Abstract
Objective To explore whether MDM2 transfection can alter the MDM2-p53 autoregulatory feedback loop so as to change the sensitivity of ovarian cancer cell lines to cisplatin.
Methods The ovarian cancer cell line A2780 expressing wild-type P53 and the ovarian cancer cell line SKOV-3 with the p53 null type were stably transfected with pCMV-MDM2 or pCMV as a control. The blocked expression of P53 was determined by Western blots. Cytotoxicity was assessed using the MTT assay and the trypan blue exclusion assay. Flow cytometry was used to detect changes in the cell cycle and removal of platinum -DNA adducts was measured by atomic absorption spectroscopy.
RESULTS (1) Parental A2780 and A2780-V cells (IC50=15.14±1.39 μmol) have similar cisplatin sensitivities, whereas sensitivity to cisplatin in A2780-M cells (IC50=7.98±1.32 μmol) was 2 to 3 fold greater (P=0.001). The trypan blue exclusion assay demonstrated that cisplatin killed a higher percentage of A2780-M cells compared to A2780-V cells. There was no significant change following MDM2 transfection in SKOV-3 cells. (2) After cisplatin treatment, A2780-M cells showed a pronounced S-phase arrest, however, A2780 cells with the intact wild-type P53, arrested primarily at the G2/M transition. (3) Platinum uptake was similar for all of the A2780 cell lines after ciaplatin treatment, but the removal of platinum-DNA adducts was reduced in the A2780-M cells compared with A2780-V cells.
Conclusion MDM2 increases cisplatin cytotoxicity in ovarian cancer cells by blocking the expression of p53 through the MDM2-p53 autoregulatory feedback loop.
keywords
The main obstacle to solve the puzzle of ovarian cancer is that most patients will develop resistance to chemotherapy during the course of their treatment. The statuses of p53 and of MDM2 are determinants of in vitro tumor cell chemosensitivity.[1] In response to DNA damage, the p53 gene encodes a nuclear phosphoprotein which activates G1 cell-cycle arrest and regulates a gene product (GADD 45) to stimulate nucleotide-excision repair. Therefore, p53 extends the time available for DNA repair before entry into the critical S phase and thereby prevents cell death. Thus, disruption of p53 has been shown to sensitize cell lines to cisplatin [2] and resistant cell lines often over-express p53.[3] In addition, p53 can also activate an apoptotic response to DNA damage. Thus, in cell types programmed for apoptosis, disruption of p53 functions decreases sensitivity to DNA-damaging agents. Thus, it depends on the cell type as to whether p53 disruption decreases or increases chemosensitivity. Whereas the influence of p53 on chemosensitivity has been extensively studied, relatively little is known about MDM2, its functional antagonist, although it has been shown that P53 and MDM2 form an autoregulatory feedback loop. Thus, we transfected a stably-integrated human MDM2 plasmid into the wild-type p53-expressing cell line A2780, and into the p53-null-expressing cell line, SKOV-3. We investigated platinum drug cytotoxity as well as the effects on cell cycle status and DNA repair in the transfected cell lines. We showed in this model system that a high level of MDM2 in the A2780 cells led to the loss of p53 expression and enhanced cisplatin chemosensitivity due to the loss of Gl/S checkpoint control and DNA repair. However, a change in the sensitivity to cisplatin was not observed in SKOV-3 cells with effective MDM2 transfection.
Materials and Methods
Materials
The A2780 and SKOV-3 human ovarian cancer cell lines were supplied by Professor Robert Zeillinger, Department of Obstetrics and Gynecology, University of Ulm Medical School, Germany, and the pCMV-MDM2 plasmid was provided by Professor Moshe Oren, Department of Molecular Cell Biology, the Weizmann Institute of Science, Isreal.
Cell culture and stable transfection
The cell lines were cultured in RPMI 1640 containing 10% FCS, 50 units penicillin, and 50 μg/ml streptomycin. For stable transfection, 1.5 × 106 cells were seeded in 10-cm diameter Petri dishes 24 h before transfection. Cells were transfected with either the pCMV expressing neomycin gene alone (empty vector) or pCMV-MDM2 expressing MDM2 and neomycin, using the lipofectamine reagent (Life Technologies, Inc., USA). Transfectants were selected in a 500 μg/ml G418 sulfate solution. Approximately 14 days later, G418 resistant-colonies were selected and expanded.
Radiation treatment
The induction of p53 protein was determined at 4 h after 5-Gy irradiation. Radiation was delivered using a 60Co source with a source-to-flask distance of 40 cm and a dose rate of 1.36 Gy/min.
Western blot analysis
Cells were broken in lysis buffer for 30 min on ice, centrifuged, and the supernatant recovered. Protein concentration was determined using a protein assay kit (GIBCO, USA). Total cell protein (100 jxg) was loaded onto 8% SDS-polyacrylamide gels and then transferred electrophoretically to Immobilon membranes and then blocked overnight in 5% nonfat milk at 4°C. Immunodetection of the MDM2 and p53 proteins was performed using a monoclonal MDM2 Ab-1 antibody and a monoclonal p53 antibody DO-1 (NeoMarkers, USA). The antibody reactions were determined using enhanced chemiluminescence (Bio Basic, Canada).
Drug sensitivity assays
Cisplatin (Qi Lu Pharmacy, China) was prepared in 0.9% saline at a concentration of 1 mM. Cytotoxicity was assessed using the MTT assay.[4] Briefly, cells were seeded onto each well of 96-well plates and allowed to attach overnight. Serial dilutions of platinum drugs were added to quadruplicate wells for 72 h. At this time, viability was assessed by the ability of cells to convert the soluble salt of MTT into an insoluble formazan precipitate. Differences in drug sensitivity of the respective cell lines were determined from at least 4 independent experiments with continuous drug exposure and are reported as the concentration required to suppress proliferation by 50% (IC50). Cytotoxicity was also assessed using the trypan blue exclusion assay. Exponentially growing cells were incubated for 2 h with cisplatin, washed once with PBS, and exposed to fresh medium. Cells were removed from duplicate flasks at 24, 48, and 72 h after the addition of cisplatin. Trypan blue (final concentration, 0.8%) was added to the detached cells, and after 5 min, the trypan blue-excluding cells were counted by hemocytometry.
Flow cytometry
Cells were incubated for 2 h with 25 μM cisplatin, washed with PBS, and then grown under standard conditions. The cells were fixed in ice-cold 70% ethanol at 24 h after initial cisplatin treatment, washed with PBS, treated with RNase at 50 μg/ml for 15 min, and stained with 50 μg/ml propidium iodide. Cell cycle analysis was performed using a fluorescence-activated cell analyzer.
DNA-platinum (DNA-Pt) binding and removal
Exponentially growing cells were exposed to 100 μM cisplatin for 2 h, washed once with PBS, and further incubated in normal medium for 0, 3, or 22 h. DNA was extracted by a high salt extration method.[5] Then the DNA was ethanol-precipitated, washed, and redissolved in 350 μl of DNase-free water and quantified using A260 absorption. Platinum content was determined by atomic absorption spectroscopy.
Significance test
Levels of significance were determined by SPSS software using the 2-tailed paired Student’s test.
Results
Characterization of MDM2 and vector transfectants of A2780 and SKOV-3 cells
Stable MDM2 transfected cells exhibited increased levels of the MDM2 protein both in A2780 and SKOV-3 cells as determined by immunoblotting (Fig. 1A). The A2780-V vector control cells and the A2780-MDM2 cells were screened for either p53 protein induction or lack of expression after irradiation. Wild-type p53 protein is normally present in low concentrations in cells but can be induced and stabilized after DNA damage.[6] As shown in Fig.1B, constitutive levels of p53 protein were barely detectable in parental A2780, A2780-V, or A2780-M cell lines. However, 4 h after 5-Gy irradiation, levels of p53 protein were increased to a similar extent in parental A2780 and A2780-V cells. By contrast, there was no detectable p53 protein observed in A2780-M after this treatment. Thus, Fig.1A and Fig.1B together provide a model to study MDM2 overexpression and its effect on drug sensitivity in human ovarian cancer cells.
Characterization of A2780 and SKOV-3 cell lines after MDM2 transfection. A: representative Western blot showing stable MDM2 transfectant cells exhibit increased levels of MDM2 protein. B: a typical immunoblot showing induction of p53 expression after irradiation in A2780 and A2780-V cells.
Sensitivity to cisplatin
The sensitivities of parental A2780, A2780-M, and A2780-V cells to cisplatin were determined by the MTT assay. The IC50 value after 72 h of continuous exposure to cisplatin was similar for both parental A2780 and A2780-V cells. However, A2780-M showed a significant increase in sensitivity (P=0.00l). This increased sensitivity was confirmed by the trypan blue exclusion assay. In SKOV-3 cells, there were no significant changes (Table 1, Fig.2).
Sensitivity of A2780-M and A2780-V cells to killing by cisplatin determined with the trypan blue exclusion assay. Each point represents the mean ± S.E. of at least 4 independent experiments.
Distribution of cells in the cell cycle after cisplatin treatment
The cellular distribution was not detectably different between the 3 A2780 cell lines in the absence of DNA-damaging treatments. Cisplatin-treated cells exhibited marked differences between A2780-wt-p53 cells and A2780-MDM2 cells. While there was a great increase in A2780-wt-p53 cells in the G2/M transition after cisplatin treatment, A2780-MDM2 tranfectants exhibited a pronounced S-phase arrest as shown in Table 2.
Platinum removal from DNA after cisplatin treatment
Similar levels of DNA-Pt binding were seen in A2780-M and A2780-V cells immediately after cisplatin treatment. However, at 5 and 24 h after the start of treatment, a greater amount of DNA-Pt binding was seen in the A2780-M cells compared with A2780-V cells, suggesting compromised DNA repair kinetics in A2780-M cells (Fig.3).
DNA-Pt binding after 100 μM cisplatin exposure in A2780-M and A2780-V cells.Values are meant± S.E. from 3 independent experiments.
Discussioin
In this study, we observed that A2780-M cells were more sensitive to cisplatin than the p53 wild-type control cells. In the SKOV-3 cell line with p53 null expression, there was no significant chemosensitivity change after the introduction of MDM2.
As shown in Fig.1A, a higher level of MDM2 expression in A2780 and SKOV-3 transfectants was observed. To demonstrate that our thansfection was effective, we examined the p53 protein in the A2780 cells after radiation treatment. In normal cells, the WT-p53 protein is present at a very low concentration. However, in response to various stimuli, such as DNA damage, hypoxia, or metabolic changes, it can be induced and stabilized.[7] So as showen in Fig.1B, constitutive levels of p53 protein were barely detectable in parental A2780, A2780-MDM2 and A2780-Vector cells. However, 4 h after 5 Gy irradiation, levels of p53 protein were increased to a similar extent in parental A2780 and A2780-Vector cells. By contrast, there was no detectable p53 protein observed in A2780-MDM2 transfectants after this treatment. These results suggested that MDM2 had been successfully transfected and had reached an effective level to block the expression of p53 in the A2780 cells. In the SKOV-3 cell line, there was high MDM2 expression compared to their parental cells which had no MDM2 expression before transfection. Therefore, we established 2 human ovarian cell lines that differ only in p53-MDM2 feedback loop status and investigated the chemotherapeutic effects of cisplatin in this model.
A2780-MDM2 cells showed an increased sensitivity to cisplatin in which high levels of MDM2 blocked the expression of p53. But in SKOV-3-MDM2 transfectant cells whose MDM2 expression was also high, no significant difference of chemosensitivity was observed. This suggested that MDM2 increased the sensitivity of cisplatin in human ovarian cells only through the MDM2-p53 feedback loop although there have been clear suggestions that the MDM2 protein may have p53-independent functions.[8]
In A2780 cells, transfection of MDM2 leads to the blocked expression of p53 and increased chemosensitivity. To examine the mechanism of this response, we determined the distribution of cells in the cell cycle and platinum removal from DNA after cisplatin treatment. p53 plays a key role in Gl/S cell cycle arrest after DNA damage.[9] After MDM2 transfection, the p53 effect was blocked and may not have this function. We observed that MDM2 transfectants exhibited no overt alteration of the cell cycle in untreated cells. However, differences in the distribution of cells in the cell cycle after cisplatin treatment were seen between A2780-M and A2780-V cells.The presence of fewer A2780-MDM2 cells in the G1 phase and more A2780-MDM2 cells in the S phase was indicative of a loss of G1/S checkpoint integrity in this p53-deficient cell line. p53 is also involved in facilitating a G2 arrest.[10] A2780-V cells became arrested primarily in the G2 phase after cisplatin treatment. The G2 checkpoint would delay entry of DNA-damaged cells into mitosis, thereby allowing more time for constitutive DNA repair processes to remove DNA lesions before the critical chromosome segregation process.[11]
The cytotoxicity of cisplatin is thought to be due to the formation of intrastrand and interstrand cross-links in the DNA, which may induce cell cycle arrest and apoptosis. Cisplatin-DNA adducts have been shown to be repaired predominantly by nucleotide-excision repair(NER).[12] Increased levels of the p53 protein result in transactivation of GADD-45 and p21WAFl/CIPl which have a key role in NER. The p53 protein also interacts directly with transcription factors involved in NER. MDM2 inhibits a number of p53-mediated biological activities. However, because the p53 pathway is complex, it was not clear whether MDM2 overexpression would inhibit p5 3-associated DNA repair. To compare the ability of A2780-M and A2780-V cells to remove DNA-Pt adducts after cisplatin treatment, we looked at levels of platinum bound to DNA. Similar levels of DNA-Pt binding were seen in A2780-M and A2780-V cells immediately after cisplatin treatment, as would be anticipated from the comparable rates of uptake. However, greater DNA-Pt binding was seen in A2780-M cells at later time points, suggesting compromised DNA repair kinetics in A2780-M cells. This method does not distinguish between decreased DNA-Pt binding as a consequence of DNA repair versus DNA synthesis. However, at the high concentration of cisplatin used (100 μM), it is unlikely that DNA synthesis was occurring. The results demonstrate that MDM2 inhibits DNA repair as a consequence of p53 inhibition.
In conclusion, we have demonstrated that transfection of MDM2 into the A2780 human ovarian cancer cells with wild type p53 status results in increased sensitivity to cisplatin. But MDM2 had no effect on the chemosensitivity in SKOV-3 cells which have null p53 expression. Our results suggest that MDM2 can change the sensitivity in human ovarian cancer cells through the MDM2-p53 feedback loop.
Acknowledgements
We thank Professor Robert Zeillinger from the Department of Obstetrics and Gynecology, University of Ulm Medical School, Germany, for the generous gift of the A2780 and SKOV-3 human ovarian cancer cell lines and Professor Moshe Oren from the Department of Molecular Cell Biology, the Weizmann Institute of Science, Isreal, for the pCMV-MDM2 plasmid.
- Received November 20, 2006.
- Accepted April 4, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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