Comparative analysis of caspase activation and apoptosis in renal tubular epithelial cells and renal cell carcinomas.
OBJECTIVE
Treatment of renal cell carcinoma (RCC) is limited by its resistance to conventional chemotherapies. This may occur, in part, from resistance to apoptosis. The role of caspase activation in apoptosis resistance in treated RCCs was investigated.


METHODS
Two human RCC cell lines (ACHN and SN12K1) and renal tubular epithelial cells (HK2) were treated with 5-fluorouracil (0.2-20 microg/ml) or cisplatin (1-100 microM). Activation of caspase-3 and -2 was analysed and compared with levels of apoptosis. Caspase function was analysed using pan-caspase inhibition (z-VAD-fmk) and caspase-2 inhibition (z-VDVAD-fmk).


RESULTS
RCC apoptosis was significantly lower (p < 0.05) than in HK2s after treatment, confirming their chemoresistance. Pro-caspase-3 (32 kDa) was detected in all cell lines. Cleaved caspase-3 (19 kDa) was not detected by Western immunoblots in treated RCCs and only minimal activated caspase-3 was detected in treated RCCs using immunohistochemistry. All cells had pro-caspase-2 (48 kDa) and the activated form (33 kDa) appeared in all treated cells. Caspase inhibition caused a reduction in, but not negation of, therapy-induced apoptosis in HK2s and RCCs (p < 0.05 for HK2s and ACHN cells), indicating that a caspase activation pathway must occur in RCC apoptosis but this pathway does not act via caspase-3 cleavage. Inhibition of caspase-2 reduced apoptosis only in HK2s, indicating that the activated caspase-2, identified in treated RCCs, was not responsible for their apoptosis induction.


CONCLUSIONS
Specific differences in caspase-3 and -2 activation were identified in renal tubular epithelium and RCCs after chemotherapy. Identification of RCC-specific caspase inactivation or redundancy may explain, in part, the resistance of RCCs to cancer therapies and may be useful in targeting apoptotic pathways to overcome RCC resistance to treatment.
Davidson
Keryn
K
Molecular and Cellular Pathology, School of Medicine, University of Queensland, Herston, Australia.
Percy
Christine
C
Rennick
Alina J
AJ
Pat
Betty K
BK
Li
Jun
J
Nicol
David
D
Johnson
David W
DW
Gobe
Glenda C
GC
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002292:Carcinoma, Renal Cell / Q000201:enzymology / Q000503:physiopathology*; D053143:Caspase 2; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000201:enzymology / Q000502:physiology; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007680:Kidney Neoplasms / Q000201:enzymology / Q000503:physiopathology*; D007684:Kidney Tubules / Q000201:enzymology / Q000502:physiology*; D008938:Mitosis / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; C505407:CASP3 protein, human; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin; D005472:Fluorouracil