Differentiation Response of Acute Promyelocytic Leukemia Cells and PML/RARalpha Leukemogenic Activity Studies by Real-Time RT-PCR

摘要

Acute promyelocytic leukemia (APL) is a human cancer generated by a chromosomal translocation t(15;17) involving the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes.The PML/RARalpha oncoprotein expressing blasts show two of the most important biological features of neoplastic progression:block of differentiation,at the promyelocytic state,and increased survival.Although PML/ RARa interferes with the normal maturation of myeloid precursors to granulocytes,pharmacological doses of retinoic acid are sufficient to restore the differentiation processes.We designed an assay based on the Real-Time reverse transcriptase polymerase chain reaction (RT-PCR) to experimentally follow the differentiation response of leukemic cells even after short-time differentiating treatments.Amplifying CDllb,CDllc,and CD14 mRNAs,as specific markers of differentiation,by the real-time RT-PCR assay we could detect both retinoic acid (RA) and vitamin D_3 and human transforming growth factor beta_1 (VitD_3/TGFbeta_1) induced cellular maturation more precociously than the canonical flow-cytofluorimetric assay.Moreover,by amplifying CD 14 mRNA it was possible to monitor the ability of PML/RARalpha oncoprotein to block VitD_3/TGFbeta_1 induced differentiation in U937-PR9 promonocytic induc-ible model systems.The proposed real-time quantitative RT-PCR approach is a reproducible and highly sensitive assay and can be considered a valid method to study both cellular maturation state and differentiation response.