Influence of the Proto-oncogene c-fos on Cisplatin Sensitivity Roger A. Moorehead* and Gurmit Singh†‡ ONTARIO CANCER TREATMENT AND RESEARCH FOUNDATION,HAMILTON REGIONAL CANCER CENTRE,HAMILTON, ONTARIO,CANADA L8N 3Z5 ABSTRACT. Cisplatin resistance has been associated with overexpression of the c-fos gene in a human ovarian carcinoma cell line. To determine whether the correlation between c-fos overexpression and cisplatin resistance was limited to this cell line or was a more generalized phenomenon, we investigated cisplatin sensitivity in rat fibroblast cells that overexpressed the c-fos gene. The cisplatin IC 50 values for two different c-fos transfectants, CMVc-fos and L1–3c-fos, were 7.6  0.8 and 5.6  1.0 M, respectively, whereas the cisplatin IC 50 value for the parental line, 208F, was 2.4  0.1 M. This represented a 3.2- and 2.3-fold resistance to cisplatin for CMVc-fos and L1–3c-fos cells, respectively. The correlation between c-fos expression and cisplatin resistance also was examined in a human ovarian carcinoma cell line, 2008, and its cisplatin-resistant variant, C13*. Expression of c-fos was elevated slightly at both the mRNA and protein levels in the C13* cells compared with 2008 cells, and c-Fos protein levels were induced in C13* cells following cisplatin treatment. In addition, it was observed that C13* cells were significantly more sensitive than 2008 cells to a c-fos antisense oligonucleotide. The IC 50 values for the c-fos antisense oligonucleotide were 19.9  5.0 pmol for C13* cells and 58.1  6.0 pmol for 2008 cells (P = 0.0012). Furthermore, combinations of c-fos antisense and cisplatin reduced the amount of cisplatin required to kill 50% of the C13* cells, although the interaction was not synergistic. These results suggest that expression of the c-fos gene can influence cisplatin sensitivity, and that c-fos antisense oligonucleotide based therapy may be effective at killing parental and cisplatin-resistant ovarian carcinoma cells, either alone or in combination with cisplatin. BIOCHEM PHARMACOL 59;4:337–345, 2000. © 2000 Elsevier Science Inc. KEY WORDS. cisplatin; c-fos; resistance; antisense oligonucleotides; combination chemotherapy; ovarian carcinoma Cisplatin is one of the most effective anticancer agents against a number of solid tumours, but it is limited by the frequent emergence of cisplatin-resistant cell populations [1, 2]. Resistance to cisplatin is particularly apparent in ovarian cancer, where the occurrence of cisplatin-resistant tumours approaches 60% [2]. Cisplatin resistance has been associated with a number of cellular changes, several of which have been investigated extensively, including: (i) enhanced scavenging of the drug through increased gluta- thione or metallothionein content, (ii) decreased accumu- lation and/or increased efflux of cisplatin, and (iii) in- creased repair of cisplatin–DNA lesions [3]. However, no single mechanism has been associated consistently with cisplatin resistance. Several papers published by Scanlon and co-workers [4 – 6] indicate that cisplatin resistance is associated with overexpression of the proto-oncogene c-fos. The c-fos gene encodes a nuclear transcription factor, c-Fos, that induces transcription of a number of genes involved in the regula- tion of cell replication, cell cycle progression, and differen- tiation through its interactions with members of the c-Jun and ATF/CREB families [7–10]. Overexpression of the c-fos gene has been observed in tumour tissue from patients that have failed cisplatin-based chemotherapy and in human ovarian carcinoma cells selected for cisplatin resistance [2]. Several additional observations have strengthened the association between cisplatin resistance and overexpression of the c-fos gene. c-fos expression can be induced by physical stresses such as reactive oxygen species, heat shock, and several DNA-damaging agents, including cispla- tin [11, 12]. Cells lacking the c-fos gene are hypersensitive to a number of DNA-damaging agents [13]. Transfection of a human ovarian carcinoma cell line with a c-fos expression vector induces cisplatin resistance [4]. Treatment of cispla- tin-resistant human ovarian carcinoma cells with cisplatin induces the expression of the c-fos gene as well as a number of genes involved in DNA repair [5]. Reducing c-fos expression in these cisplatin-resistant human ovarian car- cinoma cells through treatment with either cyclosporin A or a c-fos ribozyme results in decreased expression of the * Supported by a Medical Research Council of Canada Studentship. † Supported by a grant from the Medical Research Council of Canada (MA-10409). ‡ Corresponding author: Dr. Gurmit Singh, Hamilton Regional Cancer Centre, 699 Concession Street, Hamilton, Ontario, Canada L8V 5C2. Tel. (905) 387-9711, Ext. 67007; FAX (905) 575-6330; E-mail: singhg@fhs.mcmaster.ca Received 3 November 1998; accepted 12 July 1999. Biochemical Pharmacology, Vol. 59, pp. 337–345, 2000. ISSN 0006-2952/00/$–see front matter © 2000 Elsevier Science Inc. All rights reserved. PII S0006-2952(99)00333-0