High levels of p26BCL-2 oncoprotein retard taxol-induced apoptosis in human pre-B leukemia cells.
In human leukemic cells clinically relevant concentrations of taxol have been demonstrated to induce the biochemical and morphologic hallmarks of apoptosis (Leukemia 1993;7:563-568). Since overexpression of the bcl-2 gene has been reported to retard apoptosis due to a variety of anticancer agents, we examined and compared taxol-induced intracellular microtubular bundling and apoptosis in pre-B human leukemia 697 cells and their counterparts which have been transfected with and overexpress cDNA derived from the bcl-2 gene. Treatment with 0.1 or 1.0 mumol/l taxol for 24 h resulted in internucleosomal DNA fragmentation and morphologic features of apoptosis in 697 cells, but not in 697/BCL-2 cells. However, indirect immunofluorescent staining with anti-tubulin antibody revealed that taxol treatment produces stable microtubule bundles resistant to calcium-mediated disassembly in 697, as well as 697/BCL-2 cells. In addition, taxol-induced microtubule bundling was associated with a marked accumulation of the two cell types in the G2/M phase of the cell cycle. Following exposure to taxol, when 697 cells were washed and kept in drug-free medium, they showed rapid onset of apoptosis followed by loss of cell viability and a decline in cell numbers. In contrast, identically treated 697/BCL-2 cells kept in drug-free medium remained in a growth arrested state, but showed little evidence of apoptosis for up to 4 days. They eventually demonstrated features of apoptotic cell death and loss of viability between 5 and 7 days. This was not accompanied by a decrease in p26BCL-2 levels. Anti-phosphotyrosine or anti-MAP kinase immunoblot analyses of proteins isolated from taxol-treated 697 and 697/BCL-2 cells failed to show any difference in tyrosine phosphorylation of cellular proteins. Therefore, our findings indicate that in 697/BCL-2 cells, high levels of p26BCL-2 significantly delay taxol-induced endonucleolytic internucleosomal DNA fragmentation and apoptosis, but do not affect taxol-induced microtubule bundling or cell cycle growth arrest. The delayed onset of taxol-induced DNA fragmentation and apoptosis in 697/BCL-2 cells without down-regulation of p26BCL-2 levels suggests that an alternative mechanism of taxol-mediated apoptosis might be triggered which is unimpeded by high p26BCL-2 levels, or taxol-induced prolongation of mitotic arrest may lead to the inactivation or inhibition of that mechanism by which p26BCL-2 is able to block apoptosis.
Tang
C
C
Department of Medicine, Medical University of South Carolina, Charleston 29425.
Willingham
M C
MC
Reed
J C
JC
Miyashita
T
T
Ray
S
S
Ponnathpur
V
V
Huang
Y
Y
Mahoney
M E
ME
Bullock
G
G
Bhalla
K
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis; D066298:In Vitro Techniques; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure; D017239:Paclitaxel / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004273:DNA, Neoplasm; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014404:Tubulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017239:Paclitaxel