A plethora of experimental evidence has linked PRL action to immune cells, yet a consensus regarding its precise function as an immunomodulator remains elusive. Experimental results from our laboratory obtained in PRL-dependent rat Nb2-11 lymphoma T-cells and a PRL-independent subline (Nb2-SFJCD1) have lead us to formulate the following central hypothesis: (1) PRL is a critical mediator of lymphocyte homeostasis by a mechanism that reflects its direct regulation of apoptosis (programmed cell death). Intricate regulation of this process is critical for maintenance of the normal immune response; its dysregulation can lead to immuno-deficiency. Our results indicate that the Nb2-11 line is sensitive to activation of apoptosis. Importantly, PRL treatment completely abrogates this process. In contrast, Nb2-SFJCD1 cells resist activation of apoptosis. Other studies indicate that PRL rapidly induces expression of the protooncogene, pim-1 in Nb2-11 cells; in the Nb2-SFJCD1 subline, it is constitutively expressed. However, pretreatment of Nb2-SFJCD1 cultures with butyrate, a fatty acid that induces differentiation, attenuates pim-1 expression and reverses apoptosis resistance. In addition, we have found that stable transfection of pim-1 completely blocks apoptosis induced by disparate stimulators. These observations lead us to hypothesize that (2) modulation of lymphocyte apoptosis by PRL directly reflects its action to induce pim-1, and to propose as a corollary, (3) the protein product of pim-1 represents a novel suppressor of the apoptosis which is transcriptionally regulated by PRL. Other results indicate that ZAP-70, a tyrosine kinase coupled to T cell receptor activation, is rapidly stimulated by PRL. This observation, together with results generated from pim-1 promoter-reporter studies have lead to our final hypothesis; (4) PRL-provoked expression of pim-1 reflects activation of the ZAP-70 signaling pathway. These hypotheses will be tested by the following Specific Aims: S.A. 1: To determine whether ectopic pim-1 overexpression inhibits glucocorticoid (dexamethasone)-, anti-neoplastic agent (vinblastine)-, or heat shock-activated apoptosis in Nb2 cells; S.A. 2: To define the 5'-cis acting elements within the pim-1 gene promoter which mediate the induction of its expression by PRL; and S.A. 3: To determine whether PRL activates the ZAP-70 signalling pathway and whether its activation leads to activation of pim-1 transcription. The results from the proposed studies will definitively rule in or out a critical role for pim-1 in PRL-afforded inhibition of apoptosis and may suggest novel therapeutic approaches for the treatment of certain immunodeficiency diseases.
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