Differential changes of retinoid-X-receptor (RXR alpha) and its RAR alpha and PML-RAR alpha partners induced by retinoic acid and cAMP distinguish maturation sensitive and resistant t(15;17) promyelocytic leukemia NB4 cells.
The expression of retinoid receptors (RXRalpha, RARalpha and the chimeric form PML-RARalpha) was analysed both at the mRNA and protein level in the maturation sensitive NB4 and resistant NB4-R1 cell lines of t(15;17) promyelocytic leukemia (APL). All-trans RA and cAMP which show synergistic activity in inducing maturation of NB4 cells and maturation triggering of the RA 'primed' NB4-R1 resistant cells, distinctly modulate RXRalpha, RARalpha and PML-RARalpha mRNA. In the NB4 and NB4-R1 cells, RXRalpha mRNA was downregulated by RA, but only in RA-primed NB4-R1 cells a release from RXRalpha mRNA downregulation was obtained by cAMP treatment. RXRalpha protein (53 kDa) was decreased to the western-blot detection limit (97.5%) by RA in NB4 cells, but in NB4-R1 cells although it was frankly decreased (85%), the signal for RXRalpha protein remained very significant. More importantly, while cAMP slightly upregulated RXRalpha protein in RA-treated NB4 cells, it caused an increase of RXRalpha protein in RA-treated NB4-R1 cells bringing RXRalpha to the initial control level. RXRalpha partners in heterodimers (PML-RARalpha, RARalpha) were also analysed. In contrast to RXRalpha, RARalpha and PML-RARalpha mRNA were not modulated by RA and/or cAMP, while significant changes were observed at the protein levels. A putatively phosphorylated form of RARalpha (52 kDa) decreased during maturation of NB4 cells, but was unchanged in resistant NB4-R1 cells. Conversely, while PML-RARalpha remained stable during RA-induced NB4 maturation, RA treatment which failed to induce maturation of NB4-R1 cells significantly down-regulated the chimeric receptor (120 kDa). These differences most likely results from translational and post-translational regulation. This work reveals complex pattern of subtle changes at the protein level distinguishing RA-sensitive and RA-resistant cells. Our data show that the RA-cAMP synergistic effect on NB4 cell maturation and cooperation in triggering maturation of RA-primed NB4-R1 cells operate changes in the RXR/PML-RARalpha ratio which are both favouring RXRalpha. In both cell lines, variations of PML-RARalpha and RXRalpha may result in a decrease in the formation of the PML-RARalpha/RXRalpha heterodimers which are supposed involved in the block of maturation. This may prove crucial to embark cells on maturation.
Duprez
E
E
INSERM U-301, Génétique Moléculaire et Cellulaire des Leucémies, Centre G. Hayem, Hôpital Saint-Louis, Paris, France.
Lillehaug
J R
JR
Gaub
M P
MP
Lanotte
M
M
D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology*; D015536:Down-Regulation; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C000607071:RARA protein, human; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin; D000242:Cyclic AMP