Effects of RIa Overexpression on Cisplatin Sensitivity in Human Ovarian Carcinoma Cells
Mary Ellen Cvijic and Khew-Voon Chin1
Department of Medicine and Department of Pharmacology and Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901
Received July 24, 1998
Our laboratory has found that Chinese hamster ovary (CHO) and mouse Y1 adrenocortical carcinoma PKA mutants with a defective R subunit, but not al- tered C subunits, exhibit increased resistance to cis- platin as well as other DNA-damaging agents. The mechanism of resistance may be associated with in- creased recognition of the cisplatin-damaged DNA and protein binding to the DNA lesion, thus enhancing DNA repair in the RIa mutants. These data suggest that mutation of RIa may confer resistance to cisplatin by affecting DNA repair activity. In the present study, we overexpressed RIa in human ovarian carcinoma A2780 cells to demonstrate that RIa can modulate cel- lular sensitivity to cisplatin. Retroviral-infected A2780 cells overexpressing wild-type RIa cDNA displayed a four- to eightfold greater sensitivity to cisplatin com- pared with parental cells. Overexpression of RIa in the CP70 cisplatin-resistant derivative of A2780 also increased the sensitivity of these cells to cisplatin. Therefore, enhanced expression of the RIa subunit of PKA sensitizes cells to the cytotoxic effects of this DNA-damaging agent. These data suggest that RIa may act directly, independent of the C subunit, to in- fluence cellular sensitivity to cisplatin. Therefore, modulation of RIa expression or its functional status by pharmacological agents may potentially reverse cisplatin resistance in tumors. @ 1998 Academic Press
Binding of cAMP to the regulatory (R) subunit of the holoenzyme and the subsequent dissociation and activation of the catalytic (C) subunit has been the accepted mechanism that regulates the cAMP-depen- dent protein kinase (PKA). There has been speculation that cAMP could act through mechanisms other than the C subunit activation. One possibility is that the R subunit containing bound cAMP has functions inde- pendent of its interaction with the C subunit. How-
ever, our understanding of the role of the R subunit has been confined to its inhibition of the C subunit. Nevertheless, there have been sporadic reports which seem to indicate that the R subunit may have other physiological functions [1-7].
It has been shown that overexpression of RIa in Chi- nese hamster ovary (CHO) cells rendered growth ad- vantages in monolayer and soft agar conditions, whereas overexpression of the C subunit did not pro- duce such consequences [8]. It is apparent from these studies that the role of cAMP in CHO cell growth can- not be explained by changes in the kinase activity and thus raises the possibility that the R subunit or an unidentified cAMP receptor molecule may mediate the effects of cAMP. Similarly, overexpression of RIa, but not the C subunit, in MCF-10A cells conferred the abil- ity to grow in serum and growth-factor free conditions [9], suggesting a potential growth regulatory function for RIa. Furthermore, overexpression of wild-type RIa in CHO cells causes hypersensitivity to topoisomerase II inhibitors, implicating a function for RIa in the mod- ulation of drug resistance [10].
Using a mutant genetic approach, our laboratory has found that the CHO and the mouse Y1 adrenocortical carcinoma PKA mutants with a defective R subunit, but not altered C subunits, exhibit increased resistance to cisplatin as well as other DNA-damaging agents [11-13]. The mechanism of this resistance may be as- sociated with increased binding and recognition of the cisplatin-damaged DNA and enhanced DNA repair in the RIa mutants [11-13]. These data suggest that mu- tation of RIa may confer resistance to cisplatin by af- fecting DNA repair activity. In an effort to expand these studies into a human model system, we overex- pressed RIa in the human ovarian carcinoma A2780 cells to demonstrate unambiguously a function for RIa in modulating cellular sensitivity to cisplatin.
MATERIALS AND METHODS
Cell culture, retroviral expression vectors and infections. A2780 ovarian cancer cells and their cisplatin-resistant derivative CP70
1 To whom correspondence should be addressed at 195 Little Al- bany St., New Brunswick, NJ 08901. Fax: 732-235-7493. E-mail: chinkv@rwja.umdnj.edu.
0006-291X/98 $25.00 Copyright @ 1998 by Academic Press
(kindly provided by Dr. Kyonghee Son, Rutgers University, Piscata- way, NJ) were grown in RPMI medium supplemented with 10% fetal bovine serum, 5 mM glutamine and 100 µg/ml of streptomycin and penicillin in a humidified atmosphere of 5% CO2 at 37℃. The MT- RIa retroviral construct which contains the full length cDNA encod- ing the human RIa subunit of PKA in the Moloney murine leukemia virus-based retroviral vector OT1521 was obtained from Dr. Yoon Sang Cho-Chung, National Cancer Institute, Bethesda, MD. This retroviral expression vector contains a viral LTR-driven neomycin resistance gene and the heavy metal-inducible mouse metallothio- nein (MT-1) promoter driving the expression of the RIa subunit.
Virus was generated for the replication-defective retroviral vector by transient transfection into the BING amphotropic packaging cell line. BING cells were plated at 2 × 106 cells per 100-mm dish. The following day, culture media were replaced and the cells were trans- fected with the retroviral construct by the calcium-phosphate precipita- tion procedure [14]. After a 48 h incubation at 37℃, the virus-con- taining medium was removed and filtered to eliminate cells and debris. Infection of A2780 and CP70 cells was carried out by adding 10 ml of the retrovirus-containing supernatant to 100-mm dishes containing 5 × 105 A2780 or CP70 cells followed by a 24 h incubation at 37℃. Cells containing the RIa vector were selected in G418-containing medium (750 µg/ml) and individual colonies were expanded into cell lines. To induce expression of the RIa subunit, the infectants were grown in the presence of 70-100 AM ZnSO4, which induces the metallothionein promoter without cytotoxic effects on A2780 or CP70 cells.
Cytotoxicity and cell growth assays. Drug sensitivity was evalu- ated by MTT assay [15]. Briefly, 1 x 106 cells were plated in 96-well dishes (total volume of 100 pl) in the presence or the absence of 70 pM ZnSO4. Various concentrations of cisplatin (Sigma Chemical Co., St. Louis, MO) were added immediately after plating and the culture was incubated for 72 h at 37℃. The percentage of cells surviving drug treatment was determined by the ability of the cells to convert the tetrazolium MTT salt into a formazan product solubilized in acid- propanol. For cell growth experiments, 1 × 104 cells were plated in 24-well dishes in the presence or the absence of 1 mM 8-Br-cAMP (Sigma). At 24-h intervals, cells were trypsinized and counted using a Coulter Counter model ZM (Hialeah, FL).
Western blot analysis. To determine RIa expression, cells were incubated in the presence or the absence of various concentrations of ZnSO4 for 9 h and protein lysates (5 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes and incubated for 12 h with a 1:6,000 dilution of the polyclonal anti- RIa antibody (generously provided by Dr. Yoon Sang Cho-Chung, NCI, Bethesda, MD). Immunoreactive bands were detected with an ECL kit (Amersham Corp., Arlington Heights, IL) using a secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio- Rad, Hercules, CA).
Statistics. Statistical significance was assessed where appro- priate by a paired Student’s t test with a significant difference taken as P < 0.05.
RESULTS
To demonstrate conclusively that the RIa subunit of PKA may modulate cellular sensitivity to cisplatin, we overexpressed RIa in the human ovarian carcinoma A2780 cells and their cisplatin-resistant derivative CP70. CP70 cells are approximately 13-fold more resis- tant to cisplatin than wild-type cells (Fig. 1A) and both parental and resistant cells maintain an intact PKA signal transduction pathway, as evident from their sen- sitivity to cAMP-mediated growth inhibition (Fig. 1B). PKA assays also revealed no significant difference in
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kinase activity between wild-type and cisplatin-resis- tant cells (data not shown). A2780 and CP70 cells were infected with a retroviral vector containing the full
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length human RIa cDNA driven by the metallothionein promoter. Cells containing the RIa retroviral vector were selected in G418 and the infectants were ex- panded into cell lines.
Infected cells were screened for RIa overexpression by Western blot analysis. Results in Fig. 2 showed that there is increased RIa expression in A2780 MTRIa in- fectants compared with parental cells. The high basal expression of the uninduced cells is most likely a result of the leakiness of the metallothionein promoter. Zinc- treated samples sustained a modest increase in RIa expression compared with uninduced cells.
Since mutation in the RIa subunit and its overex- pression cause increased resistance to cisplatin [11], therefore, conversely, overexpression of wild-type RIa is expected to increase cellular sensitivity to cisplatin. Indeed, as shown in Fig. 3A, pooled colonies of RIa infected A2780 cells (A2780 MTRIa pool) displayed an 8-fold greater sensitivity to cisplatin compared with the parental cells. Upon stimulation of the metallothionein promoter with ZnSO4, a further 2.6-fold increase in sen- sitivity was observed compared with the unstimulated infected cells. As shown above, the increased cisplatin sensitivity of the uninduced infectants may have been due to the leaky expression of RIa from the MT pro- moter (Fig. 2). A2780 cells infected with the control vector (A2780 OT1521) showed no alteration in cis- platin sensitivity in comparison to wild-type cells (Fig. 3A). Furthermore, two individual clones containing the RIa retroviral vector, A2780 MTRIa Cl. 1 and A2780 MTRIa Cl. 7, also displayed a 4-fold and 7-fold increase in sensitivity to cisplatin, respectively (Figs. 3B and 3C). These results show that overexpression of wild- type RIa sensitizes cells to cisplatin.
We then questioned whether RIa overexpression may reverse drug resistance in the cisplatin resistant ovarian cells. As shown in Fig. 4, CP70 infectants con- taining the RIa vector displayed a 2-fold enhanced sen- sitivity to cisplatin, whereas control vector infected cells showed no alteration in cisplatin sensitivity com- pared with the uninfected CP70 parental control (data not shown). These results indicate that modulating the expression of RIa may partially reverse cisplatin resis- tance in resistant ovarian carcinoma cells.
DISCUSSION
Our results in this study showing that overex- pression of the human wild-type RIa sensitizes human ovarian carcinoma cells to the cytotoxic effects of cis- platin support our hypothesis that RIa may modulate cisplatin sensitivity. Previously, we have evaluated the
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Cisplatin (uM)
sensitivity of PKA mutants to cisplatin. We determined that RIa subunit mutants of CHO and Y1 cells exhib- ited increased resistance to cisplatin [11, 12]. However, CHO mutants with altered C subunits maintained a sensitivity to cisplatin comparable to wild-type cells [11]. These results led us to speculate that functional inactivation of the RI subunit may result in increased DNA repair and the acquisition of resistance to cis- platin [11, 13]. Overexpression of the human wild-type RIa in the current study in human ovarian cancers demonstrates that it can sensitize cells to the cytotoxic effects of cisplatin. More importantly, cisplatin resis- tant ovarian cells are also partially susceptible to the high level expression of RIa. It is possible that other biochemical or genetic alterations may have occurred that contributed to the cisplatin-resistant phenotype in the CP70 cell line, hence, overexpression of RIa can only partially reverse the cisplatin resistance in these cells. Nevertheless, these results suggest that modulat- ing the activity of RIa or alteration of cAMP levels may sensitize resistant cells to cisplatin.
Several studies have implicated that cAMP may func- tion as a chemosensitizer of cisplatin. For example, for- skolin, an adenylate cyclase stimulator, and dipyridam- ole and methylxanthines, which inhibit phosphodiester- ases, were shown to enhance cellular sensitivity to cisplatin in ovarian and cervical carcinoma cells [16-19]. Similarly, non-toxic doses of the methylxanthine caffeine enhanced cytocidal effects of cisplatin in the human os- teosarcoma OST cells, and this antitumor effect of a com- bination of cisplatin and caffeine was also observed in
transplanted osteosarcoma in BALB/C athymic mice [20]. Keane et al. [21] also demonstrated an in vivo poten- tiation of cisplatin efficacy through combination treat- ment with dipyridamole in a nude mouse/human bladder and testicular tumor xenograft model.
We have also shown that the RIa subunit mutant CHO cells are cross-resistant to other DNA-damaging drugs including bleomycin, melphalan and carboplatin, suggesting that this phenotype is not limited to cis- platin resistance alone and that the RIa subunit may be involved in the regulation of repair of many different types of DNA lesions [11, 12]. In corroboration with this hypothesis, other studies in the literature have shown the potentiating effects of caffeine and other cAMP-elevating agents on the toxicity of such DNA- damaging drugs as sulphur mustards, melphalan and mitomycin C [19, 22, 23]. Forskolin, dibutyryl-cAMP and theophylline synergistically enhanced the growth- inhibitory effects of mitomycin C on AH66 rat ascites hepatoma [22]. Taken together, these studies suggest that the effects of cAMP, mediated through RIa, may influence cellular sensitivity to DNA-damaging drugs in a C subunit kinase independent manner.
Despite the above findings, the mechanisms by which these cAMP analogues and cAMP-elevating agents ex- ert their potentiating effects on cisplatin cytotoxicity as well as their synergism with other DNA-damaging drugs are not clearly understood. It is conceivable that the R subunit of the PKA may interact with other pro- tein targets including those involved in DNA repair. Thus, R subunit protein-protein interaction studies may yield insights into the mechanisms of RIa medi- ated cellular sensitization to DNA-damaging chemo- therapeutic drugs.
In summary, results from this study demonstrate that overexpression of the RIa subunit of PKA in A2780 and CP70 cells enhances their sensitivity to cisplatin. These data are consistent with our previous findings that suggest that RIa may act independently of the C subunit to influence cellular sensitivity to cisplatin. Therefore, modulation of RIa expression or its func- tional alteration by pharmacological agents may poten- tially reverse cisplatin resistance in tumors.
ACKNOWLEDGMENTS
This work was supported in part by NIH Grant CA67722 (to K .- V.C.). K .- V.C. is also the recipient of Career Development Award DAMD17-94-J-4162 from the U.S. Army Medical Research and De- velopment Command.
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