Characterization of cell lines expressing novel PML/RARα mutants and their ATRA-binding activity.
(A) ATRA induces PML/RARα protein degradation in the ATRA-sensitive NB4 cells, but not in the ATRA-resistant NB4-MRA1 APL cells. Cells were treated with 1 μM ATRA for 24 hours, and 50 μg of nuclear extracts were used in Western blot analysis for the expression of PML/RARα protein. NB4 and NB4-MRA1 expressed a long PML/RARα (L) isoform (110 kDa). The lower panel shows laminin B expression to confirm protein loading. (B) Specific HPLC ATRA-binding profiles of nuclear extracts from NB4 cells (●) compared with those from the ATRA-resistant NB4-MRA1 (○) and UF-1 (▾) APL cells. Nuclear extracts were incubated with 10 nM [3H]-ATRA (●, ○, ▾) or with [3H]-ATRA in the presence of 200-fold excess of unlabeled ATRA (▿). Extracts were subjected to HPLC analysis using a 6 HR 10/30 size exclusion column.
