A Positive Effect of p21cip1/waf1in the Colony Formation from Murine Myeloid Progenitor Cells as Assessed by Retroviral-Mediated Gene Transfer

Abstract

ABSTRACT: p21cip1/waf1is a cyclin dependent kinase inhibitor. We have previously reported stimulation of p21cip1/waf1by steel factor and GM-CSF in a factor dependent cell line and of p21cip1/waf1involvement in hematopoiesisin vivoin p21cip1/waf1gene knockout (-/-) mice. To further assess a role for increased p21cip1/waf1in hematopoietic progenitor cells, we developed the retroviral vector L(p21cip1)SN to transcriptionally regulate p21cip1/waf1from the Mo-MLV LTR. L(p21cip1)SN and the control vector LXSN were used to transduce murine bone marrow progenitor cells from p21cip1/waf1(-/-) and littermate control (+/+) mice, as well as from other mouse strains. Hematopoietic colony formation by transduced cells was assessed in semi-solid culture medium with multiple growth factors. Myeloid colony formation by bone marrow cells from p21cip1/waf1(-/-) mice was significantly lower than that by (+/+) mouse cells. Transduction of cells with LXSN had no effect on colony formation; however, (-/-) cells transduced with L(p21cip1)SN formed significantly greater numbers of colonies than either LXSN-transduced (-/-) or (+/+) cells. Moreover, L(p21cip1)SN-transduced (+/+) cells formed significantly more colonies than LXSN-transduced (+/+) cells. Increased cloning efficiency of progenitors from normal strains of mice induced by L(p21cip1)SN compared to LXSN-transduced cells was seen whether unseparated or highly purified populations of Sca1+Lin-marrow cells were used. Gene transfer of L(p21cip1)SN increased the size and number of cells per colony, as well as the number of colonies compared to LXSN gene transfer. No colonies grew from non-transduced, LXSN-, or L(p21cip1)SN-transduced cells when no growth factors were added to the cultures. These results document the positive effect of p21cip1/waf1in the proliferation and/or differentiation of the murine myeloid progenitor cells that lead to colony formation.