Investigation of Intracellular Signals Mediating the Anti-Apoptotic Action of Prolactin in Nb2 Lymphoma Cells (original) (raw)

1995, Experimental Biology and Medicine

Studies were undertaken to identify intracellular mediators of prolactin inhibition of giucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (GdG,) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (2S100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the giucocorticoid receptor antagonist, RUM6 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 ILM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosls including: protein kinase C activation, arachidonic acid metabolism, poiyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13acetate, and 1,2-dioctanoyl-sn-glyceroi, f the calcium ionophore, A231 87 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11 , I 4-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with a-dlfluoromethyl ornithine or methyiglyoxal bis(guany1hydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase Inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-Induced apoptosls both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the poiyamine cascade did not inhibit prolactin action, suggesting that poiyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extracellular calcium was not required for prolactin or DEX action. [P.S.E.B.M. 1995 he Nb2 lymphoma cell line is a pre-T-cell line derived from a lymph node tumor in an estro-T gen-treated male rat (1). Nb2 cells proliferate in response to prolactin (PRL) and other lactogenic hormones which act through the mutant, intermediate form of the prolactin receptor present in these cells (2, 3). Other Nb2 cell mitogens include cytokines, interleukin-2 (IL-2) and IL-7 (4, 5). The high sensitivity of the Nb2 cell to prolactin-induced mitogenesis have made it a model system for studying prolactin mechanism of action.