Grant Number :    1Z01AG000910-02

Pricipal Investigator : Westin Eric

Project Title : Mechanisms Controlling Hematopoietic Cell Proliferation

Abstract : The program of normal human hematopoietic cell proliferation and differentiation occurs through a complex interplay of circulating and locally secreted growth factors as well as interaction of hematopoietic stem cells with the stroma cell micro-environment. For a variety of cell lineages, it is a process of continuous cell renewal that appears to be minimally affected by normal aging and a process that is disrupted in malignancies of the hematopoietic system. Therefore, insights gained from studies of the control of this process may contribute to both our understanding of apparent exceptions to the aging process as well as events critical in the genesis of leukemias. HL-60 is a human myeloid leukemia cell line that does not harbor the translocations characteristic of acute promyelocytic leukemia and yet undergoes terminal granulocytic and monocytic differentiation in response to a variety of agents including retinoids and vitamin D3 as well as DMSO and phorbol esters. The differentiation occurs through a sequence of both early and late changes in gene expression with a commitment to terminal differentiation occurring at the 48-72 hour exposure window for most agents with differentiation complete by 144 hours. This model has provided multiple insights into both pathogenesis of acute leukemia as well as therapy and is a novel model in which commitment to irreversible withdrawal from cell cycle can be examined. CDNA microarray technology has been applied to examine sequential changes in gene expression in HL-60 during terminal differentiation induced by DMSO. From this work, expression patterns have been identified that are both early (prior to withdrawal from cell cycle) and late (during the development of differentiated features and cell cycle withdrawal) as well as unique to the commitment phase at 48-72 hours. Findings from initial studies validate the use of this technique as a general approach for comparison of different differentiation pathways to identify changes specific to each inducing agent. They have also highlighted a number of specific changes at the commitment stage that can be examined mechanistically using molecular or pharmacologic approaches. These include novel changes in BRCA-1 expression as well as changes in defensin expression (recently associated with resistance to HIV disease progression) and changes in the specific induction of inducible NOS at the commitment step which may interplay with other changes within the cell to lead to commitment to terminal differentiation. Using the approach of synchronization of cells by centrifical elutriation, we have also demonstrated previously unknown cell cycle regulation of c-myb but not c-myc during the G1 phase of the cell cycle which may explain the ability of HL-60 cells to undergo differentiation in the absence of proliferation during the G1 phase of the cell cycle. Transfection of a c-myb constitutive expression construct has also been acomplished to permit dissection of the specific role of c-myb in proliferation and differentiation. Preliminary studies are underway with early results indicating suppression of the endogenous 95 kd alternatively spliced form of c-myb occurs specifically in cells expressing the c-myb 72 kd construct. Experiments examining differentiation and cell cycle effects are in progress. From early results, it is clear that cDNA microarray and other approaches will provide powerful approaches to increasing our understanding of both hematopoietic differentiation as well as the critical events surrounding the commitment step at which cells within the hematopoietic system are no longer able to divide, mimicking cellular aging. Results such as those with NOS may also provide directly testable hypotheses which are likely to not only improve our understanding of the differentiation process but may also prove useful in therapy.

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