© 1997 by Oxford University Press
Journal Of The National Cancer Institute, Vol 89, 365-373, Copyright © 1997 by Oxford University Press
M Dixit, JL Yang, MC Poirier, JO Price, PA Andrews and CL Arteaga
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. PURPOSE: 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.
ARTICLES
Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-5536, USA.
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