Expression of P21(WAF1/CIP1/SID1) cyclin-dependent kinase inhibitor in hematopoietic progenitor cells

P21(Waf1/Cip1/Sid1) is a critical component of biomolecular pathways leadin g to the G(1) arrest evoked in response to DNA damage, growth arrest signal s and differentiation commitment. It belongs to the Cip/Kip class of cyclin -dependent kinase inhibitors and is at least partly regulated by p53. P21(W af1/Cip1/Sid1) functional inactivation possibly resulting from mutations of the gene, itself or, more likely, from p53 mutations may be critical for e ither the cell fate following DNA-damaging insults or clonal evolution towa rd malignancy. In the study presented here we describe a competitive polyme rase chain reaction (PCR) strategy whose sensitivity and reproducibility en able us to attain a precise quantitation of p21(Waf1/Cip1/Sid1) expression levels in hematopoietic progenitors, the cell compartment which mostly suff ers from the side effects of genotoxic drugs in use for cancer cure. The st rategy was set in the M07 factor-dependent hematopoietic progenitor cell li ne. We confirmed that its p21(waf1/cip1/sid1). constitutive expression leve l is very low and up-modulated by DNA-damaging agents: ionizing radiations and ultraviolet light. Gene up-modulation resulted in checkpoint activation and, in particular, in a significant G(1) arrest, required for either the repair of damaged DNA sequences or apoptotic cell death. Our competitive PC R strategy was further validated in CD34(+) purified hematopoietic progenit ors from healthy donors mobilized into the peripheral blood by granulocyte colony-stimulating factor and intended for allogeneic bone marrow transplan tation. The constitutive p21(WAF1/CIP1/SID1) expression levels, measured in three separate harvests, were very low and no significant differences were apparent. Our results support the use of a competitive PCR strategy as a u seful tool for clinical purposes, to assess the individual biomolecular res ponse of early hematopoietic progenitors to antiblastic drugs. (C) 2001 Els evier Science B.V. All rights reserved.