Related Experiment Videos
Versatility of BCR/ABL-expressing leukemic cells in circumventing proapoptotic BAD effects
P Salomoni1, F Condorelli, S M Sweeney
1Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Abstract:
BAD, the proapoptotic member of the "BH3-only" subfamily of BCL-2 proteins, is inactivated by phosphorylation at serines 112 and 136 and by sequestration in the cytoplasm where it interacts with members of the 14-3-3 family. In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and mainly cytoplasmic, whereas in cells expressing BCR/ABL mutants unable to protect from apoptosis, BAD is nonphosphorylated. We show here that both the wild-type (WT) and the S112A/ S136A double mutant (DM) BAD are more potent inducers of apoptosis in parental than in BCR/ABL-expressing 32D myeloid precursor cells. Stable lines of parental cells expressing DM BAD could not be established and most clones from WT BAD retrovirus-infected parental cells lost BAD expression. On IL-3 withdrawal from parental 32D cells, BAD was rapidly dephosphorylated by the serine-threonine phosphatase 1 alpha, and localized in the mitochondria, whereas it remained phosphorylated and did not localize to the mitochondria in the cohort of BCR/ABL-expressing cells escaping apoptosis induced by WT BAD. Moreover, these cells showed high levels of BCL-2 and BCL-X(L) expression. The cohort of BCR/ABL-expressing cells resistant to apoptosis induced by DM BAD showed only high levels of BCL-2 and BCL-X(L). These findings suggest that BCR/ABL-expressing cells are more versatile than normal hematopoietic progenitors in counteracting the apoptotic potential of BAD, and raise the possibility that tumor cells activate multiple antiapoptotic pathways for survival in the face of death-inducing stimuli. (Blood. 2000;96:676-684)
Insights
BCR/ABL-expressing cells resist apoptosis by keeping BAD protein phosphorylated and in the cytoplasm, unlike normal cells where BAD triggers cell death. Tumor cells use multiple anti-apoptotic pathways for survival.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Biology
Background:
- The proapoptotic protein BAD, a BH3-only member of the BCL-2 family, is regulated by phosphorylation and cytoplasmic sequestration.
- In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and localized in the cytoplasm, contributing to resistance to apoptosis.
Purpose of the Study:
- To investigate the mechanisms by which BCR/ABL-expressing cells counteract the proapoptotic function of BAD.
- To compare the apoptotic potential of wild-type (WT) BAD and a double mutant (DM) BAD (S112A/S136A) in parental and BCR/ABL-expressing cells.
Main Methods:
- Utilized 32D myeloid precursor cells, both parental and BCR/ABL-expressing.
- Introduced WT BAD and DM BAD into these cells via retroviral infection.
- Assessed apoptosis induction, BAD phosphorylation status, subcellular localization, and expression of BCL-2 and BCL-X(L) following IL-3 withdrawal.
Main Results:
- Both WT BAD and DM BAD were less potent inducers of apoptosis in BCR/ABL-expressing cells compared to parental cells.
- Parental cells showed rapid dephosphorylation and mitochondrial localization of BAD upon IL-3 withdrawal, while BCR/ABL-expressing cells maintained phosphorylated BAD in the cytoplasm.
- BCR/ABL-expressing cells resistant to BAD-induced apoptosis exhibited high levels of BCL-2 and BCL-X(L) expression.
Conclusions:
- BCR/ABL-expressing cells possess enhanced mechanisms to resist BAD-mediated apoptosis compared to normal hematopoietic progenitors.
- Tumor cells may employ multiple anti-apoptotic pathways to ensure survival against death-inducing stimuli.