The Applicant's central question is: In hematopoiesis what molecular events control the transition from cycling myeloid precursor cells to noncycling terminally differentiated cells? The PI hypothesizes that the cyclin dependent kinase inhibitor p21(WAFl CIPI) is responsible for this transition. The p21 prevents cells from progressing into S phase of the cell cycle, and mediates G1 arrest in response to p53 activation or other environmental cues. Numerous studies have confirmed the association between differentiation commitment and arrest in Gl phase of the cell cycle, but mechanisms of the linkage are incompletely understood. Based on the PI's observation that p21 is induced in immediate early fashion by myeloid differentiation inducers in cell lines, the PI postulates that p21 mediates cell cycle exit during myeloid differentiation.
Specific aims of this proposal are: 1. To determine the stage specific expression of p21 in myelopoiesis and define the relationship of p21 expression to cell cycle stage and to the presence of CD11b, CD14, and CD15 antigen expression. This set of experiments will determine the normal variability of p21 levels as CFU GM cells differentiate and leave the mitotic compartment. Two channel FACScan analysis relating p21 expression to differentiation markers and to cell cycle status will be performed, and p21 message and protein will be measured in CD34+ cells under conditions promoting temporally coordinated granulocytic differentiation. 2. To uncover the effects of negative modulation of p21 activity on initiation and completion of the differentiation process. These experiments will address the questions: Is p21 expression required for the induction and maintenance of terminally differentiated cells? Antisense reagents and p21 mutants capable of abolishing endogenous p21 activity will be generated. The results of p21 downmodulation on myelopoiesis will be investigated first in HL60 cells. Experiments will then establish the effects of suppression of p21 on normal myeloid differentiation of CD34+ precursor cells in colony forming assays. Functional effects of p21 suppression on postmitotic differentiation also will be determined. 3. To determine whether forced expression of p21 enhances differentiation of murine and human myeloid progenitor cells; and to assess genetic and functional effects of forced expression of p21 in oncogene transformed myeloid progenitor cells exhibiting differentiation blockade. The p21 gene will be expressed aberrantly in myeloid cell lines using constitutive and inducible expression vectors, and in differentiating CD34+ cells using the MFG retroviral vector. Colony assays and FACScan analysis of differentiation markers will determine whether aberrant expression of p21 arrests cycling progenitor cells, accelerates differentiation, or otherwise disrupts normal myelopoiesis. Finally, the ability of p21 to suppress the growth and/or to permit differentiation of oncogene transformed cells will be determined. A clear understanding of p21 function in normal myelopoiesis will establish a context for determining if disruption of p21 function contributes to myelodysplastic syndrome or myeloid leukemias.
| Steinman, R A; Johnson, D E (2000) p21WAF1 prevents down-modulation of the apoptotic inhibitor protein c-IAP1 and inhibits leukemic apoptosis. Mol Med 6:736-49 |
| Steinman, R A; Yaroslavskiy, B; Kaplan, S S et al. (2000) Clonal response of K562 leukemic cells to exogenous p21WAF1. Leuk Res 24:601-10 |
| Yaroslavskiy, B; Watkins, S; Donnenberg, A D et al. (1999) Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells. Blood 93:2907-17 |
| Steinman, R A; Huang, J; Yaroslavskiy, B et al. (1998) Regulation of p21(WAF1) expression during normal myeloid differentiation. Blood 91:4531-42 |
