P21Waf1/Cip1/Sid1 is a critical component of biomolecular pathways leading to the G1 arrest evoked in response to DNA damage, growth arrest signals and differentiation commitment. It belongs to the Cip/Kip class of cyclin-dependent kinase inhibitors and is at least partly regulated by p53. P21Waf1/Cip1/Sid1 functional inactivation possibly resulting from mutations of the gene itself or, more likely, from p53 mutations may be critical for either the cell fate following DNA-damaging insults or clonal evolution toward malignancy. In the study presented here we describe a competitive polymerase chain reaction (PCR) strategy whose sensitivity and reproducibility enable us to attain a precise quantitation of p21Waf1/Cip1/Sid1 expression levels in hematopoietic progenitors, the cell compartment which mostly suffers from the side effects of genotoxic drugs in use for cancer cure. The strategy was set in the M07 factor-dependent hematopoietic progenitor cell line. We confirmed that its p21waf1/cip1/sid1 constitutive expression level 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 G1 arrest, required for either the repair of damaged DNA sequences or apoptotic cell death. Our competitive PCR strategy was further validated in CD34+ purified hematopoietic progenitors from healthy donors mobilized into the peripheral blood by granulocyte colony-stimulating factor and intended for allogeneic bone marrow transplantation. The constitutive p21WAF1/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 useful tool for clinical purposes, to assess the individual biomolecular response of early hematopoietic progenitors to antiblastic drugs.
- CD34 hematopoietic progenitors
- Cell cycle regulation
- Competitive polymerase chain reaction
- DNA damage
- Peripheral blood stem cell mobilization
ASJC Scopus subject areas