Growth factors, cytokines, and hormones promote both the proliferation and the survival of immature cells of the bone marrow that are destined to become blood cells. When these growth factors are down-regulated in normal ways to prevent overproduction of blood cells, factor-dependent immature blood cells will stop proliferating and die through a self imposed """"""""suicide"""""""" that is referred to as apoptosis or programmed cell death. The central hypothesis of this proposal is that the AP1 transcription factor, JunB, acts as an activator of apoptosis and/or a suppresser of proliferation when erythropoietin (EPO) is withdrawn from erythroid cells that require the hormone for survival and proliferation. JunB is not expressed in cells cultured in EPO, but JunB mRNA and JunB protein are induced following EPO withdrawal. In the first specific aim, JunB will be overexpressed to test if the presence of JunB in erythroid cells that do not normally express this factor a) induces apoptosis, b) regulates the expression and activity of pro- and anti-apoptotic signaling molecules Bcl-XL, Bcl-2, BAD, BAX; or c) suppresses proliferation by modulating the cell cycle. Bcl-XL is the primary anti-apoptotic regulator of survival in EPO-dependent erythroid cells. The loss of an intracellular kinase activity that allows BAD to inactivate Bcl-XL survival activity is important in triggering apoptosis. The notion that the ratio of BAD to Bcl-XL is more of a critical factor in regulating apoptosis than signals that regulate BAD phosphorylation will be tested by increasing the ratio of BAD to Bcl-XL in cells. In the second specific aim, dominant negative forms of P13-kinase in HCD57 cells will be expressed to test if an EPO-independent, P13-kinase dependent pathway promotes leukemia by inducing a reversible, pre-apoptotic state when EPO is withdrawn. The ratio of BAD to Bcl-XL expression will be compared in primary murine erythroid cells (FVA), primary human erythroid cells, and murine leukemic cells to see if the increased susceptibility of mature erythroid cells to undergo apoptosis after growth factor withdrawal is due to the increased ratio of BAD to Bcl-XL. Jun N-terminal Kinase (JNK) promotes survival and/or proliferation signals by activating the AP1 transcription factor c-Jun. In the third specific aim, a dominant negative form of JNK will be expressed to define the role of JNK in EPO signaling. EPO-dependent activation of JNK through the secretion of an autocrine factor will be tested.
