MUTANT AF-2 DOMAIN OF PML-RAR-ALPHA IN RETINOIC ACID-RESISTANT NB4 CELLS - DIFFERENTIATION-INDUCED BY RA IS TRIGGERED DIRECTLY THROUGH PML-RAR-ALPHA AND ITS DOWN-REGULATION IN ACUTE PROMYELOCYTIC LEUKEMIA

To study the molecular mechanism of the differentiation induced by ret inoic acid (RA) in acute promyelocytic leukemia (APL), we established a new HA-resistant NB4 subline, NB4/RA. The NB4/RA cells were neither differentiated by a single or a combination of RA isoforms, nor by the addition of clotrimazole (P450-inhibitor) or interferon gamma. Howeve r, the combination of RA and 8-(4 chlorophenylthio) adenosine cyclic 3 ',5'-monophosphate (a cAMP analog, 8-CPT-cAMP) induced differentiation . Immunostaining of NB4/RA cells using anti-PML antibody showed a micr ogranular pattern which was not restored even by the combination of RA and 8-CPT-cAMP, whereas the microgranular pattern in NB4 cells was ra pidly restored to the normal speckled pattern by RA, Western blot anal ysis revealed that RA alone or the combination with 8-CPT-cAMP did not down-regulate PML-RAR alpha in NB4/RA cells, which was in contrast to NB4 cells. The PML-RAR alpha fusion gene and transcript in NB4/RA cel ls were conserved as well as the RAR alpha gene and transcripts. Seque nce analysis of the PML-RAR alpha transcript in NB4/RA cells indicated a Pro (CCC) to Leu (CTG) mutation at codon 900 (type L) in AF-2 domai n, while the RAR alpha transcript had a normal sequence. These data su ggest that differentiation of APL by RA is triggered directly through PML-RAR alpha, and is associated with its degradation. Furthermore, th ere might be another mechanism of differentiation which does not requi re the down-regulation of PML-RAR alpha and the restoration of the PML -staining pattern.