DISTINCT APOPTOTIC RESPONSES IN MATURATION SENSITIVE AND RESISTANT T(15-17) ACUTE PROMYELOCYTIC LEUKEMIA NB4 CELLS - 9-CIS RETINOIC ACID INDUCES APOPTOSIS INDEPENDENT OF MATURATION AND BCL-2 EXPRESSION

Apoptosis has been investigated in NB4, a t(15;17) human promyelocytic leukemia cell line susceptible to maturation by all-trans or g-cis re tinoic acid, and in NB4-R1, a subclone resistant to differentiation, M aturation resistant NB4-R1 cells exhibited an onset of cell death afte r RA-treatment (72 h), whereas maturation responsive NB4 cells showed no such apoptosis, cell death being considerably delayed after cell ma turation. Only a few NB4-R1 cells underwent apoptosis in response to l ow doses of RA (below 0.1 mu M), the surviving cells became refractory to higher doses of RA. While these cells became 'resistant' to apopto sis they became competent for maturation. Typically, these RA-'primed' cells responded to cAMP by maturation, then apoptosis followed rapidl y. This model furnishes situations where cells are either resistant or susceptible to apoptosis, depending on whether they can or cannot und ergo maturation, The potential role of the Bcl-2 protein in the regula tion of apoptosis was analyzed. In NB4 and NB4-R1 cell lines, a high e xpression of the Bcl-2 protein was detected by immunocytology and West ern blotting. NB4 cells treated with either all-trans or g-cis retinoi c acid (1 mu M) were induced to differentiate and the level of Bcl-2 p rotein decreased to undetectable levels during terminal maturation whe n only a few apoptotic cells were detected, In NB4-R1 cells, while tre atment with retinoids does not induce maturation, as much as 64% of ce lls became apoptotic, and immunocytological labelling of NB4-R1 showed a strong cytoplasmic labelling of Bcl-2, Although the expression of B cl-2 remained high, cells were not protected from apoptosis. To assess whether Bcl-2 expression could be modulated as a consequence of diffe rentiation, NB4-R1 cells previously 'primed' for maturation were trigg ered with cAMP, Downregulation of Bcl-2 protein occurred concomitant w ith maturation, followed by apoptosis. Clearly, NB4 and NB4-R1 cells s how reciprocal behavior with regards to proliferation, maturation, Bcl -2 regulation and apoptosis in response to RA. Our results suggest, fi rst, that the Bcl-2 downregulation in NB4 cells belongs to the maturat ion program rather than to apoptosis, and second, that neither a high Bcl-2 expression in NB4 cells is sufficient to protect cells from g-ci s RA induced apoptosis, nor is its full downregulation sufficient to p roduce apoptosis, Finally, this work suggests that apoptosis and matur ation programs include events which cannot occur simultaneously,