Differentiation response of acute promyelocytic leukemia cells and PML/RARa 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 (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/RARalpha 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 D3 and human transforming growth factor beta1 (VitD3/TGFbeta1) 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/RARalpha oncoprotein to block VitD3/TGFbeta1 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.
Center of Biotechnology, University of Urbino, Fano, Italy.
Sara
S
Caprodossi
Marika
M
Pedinotti
Consuelo
C
Amantini
Giorgio
G
Santoni
Saverio
S
Minucci
Pier Giuseppe
PG
Pelicci
Mirco
M
Fanelli
D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002762:Cholecalciferol / Q000494:pharmacology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D000071156:Promyelocytic Leukemia Protein; D011506:Proteins / Q000494:pharmacology; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D000072482:Retinoic Acid Receptor alpha; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics*; D014212:Tretinoin / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011506:Proteins; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D002762:Cholecalciferol; D014212:Tretinoin