br Abbreviations list HPV human papillomavirus UV ultraviole
Abbreviations list: HPV, human papillomavirus; UV, ultraviolet radiation; E6AP, E6 associated protein; RB, retinoblastoma protein; TLS, translesion synthesis; NER, nucleotide excision repair; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
E-mail addresses: [email protected]ﬃth.edu.au (N. McMillan), [email protected] (N.A. Wallace).
1 Current address: Australian Institute for Bioengineering and Nanotechnology (Building 75), The University of Queensland, Cooper Rd., St Lucia, Brisbane QLD 4072, Australia.
2001) correlating with limited NER and more mutations after UV ex-posure (El-Mahdy et al., 2000; Havre et al., 1995; Rey et al., 1999). Both HPV oncogenes hinder the homologous recombination pathways that respond to double strand DNA breaks that can result from UV-induced replication fork collapse (Wallace et al., 2017). The repair mechanisms that respond to UV are also critical for repairing lesions caused a number by genotoxic drugs that cause DNA crosslinks (Alt et al., 2007; Furuta et al., 2002; Ho et al., 2006). It is not surprising then that HPV E6 and E7 sensitize HG 9 91 01 to this class of chemotherapeutics (Koivusalo et al., 2002; Liu et al., 2000; Wallace et al., 2012). The dependence of cervical cancers on these viral oncogenes suggests that these drugs would be particularly lethal to cervical malignancies.
However, HPV oncogenes also activate a plethora of DNA repair proteins including those in the pathways that respond to UV damage; nucleotide excision repair (NER), Fanconi anemia repair (FA) and translesion synthesis (TLS) (Alan and D'Andrea, 2010; Knobel and Marti, 2011; Schärer, 2013). HPV depends on DNA repair pathways to replicate its genome (Anacker et al., 2014; Gillespie et al., 2012; Hong et al., 2015; Moody and Laimins, 2009; Spriggs and Laimins, 2017a). In addition to creating an environment that is seemingly primed to re-spond to crosslinked DNA, HPV oncogenes inhibit the apoptosis in-duced by these lesions (Garnett et al., 2006; Garnett and Duerksen-Hughes, 2006; Jackson et al., 2000; Leverrier et al., 2007). Thus in at least certain scenarios, HPV E6 can protect against DNA crosslinking agents (Koivusalo et al., 2002).
The balance between chemo-sensitization and chemo-resistance is critical in the management of any cancer. DNA repair and damage tolerance pathways are particularly important with regard to cervical cancer as a DNA crosslinking agent, cisplatin, is currently the standard of care (Lorusso et al., 2014). Resistance to cisplatin in advanced and recurrent cervical cancers results in only a 10–20% chance of living through the year (Diaz-Padilla et al., 2013). As a result, substantial eﬀort has been made to identify potential mechanisms of cisplatin re-sistance as well as ways to re-sensitize cervical cancer cells (Kilic et al., 2015; Roy and Mukherjee, 2014; Zhu et al., 2016). The eﬃcacy of cisplatin is at least partially determined by gene expression patterns in cervical cancer cells, with multiple DNA repair genes being identified as important predictors of cisplatin sensitivity (Garzetti et al., 1996; Hasegawa et al., 2011; Henríquez-Hernández et al., 2011; Kitahara et al., 2002; Saito et al., 2004; Zhu et al., 2016). Because cisplatin in-duces DNA damage, increased repair capability likely accounts for the resistance (Woods and Turchi, 2013).
Conversely, if HPV oncogenes block repair in a predictable manner, these defects could be leveraged therapeutically. The sensitivity of BRCA1 or BRCA2 deficient breast and ovarian cancers is the most publicized link between known repair deficiencies and improved cancer treatments (D'Amours et al., 1999; Jasin, 2002). A similar relationship exists for cisplatin sensitivity. Specifically, the response of TLS and NER to UV predicts the lethality of cisplatin exposure (Gueranger et al., 2008; Marteijn et al., 2014; Rosell et al., 2003; Srivastava et al., 2015). In this manuscript, we characterize the interplay between HPV onco-gene-induced sensitivity to crosslinking agents and their protection from these genotoxins in the context of cervical cancer cell lines. We show that, compared to control cells, cervical cancer cells are more sensitive to sub-erythemal UV exposure and low doses of cisplatin. We demonstrate that these cells have a greater tendency to undergo apoptosis after UV. Interestingly, HPV E6 and E7 also block apoptosis after UV. We generate UV- and cisplatin-resistance cervical cancer cell lines and define their cross sensitivity. Finally, we determine the toxi-city of a chemotherapeutic agent (olaparib) with a diﬀerent mechanism of action (PARP1 inhibition) in cervical cancer cells that are resistant to Gene 688 (2019) 44–53
2. Methods and materials
LD50s were determined by MTT assay. HeLa cells were seeded in a
96 well plates and treated with UV, cisplatin, or olaparib at specified doses. 48 h after treatment, MTT solution was added to the cells and solubilized 24 h later. Plates were then read for optical density by spectrophotometer with a dual reading at frequencies 550 and 655 nm.