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

Relationship between exposure to TPA and appearance of transformed cells in MNNG‐initiated transformation of BALB/c 3T3 cells

In the BALB/c-3T3-cell transformation system, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure on the appearance of transformed cells was examined in order to investigate the mechanisms of in vitro tumor promotion. Optimal duration of TPA exposure on N-methyl-N′-nitro-N-nitrosoguanidine(MNNG)-initiated cells was at least 11 days. To investigate the effect of transformation frequencies of altering inoculating cell density at the replating of MNNG-exposed cells and of altering the time of starting TPA exposure, MNNG-exposed cells were replated at various inoculum sizes. With lower inoculum sizes (1 × 103 to 3 × 104 cells/dish), maximum TPA-induced transformation occurred for TPA commencement at confluence, while with higher inoculum size (1 × 105 cells/dish), maximum transformation frequency was observed when TPA exposure was started on day 7 after replating, being some 2 days after confluence. This may suggest that there are different mechanisms involved, depending on inoculum size, and that these may involve cell-cell interactions (at lower inoculum) and mutation expression periods (at higher inoculum). By means of redispersion experiments, it was demonstrated that the appearance of transformed cells begins on about day 7 after replating at a cell density of 1 × 104 cells/dish. These results suggest the usefulness of the replating method for optimizing transformation in the BALB/c-3T3-cell transformation assay, and provide insight into the time frame of expression of MNNG-initiated transformants and TPA-induced expansion of these transformants. Int. J. Cancer 73:271–276, 1997. © 1997 Wiley-Liss, Inc.

Relationship between exposure to TPA and appearance of transformed cells in MNNG-initiated transformation of BALB/c 3T3 cells.

In the BALB/c-3T3-cell transformation system, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure on the appearance of transformed cells was examined in order to investigate the mechanisms of in vitro tumor promotion. Optimal duration of TPA exposure on N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cells was at least 11 days. To investigate the effect of transformation frequencies of altering inoculating cell density at the replating of MNNG-exposed cells and of altering the time of starting TPA exposure, MNNG-exposed cells were replated at various inoculum sizes. With lower inoculum sizes (1 x 10(3) to 3 x 10(4) cells/dish), maximum TPA-induced transformation occurred for TPA commencement at confluence, while with higher inoculum size (1 x 10(5) cells/dish), maximum transformation frequency was observed when TPA exposure was started on day 7 after replating, being some 2 days after confluence. This may suggest that there are different mechanisms involved, depending on inoculum size, and that these may involve cell-cell interactions (at lower inoculum) and mutation expression periods (at higher inoculum). By means of redispersion experiments, it was demonstrated that the appearance of transformed cells begins on about day 7 after replating at a cell density of 1 x 10(4) cells/dish. These results suggest the usefulness of the replating method for optimizing transformation in the BALB/c-3T3-cell transformation assay, and provide insight into the time frame of expression of MNNG-initiated transformants and TPA-induced expansion of these transformants.

Sustained Activation of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Is Required for Megakaryocytic Differentiation of K562 Cells

The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/MAP kinase pathway in megakaryocytic differentiation of K562 cells, the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on ERK activation were determined. Both TPA and bryostatin are known to activate PKC but paradoxically have opposing effects on megakaryocytic differentiation. TPA, a differentiation inducer, caused sustained activation of ERK (>24 h), whereas bryostatin, a differentiation blocker, only transiently activated ERK ( approximately 6 h) and attenuated the activation of ERK by TPA. To confirm a requirement for sustained ERK activation for megakaryocytic differentiation, PD098059, a synthetic inhibitor of the MAP kinase kinase 1 (MEK1) was employed. Introduction of PD098059 at any time during the first 18 h of TPA treatment completely abrogated megakaryocytic differentiation of K562 cells. After 24 h of TPA treatment, introduction of PD098059 failed to block differentiation. Differentiation blockade by PD098059 occurred via inhibition of MEK because transfection of a constitutively active mutant of MEK2 could override the PD098059 blockade. Experiments with conditioned media suggested that sustained activation of the ERK/MAP kinase pathway promoted the autocrine secretion of megakaryocytic lineage determination factors.

Modulation of serotonin transporter activity by a protein kinase C activator and an inhibitor of type 1 and 2A serine/threonine phosphatases.

We studied the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and calyculin A (CLA), an inhibitor of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS-7 cells expressing recombinant serotonin transporter (SET). In BeWo cells, treatment with TPA decreased imipramine-sensitive serotonin uptake with a reduction in Vmax without affecting Km. CLA also decreased imipramine-sensitive serotonin uptake in a manner similar to that of TPA. TPA and CLA also decreased the uptake activity of recombinant SET expressed in COS-7 cells as seen in BeWo cells. These effects of TPA and CLA were reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phosphorylation of SET by PKC, site-directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was also down-regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct phosphorylation of SET by PKC.

Inhibition of the glucocorticoid receptor expression by diverse tumor promoters in SENCAR mouse epidermis

Glucocorticoid hormones are very potent inhibitors of keratinocyte proliferation. Their function is mediated by the glucocorticoid receptor (GR) which is highly expressed in mouse epidermis. In the study reported here we compared the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and non-phorbol ester tumor promoters such as okadaic acid, chrysarobin, and benzoyl peroxide on the levels of GR protein and mRNA in SENCAR mouse epidermis. Glucocorticoid binding assay and Northern blot analysis revealed that all four tumor promoters decreased both GR protein and mRNA levels in keratinocytes in vivo. We also found that TPA and okadaic acid inhibited GR expression in keratinocyte cell line. These results suggest that GR inhibition may play an important role in mouse skin hyperplasia and promotion of skin carcinogenesis.

SHORT COMMUNICATION: Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and benzoyl peroxide (BoP) on gap junctional intercellular communication (GJIC) and the amount and localization of E-cadherin was studied in initiated mouse epidermal cells (3PC) and in carcinoma cells (CA3/7) originating from the same cell type. In addition, the localization and phosphorylation of connexin43 was studied in both cell lines and in primary keratinocytes. GJIC inhibition by TPA and BoP was stronger in primary keratinocytes compared with both cell lines. BoP strongly decreased the amount of E-cadherin protein and the level occurring in the membranes in both cell lines, whereas TPA caused a translocation of E-cadherin from the membrane towards the cytosol, without decreasing the total amount of E-cadherin present. The effect of both tumor promoters on connexin43 phosphorylation and localization was agent as well as cell dependent. These results show for the first time that tumor promoters can decrease the quantity and membrane localization of E-cadherin in different cell types.

Cell cycle progression and phenotypic modification of Ki67 antigen-negative G1- and G2-phase cells in phorbol ester-treated Molt-4 human leukemia cells.

To elucidate the relationship between the level of cellular Ki67-reactive antigen and cell proliferation, the effects of 12-O-tetra-decanoylphorbol 13-acetate (TPA) on Ki67 expression, cell cycle progression, and surface phenotypes of human T-lymphoblastic leukemia Molt-4 cells were investigated by multiparameter flow cytometry. The Ki67 antigen is constitutionally expressed in almost all untreated exponentially proliferating Molt-4 cells. Treatment with 10 nM TPA prolonged the duration of the cell cycle time and resulted in a progression arrest of cells in G1- and G2-phases, during which Ki67 expression was decreased to an undetectable level. However, in TPA-treated cultures, the Ki67-positive fraction was invariably smaller than the growth fraction as estimated from continuous 5-bromodeoxyuridine (BrdUrd) labeling curves. This discrepancy could be explained by the finding that some Ki67-negative G1 cells do not enter the resting state but instead remain in the cycling compartment. These results show that Ki67 expression of tumor cells with relatively long G1 duration is downregulated to undetectable levels in late G1-phase and the difference in the level of Ki67 expression between late G1 cells and resting G1 cells is undetectable by conventional immunological methods. Although TPA induced differentiation of Molt-4 cells into mature suppressor T cells, the phenotypic modification was not correlated with cell cycle position and Ki67 reactivity of the cells. These results suggest that growth arrest and phenotypic differentiation of Molt-4 cells are independent effects of TPA.

Transcriptional vs. posttranscriptional control of neuropeptide Y gene expression.

Human neuropeptide Y (NPY) gene expression occurs exclusively in the central and peripheral nervous systems requiring complex cell-specific regulation. In this study we have examined the effect of modulating the second messenger systems involving protein kinase A and protein kinase C on the expression of the NPY gene in different neuronal cell types. We report that the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin on a neuroblastoma cell line (LA-N-5) and a pheochromocytoma cell line (PC12) are mediated through both increased transcription of the NPY gene and through stabilization of NPY messenger RNA (mRNA). After 8 h of treatment TPA and forskolin increase the steady-state level of NPY mRNA 10- and 12-fold in LA-N-5 and PC12 cells, respectively. This response in neuroblastoma cells is due to an increase in the half-life of NPY mRNA. The response in PC12 cells is mediated by both increased mRNA stability and increased transcription. Transient transfection analyses using PC12 cells indicate that only 51 base pairs 5' to the transcription start site are necessary for the TPA and forskolin induced transcriptional response. Thus, these experiments demonstrate that TPA and forskolin effect the regulation of the NPY gene via transcriptional and posttranscriptional mechanisms in a cell-specific manner.

Increased Expression of Acidic Ribosomal Protein (P0) mRNA After Phorbol Ester Treatment of Cultured Rat Thyroid (FRTL-5) Cells

P0, an acidic protein component of the ribosomal protein in eukaryotic 60 S ribosomal subunit, plays an important role in polypeptide chain elongation during translation. To investigate the role of protein kinase C in thyroid cell protein synthesis, we examined the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of P0 mRNA and protein. RNA slot blot hybridization revealed that TPA induced the accumulation of P0 mRNA in FRTL-5 cells in a time- and dose-dependent manner. A maximal increase of 2-fold was observed 18 h after addition of TPA. Cycloheximide markedly inhibited the TPA-induced accumulation of P0 mRNA. Nuclear runoff transcription assays using nuclei prepared from TPA-treated FRTL-5 cells revealed that TPA increased the transcription of P0 mRNA but not of β-actin. Immunoblotting experiments using anti-P protein antibody showed that TPA also increased the protein amount of P0. These results suggest that TPA activates protein synthesis in thyroid cells by inducing the expression of ribosomal proteins.

Glucocorticoid-mediated Repression of Intercellular Adhesion Molecule-1 Expression in Human Monocytic and Bronchial Epithelial Cell Lines

Increased expression of the intercellular adhesion molecule-1 (ICAM-1) on cells present in the airways has been suggested to play a role in the pathogenesis of asthma by enhancing airway inflammation. We used the monocytic U937 cell line, both undifferentiated and differentiated to a macrophage-like phenotype, and the bronchial epithelial cell line NCI-H292 as cellular model systems for human monocytes/macrophages and bronchial epithelial cells, respectively, and studied the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and dexamethasone on ICAM-1 expression. Both cell lines expressed the ICAM-1 protein constitutively. In addition, TPA- or 1,25-dihydroxyvitamin D3-mediated differentiation of the U937 cell line into a macrophage-like phenotype was associated with increased expression of ICAM-1. In both cell lines, two ICAM-1 mRNA transcripts were found, and expression was stimulated to a similar degree within 1 to 2 h after addition of TPA. In both cell lines, the anti-inflammatory corticosteroid dexamethasone repressed both constitutive and TPA-stimulated ICAM-1 expression, within 3 h of its addition. In the presence of cycloheximide, a marked superinduction of ICAM-1 was observed, while the repressive effect of dexamethasone remained, supporting the hypothesis that dexamethasone acts directly at the transcriptional level.

Analysis of the 5'-Upstream Promoter Region of Human Involucrin Gene: Activation by 12-O-Tetradecanoylphorbol-13-Acetate

Involucrin is one of the precursor proteins of keratinocyte cornified envelope. Although the formation of the cornified envelope is induced by tumor-promoting phorbol esters, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the involucrin gene expression remains unknown. We have isolated a 5'-upstream region of human involucrin gene and examined its TPA-dependent promoter activity. The involucrin upstream region with the untranslated first exon was connected to chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector (INV-CAT) and was transfected into fetal rat keratinizing epidermal (FRSK) cells. The INV-CAT-transfected FRSK cells showed considerable CAT activity that was significantly augmented by the treatment of cells with TPA. FRSK cells that were transfected with a reversely oriented 5'-upstream sequence revealed little CAT activity and did not respond to TPA. The effect of TPA was significantly inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also induced the INV-CAT promoter activity, whereas 4-O-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. Analysis of the nucleotide sequence of the 5'-upstream region detected several 5'-TGANTCAA-3' sequences that are highly conserved TPA-response elements (TRE). Cotransfection of both c-jun and c-fos expression vectors with the INV-CAT vector into FRSK cells resulted in increased CAT activity. Cotransfection of either the c-jun or c-fos vector singly with the INV-CAT vector into FRSK cells had negligible effects. Dexamethasone significantly inhibited the TPA-induced promoter activity in the INV-CAT-transfected FRSK cells. These results indicate that involucrin gene expression is positively controlled by TPA through the activation of the protein kinase C/TRE system.

Effects of five phorbol esters on gap junctional intercellular communication, morphological transformation and epidermal growth factor binding in Syrian hamster embryo cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPP A), sapintoxin D (SAP D) and sapintoxin A (SAP A) on the decrease in [125I]epidermal growth factor (EGF) binding (indicating protein kinase C activation), suppression of gap junctional intercellular communication (GJIC) and induction of morphological cell transformation (MCT) in Syrian hamster embryo (SHE) cells were investigated. All five phorbol esters were found to reduce [125I]EGF binding in early passage SHE cells at comparable concentrations. DOPP A was approximately 10-fold less potent in decreasing GJIC compared to the other phorbol esters in early passage SHE cells, while the compounds showed less difference in suppressing GJIC in the phorbol ester sensitive SHE cell line BPNi. The decreases in [125I]EGF binding and GJIC were found to be transient in the continuous presence of phorbol esters. All phorbol esters induced MCT in early passage SHE cells, but DOPP and DOPP A were approximately 10-fold less potent than TPA, SAP D and SAP A. Thus, there seems to be some degree of correlation, but not to a full extent, between the ability of the phorbol esters to activate PKC, decrease GJIC and to induce MCT. The results do not suggest a simple relationship between PKC activation, inhibition of GJIC and the reported tumor-promoting activities of the compounds.

Regulation of proliferation by vasopressin in aortic smooth muscle cells: function of protein kinase C.

To investigate the effect of arginine vasopressin-stimulated prostaglandin synthesis and the activation of protein kinase C on DNA synthesis in rat aortic smooth muscle cells. The effects of arginine vasopressin on the release of arachidonic acid and the synthesis of prostaglandin (PG) E2 and prostacyclin (PGI2) were determined. The effects of 12-o-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, and of 1-oleoyl-2-acetylglycerol, a specific activator of protein kinase C, were evaluated in cultured rat aortic smooth muscle cells. The effects of arginine vasopressin and prostaglandins on the progression from the late G1 to the S phase of the cell cycle were evaluated by measuring the DNA synthesis, and the effects of TPA on them were evaluated. Arginine vasopressin dose-dependently stimulated arachidonic acid release. TPA and 1-oleoyl-2-acetylglycerol dose-dependently increased the vasopressin-induced arachidonic acid release. Vasopressin stimulated the synthesis of both PGE2 and PGI2. TPA increased the vasopressin-stimulated prostaglandin synthesis as well as the arachidonic acid release. Vasopressin, added at the G0/G1 phase of the cell cycle, stimulated DNA synthesis of aortic smooth muscle cells. Exogenous PGE2 and PGI2 inhibited the DNA synthesis and showed maximum inhibition when added at the late G1 phase. TPA alone, added at the late G1 phase, reduced the DNA synthesis stimulated by vasopressin at the G0/G1 phase to about 45%, but vasopressin alone, added at the late G1 phase, had little effect. However, with TPA pretreatment, vasopressin significantly suppressed the DNA synthesis by about 70%. Staurosporine, a protein kinase C inhibitor, reduced the suppression by TPA alone or by vasopressin with TPA pretreatment almost to the control level. Indomethacin, a cyclo-oxygenase inhibitor, reduced the suppression by vasopressin with TPA pretreatment almost to the level of TPA alone. These results suggest that arginine vasopressin has a suppressive effect on DNA synthesis in rat aortic smooth muscle cells by inhibiting progression from the late G1 into the S phase of the cell cycle through the synthesis of PGE2 and PGI2, and that protein kinase C acts as an amplifier of this mechanism.

Glomerular hypertension

Glomerular capillary hyperfiltration and hypertension occur in certain disease states and also in response to a reduction in the number of functional nephrons. Experimental studies have shown that these glomerular hemodynamic changes are maladaptive, and ultimately damaging to the kidney. Amelioration of glomerular hyperfunction by dietary protein restriction or antihypertensive therapy lessens glomerular injury in several experimental models of chronic renal disease. Hyperfiltration may similarly occur in humans with diabetes mellitus, a solitary or remnant kidney, or acquired renal disease. There is evidence to suggest that these people may therefore be at increased risk for the development of renal injury. Clinical studies have shown that dietary protein restriction and antihypertensive therapy may beneficially affect the course of chronic renal failure in humans. Large multicenter trials are currently underway on the effects of these therapeutic maneuvers on the progression of chronic renal disease.

Effect of TPA, okadaic acid and 1 alpha,25-dihydroxyvitamin D3 on neoplastic transformation induced by 60Co gamma-rays or 252Cf fission neutrons in Balb/c 3T3 cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] and okadaic acid were individually examined on the neoplastic transformation of Balb/c 3T3 cells which were exposed to either 60Co gamma-rays or 252Cf fission neutrons. The addition of 1 alpha,25(OH)2D3, or TPA, enhanced the transformation induced by gamma-rays or low doses of fission neutrons. No enhancement was observed by the addition of okadaic acid except at toxic concentrations (5 ng/ml) and with higher doses of radiation. Moreover, the enhancement of transformation by either 1 alpha,25(OH)2D3 or TPA decreased as the radiation dose was increased. The enhancement ratio, calculated by least-square analysis from 0 Gy to 1 Gy, was greater for 1 alpha,25(OH)2D3 than for TPA, and also greater for gamma-ray irradiation than for neutron irradiation. These results suggest that the promotion of radiation-induced transformation depends on the level of the initial damage caused by radiation; and that the differences in the enhancement properties of different chemicals may be due to different individual triggering mechanisms involved in the transformation process.

Phorbol ester-mediated protein kinase C interaction with wild-type and COOH-terminal truncated insulin receptors.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin were compared in wild-type human insulin receptors (HIRc cells) and human insulin receptors lacking 43 COOH-terminal amino acid residues (HIR delta CT cells). TPA increased total phosphorylation of the wild-type insulin receptor and inhibited insulin-stimulated autophosphorylation by 32 +/- 10% in HIRc cells. TPA inhibited insulin-stimulated autophosphorylation by 46 +/- 14% in HIR delta CT cells and also caused a 65% decrease in basal phosphorylation. Insulin-stimulated tyrosine kinase activity for poly(Glu4/Tyr1) was inhibited by TPA in HIRc and HIR delta CT cells by 50 and 40%, respectively. TPA decreased insulin-stimulated glucose incorporation into glycogen by 50% in HIRc cells and to near basal levels in HIR delta CT cells; this inhibitory effect of TPA was reversed in both cell lines by staurosporine. In conclusion, 1) TPA-induced inhibition of insulin receptor tyrosine autophosphorylation was linked to concomitant inhibition of the biological effects of insulin in cells expressing either wild-type or COOH-terminal truncated insulin receptors; and 2) the inhibitory effects of TPA were not dependent upon phosphorylation of COOH-terminal residues and furthermore appeared to be independent of phosphorylation of any insulin receptor serine/threonine residues. These findings suggest a novel protein kinase C mechanism that results in altered insulin receptor function without increasing phosphorylation of the receptor.

Activation of High‐Affinity Uptake of Glutamate by Phorbol Esters in Primary Glial Cell Cultures

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, on high-affinity Na(+)-dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with TPA led to concentration- and time-dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022. The effects of TPA were potentiated by the Ca2+ ionophore A23187. Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons. We conclude that PKC activates the sodium-dependent high-affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.

Post-transcriptional destabilization of estrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of the estrogen receptor (ER) was investigated in this study. After treatment with 100 nM TPA the concentration of receptor protein was measured using an enzyme immunoassay. By 24 h the receptor protein declined by about 80% from a level of approximately 236 fmol of ER/mg of protein in control cells to 50 fmol of ER/mg of protein in cells treated with TPA. Similar results were obtained with an estrogen receptor ligand binding assay. After removal of TPA, the level of ER returned to control values. 4-alpha-Phorbol, a compound related to TPA, had no effect on ER. The effects of TPA on ER expression appear to be mediated by activation of protein kinase C as H-7, an inhibitor of protein kinase C, blocks these effects. In addition to the effect on ER protein, TPA treatment also resulted in a decrease in the steady-state level of ER mRNA as determined by a RNase protection assay. The metabolic inhibitor cycloheximide was unable to prevent the TPA-induced decrease in ER mRNA. Transcription run-off experiments demonstrated that TPA had no effect on ER gene transcription. A half-life study demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treated cells. These data suggest that the decline in ER expression is mediated by post-transcriptional destabilization of ER mRNA.

Phorbol ester, not growth hormone releasing factor, consistently stimulates growth hormone release from somatotroph adenomas in culture

In order to study the mechanism of GH secretion from somatotroph adenoma cells, we have compared the effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) with that of growth hormone releasing factor (GRF) on GH secretion from human somatotroph adenoma cells cultured in monolayer. Pituitary adenoma cells were obtained from 13 patients with acromegaly undergoing surgery. On the 7th day of culture, the cells were exposed for 2 h to secretagogues. All 13 adenoma cell cultures (100%) responded to TPA (1.6-16.0 nmol/l) with a two- to six-fold increase in GH release (240 +/- 37% increase of control: mean +/- SE). The response was detectable within 10 min, and was maximal at 2 h. Phospholipase C (7.7 mmol/l) also stimulated a two- to ten-fold increase in GH release in all four adenomas examined (100%). GH release was stimulated by GRF (2.0 nmol/l) in eight out of 12 adenoma cells (67%), but the magnitude of the responses to GRF (60 +/- 18% increase of control: mean +/- SE) were much smaller than that of TPA. Five out of 13 adenomas secreted detectable amount of PRL into the medium and these five adenomas (100%) responded to TPA (16.0 nmol/l) with a two- to six-fold increase. These observations indicate that the activation of protein kinase C is the consistent stimulator in GH and PRL secretion in human somatotroph adenoma cells. However, it is not determined whether the protein kinase C is involved in the in-vivo production of GH in patients with acromegaly.

Effects of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on morphology and anchorage-independent growth of normal and established chick embryo cells.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the organization of cytoskeleton and growth of normal and established chick embryo cells (CEC) were studied. The cytoskeleton of normal CEC formed stress fibers, while that of the CEC lines established in our laboratory formed no stress fibers. TPA treatment of normal CEC resulted in disorganization of the stress fibers into amorphous structure, while that of the established CEC lines induced no reorganization of the cytoskeleton. TPA had no promotional effect in vitro or in vivo on tumor growth in normal or the established CEC.