Platelets are produced in the bone marrow by an unusual precursor cell, the megakaryocyte, which is characterized by its rarity in the bone marrow, large size and a polyploid nucleus that results from endomitosis. This later process is lacking in many of the leukemia cell lines that possess megakaryocytic markers. The molecular analysis of cell cycle factors that regulate endomitosis in megakaryocytes has not yet been performed. The objective of the proposed study is to define important gene products committing megakaryocytes to enter S phase and skip mitosis, thus leading to the formation of polyploid cells. The study will be guided by investigations in yeasts and human cells that revealed different cyclins controlling DNA synthesis and mitosis during the cell cycle of diploid (2N) cells. For the purpose of the proposed study, we will aim to develop immortal megakaryocytic cell lines that can be triggered to undergo a complete process of endomitosis and maturation, forming 8N to 64N cells. One approach will involve infection of lipoid megakaryocytes with retrovirus vector containing genes encoding and oncogene and a growth factor receptor. We will also target expression of a conditional allele of myc oncogene to megakaryocytes in transgenic mice, via, the megakaryocyte-specific platelet factor 4 (PF4) promoter. Preliminary studies involving targeted expression of the conditional allele simian virus 40 large T antigen into megakaryocytes of transgenic mice have yielded immortal megakaryocytes (MegT37) that can be induced to become 8N and to a smaller extent 16N cells. These cells as well as primary bone marrow megakaryocytes will be used to determine whether the DNA replication int he polyploid cells is continuous or undergoes a G1/S cycle. MegT37 cells and subsequently other newly developed lines will be used to isolated cDNAs encoding cell cycle regulatory proteins induced in polyploid cells, either by substraction hybridization, using mRNA from lipoid and polyploid cells, or by amplifying mRNA by the polymerase chain reaction. The distribution of the potentially novel cell cycle regulatory factor(s) and of megakaryocytic cyclins homologs to yeast/human cyclins will be compared in diploid and polyploid mouse megakaryocytes at different stages of the cell cycle. The confirmation of the new regulator protein(s) will be done by testing for kinase activity, binding to p34cdc2 kinase and by complementation of cell cycle mutations in yeast. The role of different cyclins in promoting normal endomitosis or cellular transformation will be evaluated by creating separate megakaryocytic cell lines with defined cyclins, either by overexpression or by suppression with antisense oligonucleotides, via the PF4 promoter, by transfection experiments in-vitro and in a transgenic mouse model. These interrelated projects should projects should yield novel information shading light on the genetic mechanisms controlling normal endomitosis or abnormal megakaryocyte proliferation, events that determine platelet production.
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