Sensitization of immunoresistant prostate carcinoma cell lines to Fas/Fas ligand-mediated killing by cytotoxic lymphocytes: independence of de novo protein synthesis.
BACKGROUND
We recently reported that drug-resistant prostate tumor cells (DU145, PC-3) are resistant to Fas-mediated killing by cytotoxic lymphocytes, and that this resistance can be overcome by treatment with subtoxic concentrations of chemotherapeutic drugs. Fas belongs to the tumor necrosis factor (TNF) family of receptors. Since resistance to TNF-alpha-mediated killing has been shown to be due, in part, to the presence of protective factors and that inhibitors of protein synthesis can sensitize cells to TNF-alpha killing, we hypothesized that resistance to Fas-mediated killing may be due to similar mechanisms. Since sensitization is achieved with chemotherapeutic drugs, and some chemotherapeutic drugs can also inhibit protein synthesis, we tested whether sensitization of prostate tumor cells to Fas ligand (Fas-L) occurred through inhibition of protein synthesis in a manner analogous to that of TNF-alpha.


METHODS
The effect of chemotherapeutic drugs on protein synthesis in DU145 and PC-3 cells was characterized by (3)H-leucine incorporation assays. We also determined the ability of inhibitors of protein synthesis and chemotherapeutic drugs to sensitize Fas and TNF-resistant DU145 cells to killing. The ability of RNA (actinomycin-D, Act-D) and protein synthesis inhibitors (cyclohexamide (CHX), emetine) to block drug-mediated sensitization to Fas-L killing was analyzed. Sensitivity to Fas-L killing was determined by the (51)Cr-release assay using human lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocyte (TIL) effector cells and the murine Fas-L-expressing PMMI cells.


RESULTS
The drugs cis-diamminedichloroplatinum (II) (CDDP), adriamycin (ADR), and etoposide (VP-16) sensitized DU145 and PC-3 cells to Fas killing. CDDP and ADR, which sensitized DU145 and PC-3 cells to Fas-L- and TNF-mediated killing, inhibited de novo protein synthesis in both cell lines, while VP-16 only inhibited protein synthesis in DU145 cells. Further, neither CHX nor emetine sensitized DU145 or PC-3 cells to Fas-L-mediated killing, despite blocking >90% de novo protein synthesis. In contrast, CDDP, VP-16, and the protein synthesis inhibitors, Act-D and CHX sensitized DU145 cells to TNF-alpha killing. Finally, pretreating cells with protein synthesis inhibitors (CHX, emetine) did not abrogate drug-mediated sensitization to Fas-mediated killing.


CONCLUSIONS
These findings demonstrate that downregulation of protective factors by protein synthesis inhibition may not be the primary mechanism of drug-mediated sensitization to Fas-L killing in prostate cell lines. These findings also suggest that drug-mediated sensitization to Fas-L killing may be due to modifications of preexisting gene products that participate in Fas-L-mediated apoptosis.
Frost
P J
PJ
Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California, USA.
Belldegrun
A
A
Bonavida
B
B
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D053222:Fas Ligand Protein; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000276:immunology*; D016246:Lymphocytes, Tumor-Infiltrating / Q000187:drug effects / Q000276:immunology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D011471:Prostatic Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism*
D000970:Antineoplastic Agents; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor