Differentiation response of acute promyelocytic leukemia cells and PML/RAR alpha leukemogenic activity studies by real-time RT-PCR

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 (RAR alpha) genes. The PML/RAR alpha 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/ RAR alpha. 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 CD11b, CD11c, 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)/TGF beta(1)) induced cellular maturation more precociously than the canonical flow-cytofluorimetric assay. Moreover, by amplifying CD14 mRNA it was possible to monitor the ability of PML/RARa oncoprotein to block VitD(3)/TGF beta(1) induced differentiation in U937-PR9 promonocytic inducible 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.