The long-term goals of this proposal are to identify the molecular and biochemical control mechanisms operative during the polyploidization of megakaryocytes. In order to examine such questions, we developed a model system in which phorbol-diester (e.g. PMA) stimulated human erythroleukemia (HEL) cells undergo megakaryocyte differentiation (JCI 85:l084, 1989). As a result, these cells become endomitotic resulting in reproducible polyploidy during phorbol-diester induced megakaryocyte differentiation. We hypothesize that two control points exist at which the megakaryocyte cell cycle must be altered in order to achieve polyploidization: (1) prolongation (or repetition) of the period of DNA synthesis, and (2) inhibition of the events leading to mitosis (i.e,. nuclear division) and subsequent cytokinesis. This proposal focuses on the biochemical and molecular alterations in cell-cycle associated proteins involved with these control points. Specifically, we well examine the subunit structure, post-translational modifications, and alterations in function of cyclin-dependent kinase (CDK) complexes in normally proliferating and endomitotic cells. These studies use purified subpopulations of cells isolated by cell cycle status. Control cells consisting of non-induced HEL cells as well as PMA-resistant HEL cells will be separated into G0/G1, S-phase, and G2/M DNA content and compared to endomitotic cells of 2C, 4C, 8C, and 16C DNA content (where 2C is the G0/G1 DNA content). The CDK complex(es) is examined for alterations in subunit structure and function using protein-affinity and immunoprecipitation. Cell cycle specific kinase(s) operative during endomitosis and their association with new members of the cyclin gene family will be identified. Altered control over kinase and cyclin gene expression will be determined by Northern analysis of the relative abundance, transcription rate, and half-life of specific mRNAs. Another set of investigations will explore the role of specific kinase and cyclin genes in causing, or predisposing cells to endomitosis. In these studies, specific genes will be transfected into HEL cells using vectors containing inducible promoters thus allowing precise and selective control over expression. Finally, the information derived from this model will be applied to normal, human bone marrow megakaryocytes. The studies in this proposal are focused on examining the differences in normal cell cycle control and a novel form of DNA synthesis (endomitosis) in which DNA is replicated multiple times within a single nucleus. As such, they focus on an early event in megakaryocyte differentiation: the acquisition of a polyploid nucleus. Also, the detailed biochemical and molecular studies also should generate new and, perhaps novel, data on the general control of cell cycle events, particularly S-phase.
