The p21cip1/waf1 cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells
Introduction
Chemoresistance is a major concern in cancer chemotherapy. Ovarian carcinoma is an example of cancer in which intrinsic and acquired resistance are clinically apparent. Aggressive treatment of the patients with platinum-based combination chemotherapeutic regimens produces a high primary rate of complete clinical complete response to the drug. However a large proportion of patients relapse, become refractory to the initial drugs used, and eventually die of their disease within 5 years. Several mechanisms of resistance to platinum compounds have been identified, including decreased intracellular drug accumulation, enhanced detoxification mechanisms, DNA repair, and tolerance toward platinum adducts [1]. Since most chemotherapeutic agents exert their cytotoxic effect on tumor cells by inducing apoptotic cell death [2], defects in the apoptosis program have also been suggested to be responsible for chemoresistance in malignant cells [1], [3]. Having established an in vitro cellular model of chemoresistance in human ovarian carcinoma cells by mimicking a clinical protocol of cisplatin treatment, we found that the acquisition of chemoresistance was not directly linked to a defect in apoptosis induction, but rather to a defect in cell cycle control favoring the proliferation of a few recurrent cells that had acquired a chemoresistant phenotype [4].
The most common genetic alteration in human cancer, including ovarian cancer, involves the p53 tumor suppressor gene [5], [6]. This results in defective control of cell cycle arrest and cell death following exposure to DNA-damaging agents [7], and thereby in genomic instability. p53 encodes a transcriptional factor for a set of genes involved in the regulation of cell cycle progression, DNA repair, and apoptosis. One of the targets for p53-dependent transcriptional activation is the cyclin-dependent kinase (cdk) inhibitor p21cip1/waf1, a negative regulator of cell cycle transitions following DNA damage [8], [9], [10]. p21 is also inducible in a p53-independent manner [11], [12]. The cell cycle inhibitory effects of p21 may be attributed to its ability to bind cdks as well as the proliferating cell nuclear antigen (PCNA) [13], [14], resulting in inhibition of progression from the G1 to the S phase, of DNA replication, and of progression through G2 phase [15]. In normal cells, p21, cyclin, cdk, and PCNA can form a tetrameric complex in which the binding of more than one p21 molecule is needed to inhibit the cdk activity.
Gene transfer of p21 inhibits tumor cell growth in vitro and in vivo, as reported for melanoma cells [16], breast carcinoma cells [17], [18], lung cancer cells [19], colorectal carcinoma cells [20], and oncogene-transformed fibroblasts [21]. Having uniformly demonstrated the negative effect of p21 on malignant cell proliferation, all these data stress the potential of p21 to serve as a tumor suppressor for therapeutic purposes. However, inhibition of cell growth may not be sufficient to eradicate efficiently a tumor and should be associated with enhanced tumor cell death. The effect of p21 upregulation on apoptosis induction is controversial. p21 failed to induce apoptosis in normal and tumor cells of lung and mammary epithelial origin [17], whereas it induced the formation of giant cells that ultimately succumbed to apoptotic death in breast carcinoma [18] and colorectal carcinoma [22]. Other studies have also reported that p21 appears to protect from apoptosis [23], [24], [25], [26], and p21-deficient cells are more sensitive to killing by irradiation than their p21-expressing counterparts [27].
Based upon data on the growth inhibition efficiency of p21 on a variety of malignant cell types, the aim of the present work was to determine the potential effect of p21 on ovarian carcinoma cell growth in vitro, alone or in combination with cisplatin.
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Cell lines
The human ovarian adenocarcinoma cell lines SKOV3, OAW42, and OVCAR3 were grown as monolayers at 37°C in a 5% CO2 atmosphere. SKOV3 was grown in Mac Coy's medium supplemented with 10% fetal calf serum (Gibco BRL, Cergy-Pontoise, France) and 2 mM l-glutamine; OAW42 in DMEM supplemented with 10% fetal calf serum, 2 mM l-glutamine, 1 mM sodium pyruvate, and 2 μg/ml bovine insulin (Gibco BRL); and OVCAR3 in RPMI 1640 supplemented with 20% fetal calf serum, 2 mM l-glutamine, 2.5g/l d-glucose, 1 mM
Cisplatin effects on growth inhibition and apoptosis
CDDP exerts its cytotoxic effect by inducing apoptosis [2]. In both SKOV3 and OAW42 cell lines, the number of viable cells decreased as a function of drug concentration (Fig. 1a). The drug concentrations inhibiting cell growth by 50 and 100% after 2 days were 5 and 20 μg/ml, respectively, for OAW42 cells, and 10 and 100 μg/ml CDDP, respectively, for SKOV3 cells. SKOV3 cells appeared, therefore, to be more resistant than OA4W2 cells to CDDP. The decrease of viable cell number was related to the
Discussion
The seriousness of ovarian cancer, which is basically related to the acquisition of chemoresistance after conventional therapy, combined with extensive data unanimously indicating the tumor suppressor effect of p21cip1, prompted us to explore the effect of ectopic expression of p21 in ovarian carcinoma cells. In the present report, we show that waf1/cip1gene transfert enhanced the cytotoxic effect of CDDP and hindered the proliferation of recurrent ovarian carcinoma cells.
p21 is a universal and
Acknowledgements
We thank Professor J.F. Héron, head of the Centre François Baclesse for his constant support and M. Michel for artwork. H.L. and P.L. are recipients of a fellowship of the Ligue Nationale de Lutte Contre le Cancer (Comité du Calvados), which also supports this research, together with the INSERM, the Ministère de la Recherche et de l'Enseignement Supérieur and the Université de Caen.
References (40)
- et al.
Cisplatin resistance in a murine leukemia cell line is associated with a defective apoptotic process
Exp. Cell Res.
(1995) - et al.
High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes
J. Invest. Dermatol.
(1994) - et al.
Analysis of the Rb gene and cyclin-dependent kinase 4 inhibitor genes (p16INK4 and p15INK4B) in human ovarian carcinoma cell lines
Exp. Cell Res.
(1997) - et al.
Administration of wild-type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene
Cancer Lett.
(2000) - et al.
The biology of ovarian cancer
Semin. Oncol.
(1998) Apoptosis induced by anticancer drugs
Cancer Metastasis Rev.
(1992)- et al.
Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control
Int. J. Cancer
(1998) - et al.
p53 mutation in human cancers
Science
(1991) - et al.
p53 mutation is a common genetic event in ovarian carcinoma
Cancer Res.
(1993) - et al.
P53 and ATM: cell cycle, cell death and cancer
WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis
Cancer Res.
p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest
Cell
p21 is necessary for the p53-mediated G1 arrest in human cancer cells
Cancer Res.
Transforming growth factor beta 1 induces Cip1/Waf1 independent of the p53 pathway in ovarian cancer cells
Cell Growth Differ.
Regulation of p21 WAF1/CIP1 expression by p53-independent pathways
Oncogene
Inhibition of cyclin-dependent kinases by p21
Mol. Biol. Cell.
The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA
Nature (Lond.)
p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells
Oncogene
The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo
Nat. Med.
Effects of a recombinant adenovirus expressing WAF1/Cip1 on cell growth, cell cycle, and apoptosis
Cell Growth Differ.
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