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
K. Kitamura et al., 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, Leukemia, 11(11), 1997, pp. 1950-1956
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.