Heterogeneous apoptotic responses of prostate cancer cell lines identify an association between sensitivity to staurosporine-induced apoptosis, expression of Bcl-2 family members, and caspase activation.
BACKGROUND
The goal of this work was to identify mechanisms for the inability of metastatic prostate cancer cells to engage the apoptotic pathway following hormonal or cytotoxic therapy.


METHODS
Genotypically diverse cell lines isolated from patients with metastatic disease were used.


RESULTS
The LNCaP and TsuPr(1) lines exhibited quintessential apoptotic features in response to the pleiotropic apoptotic inducer staurosporine (STS): rapid cytochrome c translocation to the cytosol, proteolytic processing and catalytic activation of caspase-3 and -7, proteolytic inactivation of the death substrates DNA fragmentation factor (DFF) and poly-ADP-ribose polymerase (PARP), and TUNEL-positive polyfragmented nuclei. In contrast, DU-145 and PC-3 cells exhibited few, if any, of these features, while appearing necrotic by confocal microscopy. The presence of caspase-3 and -7 without proteolytic processing suggested that the apoptotic blockade was upstream of executioner caspases in these resistant cell lines. To identify the locus of this block, Western blot analysis of cytochrome c subcellular localization and of pro- and antiapoptotic Bcl-2 family members was performed, and suggested that heterogeneous expression of these proteins might be the underlying mechanism for apoptotic resistance to STS in these cell lines. Thus, the absence of the proapoptotic Bax in DU-145 cells indicated a mechanism for apoptotic resistance of these cells. Similarly, decreased Bax expression during STS treatment, coupled with overexpression of the antiapoptotic Bcl-x(L) and inability to translocate cytochrome c to the cytosol, provided a mechanism for the insensitivity of PC-3 cells.


CONCLUSIONS
These observations suggest that activation of the apoptotic machinery in metastatic prostate cancer cell lines may be determined by expression levels of Bcl-2 family members, by the ability of cytochrome c to translocate to the cytosol, and by the ability of the caspase pathway to react in response to activation of the mitochondrial phase.
Marcelli
M
M
Departments of Medicine, Molecular and Cellular Biology, Veterans Administration Medical Center and Baylor College of Medicine, Houston, Texas 77030, USA. marcelli@bcm.tmc.edu
Marani
M
M
Li
X
X
Sturgis
L
L
Haidacher
S J
SJ
Trial
J A
JA
Mannucci
R
R
Nicoletti
I
I
Denner
L
L
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001692:Biological Transport; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism*; D002384:Catalysis; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007425:Intracellular Membranes / Q000502:physiology; D008297:Male; D008928:Mitochondria / Q000502:physiology; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D019311:Staurosporine / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D059005:Topoisomerase II Inhibitors; D051028:bcl-2-Associated X Protein; C109664:caspase-activated DNase inhibitor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D019311:Staurosporine