Regulation of Human Megakaryocytopoiesis

Gewirtz, Alan

Abstract

The long term goal of this project will be to generate a comprehensive understanding of the subcellular and molecular events which regulate normal human megakaryocytopoiesis.
Our specific aims will be to 1: Determine the role of arc family tyrosine kinases, and G alpha proteins in transducing growth factor mediated signals from cytoplasm to nucleus: We hypothesize that tyrosine kinases and G proteins may play an important role in transducing growth factor initiated signals. We will test this hypothesis by determining megakaryocyte responsiveness to known growth factors after blocking de novo synthesis of these proteins with antisense oligodeoxynucleotides, or microinjecting antibodies to G proteins into megakaryocytes; 2: Determine the temporal sequence of expression and functional significance of selected cell cycle and growth regulated genes expressed during normal human megakaryocytopoiesis: Using in situ hybridization, the polymerase chain reaction, and antisense oligodeoxynucleotides for blockade of specific gene function we will begin to gather these data for genes of particular importance in megakaryocyte development; 3) Determine the molecular mechanisms which regulate megakaryocyte polyploidization: We hypothesize that megakaryocyte nuclear endoreduplication is the result of events which occur primarily during the G2/M interphase of the cell cycle. We will test this hypothesis by selectively perturbing events which occur during this phase of the cell cycle and determining if increased megakaryocyte polyploidy in response to defined stimuli (e.g. IL-3, IL-6) still occurs. We will also construct cDNA libraries from quiescent and growth factor stimulated megakarocytes to identify genes that might be unique, differentially expressed, or conversely not expressed at all, in stimulated cells; 4) Determine the mechanism whereby platelet factor 4 (PF4) inhibits megakaryocytopoiesis: We hypothesize that megakaryocytopoiesis is inhibited by PF4 through several possible mechanisms including: i) induction of c-myc and/or c-myb protooncogenes, ii) induction of transcription factors which may play a role in maturation and/or polyploidization (e.g. c-jun, c-fos, or other TPA responsive elements), or iii) impairment of response to growth factors. We will test these alternative hypotheses using the approaches described above. The availability of recombinant hematopoietic growth factors, improved culture systems, and molecular """"""""micromethodologies"""""""" now make these difficult and important questions amenable to study. Since megakaryocyte related thrombocytopenia remains a significant problem after cancer chemotherapy, bone marrow transplantation, and in some patients with acquired immunodeficiency syndrome (AIDS) this is a clinically important area of investigation.