We propose to elucidate mechanisms underlying maturation of erythroid colony-forming units (CFU-E) into red cells. Pursuing this goal, we use two populations of highly purified murine CFU-E: Generation I CFU-E forming 32-cell colonies, generation II forming 16-cell ones. For a rationale to dissect maturation, we draw upon our recent reports that (i) generation I CFU-E don't need erythropoietin (Epo) or other cytokines to progress to begins in generation II; (ii) DNA-breakdown characteristic of programmed cell death (apoptosis) first begins in generation II; (iii) Epo and insulin-like growth factor I (IGF-1) can then each almost completely forestall apoptosis; (iv) IGF-1 soon loses effectiveness whereas Epo remains effective until mid-maturation; (v) evident persistence of Epo action after it is withdrawn suggests that enduring transduction elements may have been unregulated by Epo; and (vi) for Epo- driven maturation to be optimal, a serum factor(s) other than IGF-1 is needed, especially when Epo levels are normal and this factor(s) then adds 30% or more to erythropoiesis. To identify factors (s) needed for optimal erythropoiesis, we apply reported candidates and serum fractions to well-defined serum-poor cultures to seek growth factors which can substitute for serum. We also seeks to learn why IGF-1 prematurely loses effectiveness and ask: It is due to loss of high-affinity receptors? In a parallel study, we better defined the time in CFU-E maturation when full-length Epo receptors (EpoR) capable of transducing prevention of apoptosis replace truncated EpoR which cannot. Results of such receptor analyses figure in our main purpose which is to identify molecular mechanisms by which apoptosis and cytokine response are transduced in CFU-E. Here we draw upon (i) genes whose expression in other tissues is associated with induction or prevention of apoptosis and (ii) gene expressed in CFU-E, especially those implicated by others in maturation and most especially those whose expression changes during maturation. Having established in preliminary studies that protein kinases (PK), particularly PKC, act in the pathway by which Epo forestalls CFU-E apoptosis, we focus on candidate PK whose changing transcription might underlie the temporal changes in apoptosis and/or cytokine response which we are observed. Drawing next upon pilot experiments which show that diverse PK clones can be isolated in varying recurrence frequencies from CFU-E RNA following reverse transcriptase polymerase-chain reactions primed with PK degenerate sequence oligomers, we plan to identify candidates PK stochastically. For example, using random PK clones developed from generation I CFU-E, from generation II plus Epo, from generation II plus IGF-1 and from generation II minus-cytokines, PK which recur unequally in the four CFU-E populations become candidates. Such PK, like non-PK candidates otherwise identified, will then be examined for maturation-dependent changes in RNA expression and, ultimately, for the influence their antisense oligomers have upon apoptosis and/or cytokine response. In these and other ways, we anticipate that elements underlying CFU-E maturation and apoptosis can be unabigously identified.
