br Cisplatin is a genotoxic
Cisplatin is a genotoxic agent that is quite eﬀective at killing HPV+ cervical cancers. Cisplatin works by inducing DNA crosslinking that activate many of the same cellular responses induced by UV exposure (McKay et al., 2001). For comparison with the UV-resistant HeLa cells, cisplatin-resistant HeLa SCR7 were generated using the previously de-scribed treatment-recovery cycle (Fig. 5A). Resistance was again es-tablished using MTT assays to determine LD50 values (Fig. 5C and Table 1). This approach allowed resistant cells to be identified and selected for further analysis. UV resistant HeLa A and B as well as cis-platin resistant HeLa A were chosen because they had the most robust resistance.
3.4. Sensitivity of UV- and cisplatin-resistant HPV+ cervical cancer cells to other genotoxic agents
We postulated that resistance to one type of crosslinking agent may not necessary result in resistance to agents that similar damage DNA. To begin testing this hypothesis and to characterize the mechanisms of resistance in these cell lines, MTT assays were conducted to define the LD50 for UV for the most cisplatin-resistant HeLa lines (Fig. 6A and Table 1). Although individual matched data points were not sig-nificantly between resistant and parental cell lines, a statistical analysis comparing the full data sets to each other revealed that a significant increase in UV resistance accompanied acquired cisplatin resistance in HeLa cells (Wilcoxon Matched Pairs Test, p < 0.05). The average LD50 increased from 65.47 in parental HeLa to 108.6 in the cisplatin resistant colony demonstrating an almost 2-fold diﬀerence in sensitivity. Next, MTT assays defined the LD50 for cisplatin in the most UV-resistant HeLa lines (Fig. 6B and Table 1). This analysis demonstrated that cells that acquired UV resistance could become more sensitive to other crosslinking agents. Finally, a recent report indicated that acquired cisplatin resistance could sensitize cells to PARP inhibition (Michels et al., 2013). To determine if this was true for the cisplatin-resistant HeLa cells described in this report, MTT assays were used to measure sensitivity to olaparib, a commercially available inhibitor of PARP1
Fig. 5. Creating cross-linker resistant HeLa and SiHa cell lines. A. This image depicts the steps used to generate the cross linker resistant cell lines. Briefly, cells were treated 4 times with either 10 μM cisplatin or 5 mJ UV and given recovery periods between treatments. The resulting colonies were then isolated and transferred to individual wells of a 6-well plate for expansion. The resulting clonal populations were then tested for acquired resistance by MTT. B. This chart depicts the LC50s (amount required to kill 50% of cells) for each isolated cell line. The color gradient represents increasing resistance with black being the most resistant. C. This graph depicts UV sensitivity of the 2 most UV resistant HeLa cell lines as measured by MTT. The solid black line and circles represent the parental HeLa. The grey line, square, and triangle represent the 2 most resistance colonies. D. This chart depicts the LC50s for each isolated cell line with error bars representing the 95% confidence intervals. The color gradient represents increasing resistance with black being the most resistant. E. This graph depicts the cisplatin sensitivity of the isolated cell lines as measured by MTT. The solid black line and circle points represent Parental HeLa. The light grey line and square points represent the most resistant colony. For all, n = 3, *p < 0.05 by unpaired t-test and error bars represent mean ± SD.
(Fig. 6C and Table 1). This analysis demonstrated that the sensitivity to PARP1 inhibition in HeLa cells that acquired cisplatin resistance de-pended on the concentration of inhibitor. At lower concentrations,
cisplatin resistance was associated with increased resistance to PARP1 inhibition, while at very high concentrations of inhibitor the cells were notably more sensitive. Since this result diﬀered from published
Lethal Concentrations in UV and cisplatin-resistant HeLa and SiHa. This table depicts the toxicities in cell lines before and after acquisition of resistance to cisplatin and UV. LC50 denotes the concentration or dose required to kill 50% of the cells calculated from MTT data (Fig. 6). 95% CI denotes the 95% con-fidence intervals. * denotes significance diﬀerence compared to parental cell line determined by Student t-test (p value ≤ 0.05).
UV toxicity (mJ/cm2)
reports, we generated a pool cisplatin-resistant cervical cancer cell line using SiHa cells (Supplemental Fig. 2). Unlike clonal populations of resistant cells, this cell line is likely to have gained resistance through multiple mechanisms providing a broader representation of resistance mechanisms. SiHa cisplatin resistant pooled cells were more sensitive to PARP1 inhibition via olaparib (Supplemental Fig. 3 and Table 1), suggesting that the increased dependence on PARP1 activity varies depending on the individual tumor or more likely the mode of re-sistance.