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 receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R.


OBJECTIVE
Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity.


METHODS
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 content was assessed by 125I-EGF binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium 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 dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA.


RESULTS
Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-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 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin. Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones.


CONCLUSIONS
These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.
Dixit
M
M
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-5536, USA.
Yang
J L
JL
Poirier
M C
MC
Price
J O
JO
Andrews
P A
PA
Arteaga
C L
CL
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000503:physiopathology*; D002945:Cisplatin / Q000494:pharmacology*; D018736:DNA Adducts; D004253:DNA Nucleotidylexotransferase; D018076:DNA, Complementary; D004273:DNA, Neoplasm / Q000187:drug effects; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016372:RNA, Antisense; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018736:DNA Adducts; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D016372:RNA, Antisense; D066246:ErbB Receptors; D004253:DNA Nucleotidylexotransferase; D002945:Cisplatin