerol, and dioctanoyl diacylglycerol, known activators of PKC, on glucose metabolism in rat skeletal muscles. In contrast to results reported for other tissues, incubation of muscles with PKC activators produced only small increases in glucose transport and had minimal effects on the ability of insulin to stimulate transport. However, TPA treatment of muscles produced a significant decrease in basal glycogen synthesis. Incubation of muscles with TPA did not affect insulin binding or the tyrosine kinase activity of partially purified insulin receptors measured under basal conditions or after stimulation by insulin in situ or in vitro. Our findings do not support activation of PKC as a major mechanism for regulating glucose uptake or insulin receptor activity in skeletal muscle. However, the data do not rule out the possibility that glucose transport in skeletal muscle may respond to physiological activators of PKC.

Effects of a phorbol ester on acetylcholine‐induced Ca2+ mobilization and contraction in the porcine coronary artery.

1. The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, have been investigated on intact and chemically skinned muscle strips of the porcine coronary artery. 2. In the presence or absence of extracellular Ca2+, TPA (0.1-1 nM) slightly enhanced the amplitude of ACh (10 microM)-induced contractions but at 100 nM, inhibited the contractions by approximately 50%. 3. ACh (10 microM) reduced the amount of [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) and increased the amount of [32P]phosphatidic acid (PA) in the presence or absence of Ca2+. TPA (over 1 nM) dose-dependently inhibited the hydrolysis of PIP2 induced by ACh. 4. ACh (over 0.1 microM) dose-dependently increased the intensity of fura-2 fluorescence in dispersed single-cell suspensions. TPA (over 1 nM) dose-dependently inhibited the increase of the Ca2+ transient evoked by ACh, but it did not modify the ionomycin-induced Ca2+ transient or the resting fluorescence. These inhibitory effects of TPA occurred over a similar dose range to that which inhibited ACh-induced PIP2 break-down. 5. When the relationship between ACh-induced contraction amplitude and Ca2+ transient was observed in the presence or absence of 10 nM-TPA, TPA greatly reduced the Ca2+ transient but did not modify the amplitude of contraction. 6. In saponin-treated skinned muscle strips, TPA (10 nM) or 1,2-diolein (50 micrograms/ml) with phosphatidylserine (PS; 50 micrograms/ml) increased the amplitude of contraction evoked by various concentrations of Ca2+ (0.1-1.0 microM) without any change in the maximum amplitude of the Ca2+-induced contraction. 7. TPA (10 nM) with PS (50 micrograms/ml) increased the amplitude of contraction evoked by 10 microM-inositol 1,4,5-trisphosphate in chemically skinned muscle strips. 8. It is concluded that TPA inhibits the ACh-induced [Ca2+]i increase by inhibiting the hydrolysis of PIP2, but that it enhances the Ca2+ sensitivity of the contractile proteins. These results indicate that ACh-induced contractions are controlled by negative feed-back regulation of PIP2 hydrolysis together with a positive feed-back regulation of the Ca2+ sensitivity of the contractile proteins. This may depend on the on-going level of protein kinase C activation.

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.

Protein kinase C and parietal cell function

Ca 2+-phospholipid dependent protein kinase activity and the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on H + secretion was studied in guinea-pig parietal cells. In resting parietal cells PK-C activity was distributed almost equally in both the cytosolic and particulate cell fraction. Exposure of the cells to TPA resulted in a loss of cytosolic PK-C activity which was not accompained by a concomitant increase of particulate activity. Furthermore TPA inhibits the K + H + -ATPase and dissipates H + gradient in intact gastric vesicles by a protonophoric action. The role of these biochemical events for the antisecretory action of TPA found in the intact parietal cell are discussed.

Enhancement of Esophageal Carcinogenesis Induced in Rats by <italic>N</italic>-Amyl-<italic>N</italic>-methylnitrosamine in the Presence of 12-<italic>O</italic>-Tetradecanoylphorbol-13-acetate

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol diester, were determined with regard to the induction of esophageal cancer in Wistar rats following a low-dose initial administration of the esophageal carcinogen N-amyl-N-methylnitrosamine [(AMN) CAS: 13256-07-0]. The induction of esophageal cancer was enhanced by TPA given in drinking water after AMN administration; i.e., the incidence of developing esophageal cancers and the multiplicity (number of esophageal cancers per rat) were significantly higher in groups given TPA solution orally after an oral administration of AMN than in those given the AMN solution alone. The enhancement of carcinogenesis with TPA was not affected by the interval between the administration of AMN and the administration of TPA. However, pretreatment with TPA before AMN administration did not enhance the induction of esophageal cancer. Neoplasms were not detected in groups given only TPA or tap water. Because this approach is similar to the phenomenon of two-stage carcinogenesis in the skin, it should provide a meaningful experimental model for studying two-stage carcinogenesis in the esophagus.

Sn-1,2-diacylglycerols and phorbol diesters: uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells.

The cell-permeable diacylglycerol mediators have been shown to mimic partially the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL-60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2-dioleoyl-sn-glycerol (DiOG), 1-oleoyl-2-acetyl-sn-glycerol (OaG), 1-palmitoyl-2-acetyl-sn-glycerol (PaG), the ether-linked analog 1-palmityl-2-acetyl-sn-glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally greater than 92% 1,2-isomer) had isomerized, and rapid formation of the 1,3-isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60-min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat-inactivation of serum reduced the enzymatic formation of fatty acid by 60-70%. ePaG was also metabolized by serum enzymes, but the ether-linked alkylglycerol product was stable. The results of cell-free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time-dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing 1-acyl-2-acetyl-sn-[3H]glycerol and [3H]acyl-2-acetyl-sn-glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H-acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat fetal islets as a useful model for the study of insulin release failure.

To study the mechanism of imparied insulin secretion from rat fetal islets, the insulin responsiveness of islets from fetuses (day 21.5 of gestation) to a variety of secretagogues was compared with that of adult rat islets. Forskolin (30 microM)-induced insulin release from fetal and adult islets was 2.7-and 2.5-fold higher, respectively, than that from islets treated with 5.6 mM glucose alone. The effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (200 nM) were also similar in fetal and adult islets. Thus, the responsiveness to forskolin and TPA showed no significant difference in adult and fetal islets. A synergistic effect of combinations of various insulin secretagogues was observed in adult islets; however, a weak synergistic effect was present with gliclazide plus TPA only in fetal islets. After islets were cultured in RPMI 1640 (containing 11.1 mM glucose), gliclazide-, forskolin-, and TPA-induced insulin release reached the levels obtained in adult islets. However, the synergistic effect of gliclazide and TPA disappeared after culture of the islets. These results suggest that the poor insulin secretion from fetal islets is not due to a defect in the activating system of either cAMP or C-kinase, but to the immaturity of the interaction of those messenger systems.

Induction of human B cell proliferation and differentiation by the combination of phorbol ester and ionomycin.

Phorbol esters and Ca2+ ionophores are known to mimic intracellular messengers involved in cell activation. We studied the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) and ionomycin on tonsil and peripheral blood-derived B lymphocytes. We show that TPA and ionomycin are co-mitogenic and induce B lymphocyte differentiation. Although TPA in high concentrations is mitogenic to B lymphocytes by itself, submitogenic concentrations of TPA in combination with ionomycin trigger 50% of B lymphocytes to synthesize DNA. Stimulation of B lymphocytes with TPA plus ionomycin resulted in increased magnitude and a shift in the kinetics of c-fos and c-myc expression compared with either agent used alone. Activation markers such as the transferrin and interleukin 2 (IL2) receptors were markedly increased after 24 h incubation with TPA and ionomycin. In parallel to the rapid proliferative burst, we observed evidence for B lymphocyte differentiation with an increase in the number of cells expressing cytoplasmic immunoglobulin (Ig) and the disappearance of the B1 surface marker. Since the cells remained surface Ig+ and secreted only small quantities of Ig, our results suggest that the combination of TPA and ionomycin is a potent inducer of B cell proliferation and early differentiation; terminal differentiation to an Ig-secreting state, however, is not achieved.

A phorbol ester has dual actions on the mechanical response in the rabbit mesenteric and porcine coronary arteries.

To clarify the role of protein kinase C in the mechanical response, the effects of 12-o-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, were investigated on intact and skinned smooth muscle preparations of the rabbit mesenteric artery. TPA (0.1 microM) showed dual actions (initial enhancement followed by inhibition during long exposure) on the K+-induced contraction. The enhancement was marked in the presence of 39 mM-K+ but inhibition was the predominant effect in the presence of 128 mM-K+. Addition of 2.6 mM-Ca2+ to a Ca2+-free solution containing 2 mM-EGTA following application of A23187 (1 microM), produced contraction. TPA showed the same dual actions on this Ca2+-induced contraction. In chemically skinned muscles, TPA increased the amplitude of Ca2+-induced contractions evoked by low concentrations of Ca2+ (0.1-0.3 microM), but reduced those evoked by high concentrations of Ca2+ (1-10 microM). Both actions of TPA were facilitated in the presence of phosphatidylserine (PS). TPA with PS had no effect on the Ca2+-independent contraction evoked in relaxing solution containing 10 mM-EGTA and 4 mM-Mg ATP following application of adenosine-5-o-3-thiotriphosphate (ATP gamma S) and 0.3 microM-Ca2+. The amount of Ca2+ stored in cells estimated from the amplitude of the caffeine-induced contraction was not modified by application of TPA with PS in skinned or intact muscle tissues. The effects of TPA were investigated on the Ca2+ transient measured from the intensity of fluorescence of quin-2 in dispersed cell suspensions prepared from the porcine coronary artery. TPA had no effect on the Ca2+ transient in high K+ but enhanced the amplitude of the contraction. Amplitudes of the tonic response evoked by 39 mM-K+ in intact muscle tissues and the contraction induced by 0.3 microM-Ca2+ in skinned muscle were much the same. TPA with PS enhanced the amplitudes of both contractions to the same extent. From the above results, we concluded that TPA shows dual actions on the contractile machinery and may act on the regulatory systems of contractile proteins. Both excitatory and inhibitory actions of TPA depended on the concentration of Ca2+. However, the physiological action of protein kinase C as estimated from the action of TPA seems to be related to an excitatory action on the contractile machinery.

The tumour promoter 12-O-tetradecanoylphorbol-13-acetate increases the activities of some peroxisome-associated enzymes in in vitro cell culture

A study was conducted on the effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on peroxisomal enzyme activities in mouse embryo fibroblasts C3H/10T1/2 C18 cells and chemically transformed C3H/10T1/2 MCA16 cells. TPA is a potent tumour promoter and treatment with this compound of the two cell lines induced peroxisomal fatty acid beta-oxidation, carnitine acetyltransferase, palmitoyl-CoA hydrolase, and catalase activities after 240 h of treatment. Stimulation of the corresponding enzyme activities was dose-related and cycloheximide inhibited the TPA-induced enzyme activities, except that of carnitine acetyltransferase. The MCA16 cells appeared to be more sensitive than the C18 cells in inducing peroxisome-associated enzyme activities after TPA treatment. The activities of the microsomal marker, NADPH-cytochrome c reductase and the mitochondrial marker, glutamate dehydrogenase were not enhanced by TPA treatment. The results indicate that TPA has peroxisomal effects and may be classified as a peroxisome proliferator.

135 CHARACTERISTICS OF PROTEIN KINASE C MEDIATED PITUITARY ACTH RELEASE

Protein kinase C is a proposed mediator of pituitary ACTH secretion. To investigate this role of protein kinase C, we studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of protein kinase C activation, on in vitro pituitary ACTH release. Normal rat anterior pituitaries were epzymatically dispersed to monolayer culture. Dishes with 3.5 × 105 cells were incubated 3 days, washed, and incubated in test media for 3.5 hours. TPA stimulated ACTH(RIA) release in a dose related fashion with an ED50 of 0.7 ng/ml and a maximal stimulation of 900% over unstimulafed cells. Increasing the concentration of culture media calcium from 0 to 1 mM, 3mM and 6 mM led to a significant increase of TPA induced ACTH release from 4.80 to 6.79, 8.41, and 8.62 ng/dish respectively. Nifedipine (10 μM), a membrane calcium channel blocker, and penfluridol (10 μM), a calcium calmodulin antagonist, inhibited TPA (1 ng/ml) mediated ACTH release by 21% and 31% respectively (p < 0.01). The addition of PGE2 (0.1 μM), an inhibitor of CRF mediated ACTH release, caused a 33% decrease of TPA stimulated ACTH release. TPA (100 μg/ml) significantly increased intracellular PGE2 (RIA) and PGE2 released into culture media by 178% and 172% respectively, over unstimulated cells. In conclusion, TPA stimulated ACTH release in vitro appears to be dependent on extracellular calcium, membrane calcium influx, and calcium calmodulin activation. Also, TPA stimulates pituitary PGE2 synthesis and exogenously administrated PGE2 inhibits TPA induced ACTH release.

Effects of tumor promoters on sodium ion transport across frog skin.

Phorbol esters are tumor promoters and mitogens whose effects may be mediated by changes in ion transport across membranes. Clarification of the transport effects of these agents should be facilitated by using a well-characterized model epithelial system whose intracellular and transmural parameters are readily measurable. The current results constitute a preliminary study of the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBU), and phorbol on the short-circuit current (Isc) across frog skin. TPA produced two effects: a stimulation of Isc of variable magnitude and a far more constant inhibition of the natriferic action of vasopressin. These effects appear related to the action of TPA as a tumor promoter insofar as PDBU (an active ester) also inhibited the natriferic response to vasopressin, whereas phorbol (inactive as a tumor promoter) had no significant effect. TPA is largely active from the mucosal medium, inhibits the natriferic response to adenosine 3',5'-cyclic monophosphate (cAMP) as well as that to vasopressin, and does not stimulate Isc in the presence of 10(-4) M mucosal amiloride. Inhibition of prostaglandin E1 production by indomethacin had no effect on the actions of TPA. The results indicate that frog skin is a promising model for studying the transport effects of the phorbol esters. The data further suggest that TPA acts on frog skin by activating the physiological amiloride- and cAMP-sensitive channels gating apical Na+ entry from the mucosal medium into the epithelial cells.

The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate.

The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on survival, proliferation, and differentiation of normal and preneoplastic rat tracheal epithelial (RTE) cells. Normal RTE cells obtained from 8-week-old rats were plated into medium containing TPA at concentrations ranging from 0.1 to 100 ng/ml. A dose-related increase in colony-forming efficiency (CFE) was observed at concentrations above 0.1 ng/ml. At 100 ng/ml the CFE was increased 5-fold above that observed in controls. Similar studies were carried out with immortal, preneoplastic RTE cells derived from carcinogen-exposed primary cultures. Upon exposure to 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decrease in CFE ranging from a 70 to a greater than 99% reduction. Two out of 11 cell lines showed only a minor reduction in CFE at this TPA concentration. Teleocidin B was as potent as TPA in causing inhibition of CFE in preneoplastic RTE cells; the potency of 4-O-methyl TPA was approximately 1/100 that of TPA and phorbol was virtually inactive. Further studies suggested that inhibition of clonal growth of the preneoplastic tracheal cells by TPA was due to rapid cell death. As early as 2 h after addition of TPA to the cultures, a loss of viable cells from the cultures as well as a reduction in CFE was observed. Morphological studies support the interpretation that a large number of cells was severely damaged within 2--4 h after TPA exposure. The formation of cross-linked envelopes (CLE), a measure of keratinocyte differentiation, was determined in cultures of preneoplastic RTE cells. TPA caused only minor increases in CLE formation indicating that the cells were not dying from TPA-induced terminal keratinocyte differentiation, but rather from TPA toxicity. Thus, preneoplastic RTE cells respond differently to TPA from normal RTE cells and also from normal and transformed keratinocytes of various origins. The role of TPA toxicity in promoting preneoplastic RTE cells to the neoplastic state is being investigated.

Modulation of type-IV procollagen and laminin production in A431 human squamous epidermoid carcinoma cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF).

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) on type-IV-procollagen (basement-membrane collagen) and laminin synthesis, turnover, and secretion was studied in human A431 squamous epidermoid carcinoma cells. Type-IV procollagen and laminin were biochemically and immunologically identified in the medium and cell extracts using immuno-precipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. EGF or TPA produced a sixfold increase in type-IV-procollagen and laminin secretion within 2 h; this was accompanied by a three- to fourfold increase in the levels of cell-associated type-IV procollagen and laminin, respectively. The level of type-IV-procollagen and laminin synthesis and secretion remained elevated for at least 16 h after the administration of either EGF or TPA. A combination of EGF and TPA was more effective than either agent alone in promoting the secretion of laminin but not of type-IV procollagen. EGF and/or TPA did not, however, produce a selective increase in the synthesis of collagen and laminin, since total protein synthesis was also increased to the same degree by these agents. As determined by labeling and chase studies, neither EGF nor TPA had any appreciable effect upon type-IV-procollagen or laminin degradation. These results indicate that the synthesis of components associated with the basement membrane in A431 cells (i.e., type-IV procollagen and laminin) can be rapidly modulated by EGF and a tumor promoter, i.e., TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 12-O-tetradecanoylphorbol-13-acetate on cell-surface glycosaminoglycans in human diploid fibroblasts.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the loss of glycosaminoglycans (GAGs) from the pericellular compartment of human skin fibroblasts was studied. GAGs from this compartment were analyzed by anion-exchange chromatography after cultures had been labelled with both [3H]glucosamine and [35S]sulfate and then chased in either the presence or absence of TPA. In both control and TPA cultures radioactivity was found in glycopeptides, hyaluronic acid, heparan sulfate (HS), and chondroitin sulfate. The amount of radioactivity that was found in HS was reduced in the TPA cultures, whereas the amounts in the other GAGs were essentially unchanged when control and treated cultures were compared. These results suggest that TPA stimulates the loss of HS from the surface of human fibroblasts.

Biochemical effects of 12-O-tetradecanoylphorbol-13-acetate, retinoic acid, phenobarbital, and 5-azacytidine on a normal rat liver epithelial cell line.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), all trans-retinoic acid (RA), 5-azacytidine (5-AC), and phenobarbital (PB) on the activities of seven enzymes and/or isozymes of a diploid rat liver epithelial cell line have been studied. At 0.1 microgram/ml, TPA depressed the specific activities of lactate dehydrogenase and gamma-glutamyl transpeptidase, whereas 2 mM PB depressed gamma-glutamyl transpeptidase and alkaline phosphatase. At 0.01 microgram/ml, RA markedly depressed the activity of NADH-diaphorase and lactate dehydrogenase but enhanced the activity of alkaline phosphatase. Only 2 microM 5-AC caused the most significant shift of lactate dehydrogenase isozyme toward the "muscle"-type isozyme. Histochemical studies revealed that PB and 5-AC induced focal areas of cells with glycogen deposits, but no significant changes in either ultrastructure or alpha-fetoprotein and albumin immunohistochemical staining pattern were observed to suggest hepatocytic differentiation. Although none of the enzymatic changes could be consistently correlated with the effects of these biological modifiers on the cellular growth rate, the effect of RA on NADH-diaphorase, lactate dehydrogenase, and alkaline phosphatase activities was the opposite of the changes observed during carcinogenesis of these rat liver epithelial cells by multiple treatments with N-methyl-N'-nitro-N-nitrosoguanidine. The depression of gamma-glutamyl transpeptidase activity by PB is contradictory to that observed histochemically in hepatocytes in vivo, but such discrepancy may be related to the differences in cell type, growth conditions, or duration of exposure.