Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line.
BACKGROUND
Bcl-2 and Bcl-XL are anti-apoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these anti-apoptotic proteins in tumours is associated with poor prognosis. It has therefore been assumed that in cells the essential difference between Bcl-2 and Bcl-XL involves regulation of expression and that they are otherwise functionally similar. To examine this issue, we have compared the function of the proteins and of mutants of Bcl-2 and Bcl-XL specifically targeted to different subcellular sites.


METHODS
We generated clones of the human breast cancer line MCF-7 stably expressing known amounts of Bcl-2, or Bcl-XL as determined by quantitative immunoblotting. Clones expressing equivalent amounts of wild-type and mutants of Bcl-2 and Bcl-XL with subcellular localization restricted to the cytoplasm, endoplasmic reticulum or outer mitochondrial membrane were studied in both MCF-7 and Rat-1 fibroblasts. In MCF-7 cells we measured the functional activities of these proteins in preventing apoptosis induced by four different agents (doxorubicin, ceramide, thapsigargin, TNF-alpha). Etoposide and low serum were used to compare the effect of Bcl-2, Bcl-XL and mutants located at the endoplasmic reticulum on induction of apoptosis in fibroblasts.


RESULTS
We noted both qualitative and quantitative differences in the functional activity of these two anti-apoptotic proteins in cells: Bcl-2 localized to the endoplasmic reticulum inhibits apoptosis induced by ceramide and thapsigargin but not by doxorubicin or TNFalpha, while Bcl-XL at the endoplasmic reticulum is active against all four drugs. In fibroblasts Bcl-2 localized to the ER did not prevent cell death due to etoposide whereas Bcl-XL in the same location did. Finally in MCF-7 cells, Bcl-XL is approximately ten times more active than Bcl-2 in repressing apoptosis induced by doxorubicin. This difference can be manifest as a large difference in clonal survival.


CONCLUSIONS
When examined in the same cellular context, Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways.
Fiebig
Aline A
AA
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada. fiebiga@mcmaster.ca
Zhu
Weijia
W
Hollerbach
Catherine
C
Leber
Brian
B
Andrews
David W
DW
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein / Q000378:metabolism* / Q000502:physiology*
C494811:BCL2L1 protein, human; D050505:Mutant Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051020:bcl-X Protein; D004317:Doxorubicin