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/

Purpose: Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necros is 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. Materials and Methods: Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used. Results: Cytotoxic assays w ith 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 de monstrated less than 4% cytotoxicity. Based on these results, we defin ed our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resista nt cells. To determine whether protein kinase C (PKC), which is involv ed in the signal transduction pathway of a cell, could regulate endoge nous basal TNF mRNA levels, comparative PCR analyses for TNF expressio n 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-sens itive cell SK-RC-42. With PMA, a PKC activator, for various time point s, both cell lines demonstrated an induction of endogenous TNF mRNA. T o further confirm our findings that PKC may regulate endogenous TNF ex pression, a PKC inhibitor, staurosporine, was used. When the cells wer e 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 ho urs. After factoring out the cytotoxicity of staurosporine, the TNF-me diated cytotoxicity increased to 39.3% and 28.7% for the SK-RC-42 and SK-RC-49 cells, respectively. Furthermore, by using a nuclease digesti on assay, we demonstrated that TNF activated a Ca++/Mg++ dependent end onuclease responsible for programmed cell death or apoptosis. Conclusi ons: Our data suggest that protein kinase C may play a role in protect ing renal cancer cells from undergoing cytokine-mediated apoptosis.