ABROGATION OF CISPLATIN-INDUCED PROGRAMMED CELL-DEATH IN HUMAN BREAST-CANCER CELLS BY EPIDERMAL GROWTH-FACTOR ANTISENSE RNA

Background: Epidermal growth factor receptor (EGF-R) perturbation by r eceptor ligand(s), e.g., epidermal growth factor (EGF) and transformin g growth factor-alpha (TGF-alpha), or receptor-specific antibodies acc entuates cisplatin-induced toxicity in tumor cells, This sensitization occurs only in tumor cells with high expression of EGF-R but not in t hose with low expression of EGF-R. Purpose: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. Meth ods: MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R con tent was assessed by I-125-EGF binding and EGF-R immunoblot assays, Ci splatin sensitivity was evaluated by (a) colony-forming assay in vitro , (b) xenograft growth in nude mice, (c) cell cycle distribution of pr opidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ, Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissoc iation-enhanced fluoroimmunoassay that utilizes an antibody against ci splatin-modified DNA. Results: Selected clones (MDA-468/AS-EGFR) exhib ited more than 90% loss of both I-125-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells, By use of a colony-forming assay, the 1-hour IC50 (i .e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 mu M or less for parental and vector-transf ected clones (n = 4), whereas it was 25 mu M or more for all MDA-468/A S-EGFR clones (n = 3), MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G(2) arrest 48 hours after a 1-hour incubation with 3-30 mu M cisplatin. Under th ese conditions, apoptosis and G(2) arrest mere undetectable in all MDA -468/AS-EGFR clones. An MDA-468 subline selected after long-term treat ment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: >30 mu M) compared with parental cells, This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by u se of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by at omic absorption spectroscopy, was identical in both groups of cells, I ntrastrand drug-DNA adducts, however, were statistically higher in hig h EGF-R expressors than in low-EGF-R-expressing clones. Conclusions: T hese data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-med iated apoptosis in tumor cells and suggest an inhibitory effect of thi s pathway on the repair of cisplatin-damaged DNA.