Reactivation of the p53 wild type function has been discussed as a promising therapy for cervical cancer. However, the literature published so far revealed conflicting data. Whereas some groups reported inhibition of cell growth without stimulation of apoptosis upon p53 reactivation in HPV related cervical cancer cells [18
], other groups described apoptosis as the outcome [12
]. In addition, induction of senescence upon interference with HPV oncogenes was also described [3
]. The use of different strains of long-term established cervical cancer cell lines could be responsible for these conflicting data. Those cell lines have been cultured in tissue culture for decades and contain multiple chromosomal abnormalities and functional defects, and do therefore not represent a good model for tumor cells in cervical cancer patients. In order to overcome this critical issue we established new cervical cancer cell lines, designated CC7 and CC10. Both cell lines contain integrated copies of HPV16 DNA only. To mimic the natural conditions as closely as possible only early passages of these cell lines were used in this study and compared to long-term established cell lines SiHa and HeLa.
We noted significant differences between the newly established CC cells and the long-term established cell lines with regard to the cisplatin sensitivity. For instance, whereas in CC cells the LD30 values never exceeded 12 μM, in HeLa and SiHa cells the LD30 values were ~20 μM and ~50 μM, respectively. Generally, cisplatin-induced DNA damage activates multiple signal transduction pathways including the ATM and/or ATR protein kinases resulting in phosphorylation of p53 at serine 15 and 20 [33
]. These modifications of p53 inhibit the interaction with the ubiquitin ligase Hdm2 leading to p53 stabilization, which now can coordinate repair processes, trigger cell cycle arrest or induce apoptosis. However, in HPV-positive cells the Hdm2 degradation pathway is completely switched off and degradation of p53 is taken over by the ubiquitin ligase E6AP in concert with the viral E6 oncoprotein, which is not affected by N-terminal modification of p53 [8
]. However, we observed that cisplatin was able to stabilize p53 in all cell lines investigated indicating that a fraction of p53 in all cells escaped degradation through the E6-E6AP complex. This p53 accumulation was directly linked to transcriptional induction of p21, showing that the cisplatin-induced p53 proteins are transcriptionally active. These findings are in line with other publications where cisplatin-induced p53 stabilization in HPV-infected cells has been observed [35
]. Interestingly, the molecular pattern of p53 accumulation and p21 induction was observed at similar time points and already at low cisplatin concentrations, where the difference in cell viability was largest between the novel CC cell lines and the long-term established SiHa and HeLa cells. Therefore, the kinetics of cisplatin induction of p53 and the observed cytotoxicity did not correlate providing a first hint for the dispensability of p53 in the cisplatin-induced cytotoxicity of cervical cancer cells.
To prove our initial hypothesis of a putative synergism between p53 reconstitution combined with cisplatin treatment we effectively silenced E6AP in all cell lines before cisplatin treatment. The loss of E6AP, as expected, resulted in p53 accumulation which was directly linked to the transcriptional induction of p21. In general, there was already a higher level of p53 protein in untreated CC cell lines in comparison to SiHa and especially HeLa cells. Although we observed differences in the extent of p53 stabilization and p21 induction upon silencing of E6AP there was no correlation between cisplatin-sensitivity and the capability to accumulate p53 upon E6AP loss. Long-term established SiHa and HeLa cells differed greatly in the extent of p53 reactivation and p21 induction indicating a high level of diversity among these cell lines. This observation is supported by earlier findings where RNAi mediated downregulation of E6AP in HeLa, CaSki and SiHa cells revealed only 12 genes commonly affected in all cell lines [37
]. Interestingly in the same publication, targeting E6AP expression did not affect the cell viability. Only when the siRNA concentration was increased to 200 nM, an effect towards cell viability was observed 5 to 7 days post transfection. In our study, using lower, but effective concentrations of a siRNA directed against E6AP, we did not observe reduced cell viability in all of the cell lines tested. In contrast, Hengstermann et al. reported growth suppression and stimulation of apoptosis induced by loss of E6AP [38
]. The reason for these discrepancies may be explained by the use of different assays to determine cell death. Whereas we used a WST-1 assay measuring viability of the whole cell population, other studies used in situ TUNEL assays and detected apoptosis in single cells, which may lead to an overestimation of the rate of apoptosis induction with respect to the whole cell population. We strongly believe that the results using the WST-1 assay do more appropriately reflect the situation of tumor shrinkage after treatment in the patient situation.
Surprisingly, when we combined E6AP silencing with cisplatin treatment, we did not observe synergistic or additive effects on cell killing, suggesting that reactivation of p53, although it was functional as a transcriptional regulator of the p21 gene, does not play a major role in cisplatin-induced cell death. These results disproved our working hypothesis and the question arose whether p53 in general is required for cisplatin cytotoxicity in cervical cancer cells. Hence, we knocked down p53 using siRNA followed by cisplatin exposure. Again, the cell viability of sip53-transfected cells exposed to cisplatin remained similar to that of the control-transfected cells treated with cisplatin. This outcome demonstrated that p53 might be not necessary for cisplatin-induced cell death and is in contrast to some earlier studies. Koivusalo and coworkers reported that restoration of p53 combined with different chemotherapeutic drugs protected cervical cancer cells from the cytotoxic effects of several compounds but increased the cytotoxicity of others [18
]. In the case of cisplatin they reported that activating p53 decreased chemosensitivity. On the contrary, Putral et al. reported that reactivation of p53 via targeting HPV oncogenes increased the sensitivity towards cisplatin in HeLa cells [16
]. However, this increased chemosensitivity could not be observed in vivo in a mouse model from the same group [39
]. The literature describing the relation between p53 status and sensitivity to anticancer drugs is indeed highly controversial. Whereas in some cancer cells the presence of wild type p53 is reported to render the cells more sensitive [40
], in other cancers a functional p53 renders the cells more resistant [43
]. Moreover, as it is known that p53-negative cells respond to cisplatin, a p53-independent mechanism of action must exist [45
]. In agreement with the latter assumption, it has been reported that cisplatin induced the expression of the pro-apoptotic gene Noxa in a p53-independent manner in HeLa cells [47
]. The anticancer mechanism of cisplatin is still not fully understood, but it is generally accepted that it acts through cross linking DNA thereby inducing DNA-damage associated apoptosis [27
]. However, Mandic et al., showed that cisplatin can also act outside of the nucleus and independent of DNA-damage by inducing ER-stress [50
]. Furthermore, cisplatin reacts with many other cellular components such as proteins, cytoskeletal microfilaments and thiol-containing molecules [51
]. In our analysis, we performed reconstitution of p53 via E6AP silencing or silenced p53 directly in combination with cisplatin treatment and our results strongly indicate that p53 is dispensable in cervical cancer cells for a successful cisplatin-based chemotherapy.