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 9-cis retinoic acid, and in NB4-R1, a subclone resistant to differentiation. Maturation resistant NB4-R1 cells exhibited an onset of cell death after RA-treatment (72 h), whereas maturation responsive NB4 cells showed no such apoptosis, cell death being considerably delayed after cell maturation. Only a few NB4-R1 cells underwent apoptosis in response to low doses of RA (below 0.1 microM), the surviving cells became refractory to higher doses of RA. While these cells became 'resistant' to apoptosis they became competent for maturation. Typically, these RA-'primed' cells responded to cAMP by maturation, then apoptosis followed rapidly. This model furnishes situations where cells are either resistant or susceptible to apoptosis, depending on whether they can or cannot undergo maturation. The potential role of the Bcl-2 protein in the regulation of apoptosis was analyzed. In NB4 and NB4-R1 cell lines, a high expression of the Bcl-2 protein was detected by immunocytology and Western blotting. NB4 cells treated with either all-trans or 9-cis retinoic acid (1 microM) were induced to differentiate and the level of Bcl-2 protein decreased to undetectable levels during terminal maturation when only a few apoptotic cells were detected. In NB4-R1 cells, while treatment with retinoids does not induce maturation, as much as 64% of cells became apoptotic, and immunocytological labelling of NB4-R1 showed a strong cytoplasmic labelling of Bcl-2. Although the expression of Bcl-2 remained high, cells were not protected from apoptosis. To assess whether Bcl-2 expression could be modulated as a consequence of differentiation, NB4-R1 cells previously 'primed' for maturation were triggered with cAMP. Downregulation of Bcl-2 protein occurred concomitant with maturation, followed by apoptosis. Clearly, NB4 and NB4-R1 cells show reciprocal behavior with regards to proliferation, maturation, Bcl-2 regulation and apoptosis in response to RA. Our results suggest, first, that the Bcl-2 downregulation in NB4 cells belongs to the maturation program rather than to apoptosis, and second, that neither a high Bcl-2 expression in NB4 cells is sufficient to protect cells from 9-cis RA induced apoptosis, nor is its full downregulation sufficient to produce apoptosis. Finally, this work suggests that apoptosis and maturation programs include events which cannot occur simultaneously.
Bruel
A
A
INSERM U-301, Centre G Hayem, Hôpital Saint-Louis, Paris, France.
Benoit
G
G
De Nay
D
D
Brown
S
S
Lanotte
M
M
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology; D015536:Down-Regulation; D004351:Drug Resistance; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014212:Tretinoin; D000242:Cyclic AMP