Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells.
OBJECTIVE
Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necrosis factor are now being considered for clinical use. In this study, we used 2 renal cancer cell lines. SK-RC-42 and SK-RC-49, and determined the cytotoxic effects of tumor necrosis factor (TNF) and the possible mechanism of TNF resistance.


METHODS
Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used.


RESULTS
Cytotoxic assays with SK-RC-42 demonstrated that TNF at 50 ng./ml. for 24 hours resulted in 19% cytotoxicity of the cells. Similar assay with SK-RC-49 only demonstrated less than 4% cytotoxicity. Based on these results, we defined our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resistant cells. To determine whether protein kinase C (PKC), which is involved in the signal transduction pathway of a cell, could regulate endogenous basal TNF mRNA levels, comparative PCR analyses for TNF expression were used. The PCR results demonstrated that the TNF-resistant cell, SK-RC-49, had a higher basal expression of TNF mRNA than the TNF-sensitive cell SK-RC-42. With PMA, a PKC activator, for various time points, both cell lines demonstrated an induction of endogenous TNF mRNA. To further confirm our findings that PKC may regulate endogenous TNF expression, a PKC inhibitor, staurosporine, was used. When the cells were treated with staurosporine prior to PMA stimulation, no increase in TNF mRNA expression was seen. To determine whether PKC is involved in providing resistance against TNF, we incubated the SK-RC-42 and SK-RC-49 cells with staurosporine at 100 nM. and TNF at 50 ng./ml. for 24 hours. After factoring out the cytotoxicity of staurosporine, the TNF-mediated cytotoxicity increased to 39.3% and 28.7% for the SK-RC-42 and SK-RC-49 cells, respectively. Furthermore, by using a nuclease digestion assay, we demonstrated that TNF activated a Ca++/Mg++ dependent endonuclease responsible for programmed cell death or apoptosis.


CONCLUSIONS
Our data suggest that protein kinase C may play a role in protecting renal cancer cells from undergoing cytokine-mediated apoptosis.
Woo
K R
KR
Department of Urology, Mount Sinai School of Medicine, New York, 10029, USA.
Shu
W P
WP
Kong
L
L
Liu
B C
BC
D017209:Apoptosis / Q000502:physiology*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D007680:Kidney Neoplasms / Q000503:physiopathology*; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000502:physiology*
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease