Inducible gene expression from multiple promoters by the tumor-promoting agent, PMA.

Phorbol ester tumor promoters affect a broad scope of changes in mammalian cells. This report describes the activation of expression of an introduced chloramphenicol acetyltransferase (CAT) reporter gene by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in a variety of fibroblast and hematopoietic cell lines. PMA-mediated activation appears to be promoter region specific, yet widespread. Enhanced gene expression is observed for four out of five promoter systems tested, and, in some cases, is dependent on the cellular environment. Further experiments indicate that PMA mediates elevated gene expression by rapidly increasing steady state levels of CAT mRNA. The broad range of promoters affected by PMA may help explain the high potency of this agent in tumor production.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Inhibition of receptor-mediated stimulation of cyclic AMP accumulation in neuroblastoma-hybrid NCB-20 cells by a phorbol ester

Activation of protein kinase C by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), modulates responsiveness of the cyclase system in many cell types. In the neuroblastoma-hybrid cell line NCB-20, PMA causes a reduction in receptor-mediated accumulation of cyclic AMP. The reduction in receptor responses by PMA occurs within 3 min and is still apparent at 40 min. This occurs in a concentration-dependent manner with an EC50 for PMA of approx. 30 nM. Accumulations of cyclic AMP that are elicited by prostaglandin E2, vasoactive intestinal peptide or 2-chloroadenosine are decreased in the presence of PMA. Accumulations of cyclic AMP that are elicited by forskolin in the absence of a receptor agonist are unaffected by the presence of PMA. Inhibition of cyclic AMP generation by dopamine is not diminished by PMA suggesting the receptor input through the inhibitory Ni-guanyl nucleotide binding protein is still functional after PMA treatment. The generalized inhibition of receptor-mediated responses by PMA could be due to a protein kinase C-mediated phosphorylation of the stimulatory Ns-guanyl nucleotide binding protein, but other mechanisms are possible.     

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.     

Suppression of cyclic AMP-dependent protein kinase activity in murine melanoma cells by 12-0-tetradecanoylphorbol-13-acetate

The effect of the potent tumor promoter 12-0-tetradeconylphorbol-13-acetate (TPA) on the cyclic AMP metabolism of B16 mouse melanoma cells was examined. TPA (10^?7M) slightly increased the growth rate and inhibited melanin production by these cells. Although TPA had little effect on basal or hormone stimulated cyclic AMP levels, it did significantly suppress cyclic AMP-dependent protein kinase activity from treated cells in a dose-dependent fashion. Other phorbol ester and non-phorbol ester tumor promoters also suppressed cyclic AMP-dependent protein kinase activity while the non-promoter, phorbol, did not alter cyclic AMP-dependent protein kinase activity.