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  • br Fig Creating cross linker resistant HeLa and


    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, Filipin III 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
    Table 1
    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.
    4. Discussion
    We investigated the response of cervical cancer cells to low dose UV exposure. Particularly, our efforts illuminate the increased likelihood of UV-induced apoptosis in these cells. HPV oncogenes seem to counter the inclination towards programed cell death as their shRNA mediated knockdown caused increased p53- and BAX-associated apoptosis, sug-gesting dying cervical cancer cells sustain a greater abundance of DNA damage. This is somewhat suspected given the ability of HPV onco-genes to impair cellular DNA repair. We also generated clonal popu-lations of cervical cancer cells that were resistant to chemical- or ra-diation-induced DNA crosslinks and measured their sensitivity to other crosslinking agents and a PARP1-inhibitor.
    4.1. HPV oncogenes have both protective and detrimental effects on a cell's response to UV