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  • br HPV oncogenes decrease sub erythemal UV

    2020-08-30


    3.2. HPV oncogenes decrease sub-erythemal UV-induced apoptosis
    This increased sensitivity is likely due to HPV E6 and E7 disrupting 205640-90-0 regulation and causing abnormal activation of DNA damage response genes. However, HPV oncogenes also mitigate the deleterious consequences of DNA damage by inhibiting apoptosis. To define the ability of HPV oncogenes to block the apoptosis in the context of a cervical cancer cell line exposed to UV, a shRNA system (PLL E61) was used to partially deplete HPV oncogenes in HeLa cells. HeLa cells with
    Fig. 4. Increased cell cycle arrest and apoptosis due to UV sensitivity in E6/E7 knockdown HeLa cells. A. Representative immunoblot showing BCL-2 related X protein (Bax) and p53 levels in shRNA 18E6-1 transduced HeLa cells with and without UV. Tubulin is the loading control. B. This graph depicts the percentage of HeLa cells in sub G1 after or without UV treatment. Cells were transduced with shRNA E6E7, lentiviral control, and BCL-2 exogenously expressed. For all, n = 3, *p < 0.05 by unpaired t-test and error bars represent mean ± SD.
    PLL E61 cells had decreased HPV E7 levels 2 and 4 days after treatment with shRNA (Fig. 2A). Because these genes share a promoter, their expression is known to be linked. Indeed, a recent study was unable to achieve knockdown of HPV E6 or HPV E7 individually (Wechsler et al., 2018). Thus, decreased HPV E7 abundance is a reliable marker of HPV E6 for which a dependable antibody does exist. To determine if this knockdown was persistent, oncogene levels were measured at 1 and 2 weeks after transfection with shRNA. Transfection with PLL E61 was able to detectably decrease the abundance of HPV E7 at these late time points (Fig. 2B). As a final measure of the PLL E61's ability to diminish HPV oncogene levels, the abundance of HPV oncogenes was determined after transfection with increasing amounts of PLL E61. Immunoblot analysis shows a dose dependent decrease in HPV E7 levels (Fig. 2C). Since there is no viable commercially available antibody to HPV E6, the degradation of its target protein, p53, is used as a surrogate for HPV E6 expression. P53 levels were determined by immunoblot after treatment with PLL E6. Notably, the reduction in oncogene abundance remains below the levels that result in apoptosis (data not shown).
    Having characterized PLL E61's ability to decrease the amount of HPV oncogenes in HeLa cells, the approach was used to define the role of HPV oncogenes in the apoptosis induced by low doses of UV. Supporting a role for HPV oncogenes in repressing UV-induced apop-tosis, both MTT and colony formation assays demonstrated that UV was more toxic to HeLa cells when HPV E6 and E7 were targeted by shRNA (Fig. 3A–B). Specifically, the knockdown of HPV oncogenes combined with exposure to a low dose of UV resulted in lower cellular viability and fewer colonies than either knockdown or UV exposure alone.  Gene 688 (2019) 44–53
    As noted, these assays do not directly measure apoptosis, so the abundance of BAX, a pro-apoptotic protein, was defined by im-munoblot. After exposure to a sub-erythemal amount of UV, HeLa cells demonstrated a mild induction of both BAX and p53 (Fig. 4A). In contrast, UV-induce increases in BAX and p53 abundance were en-hanced by HPV E6 and E7 knockdown. Notably, PLL E6.1 also increased the abundance of p53 in untreated HeLa cells further confirming the knockdown of HPV E6. To more directly detect the role of HPV onco-genes in protecting cervical cancer cells against apoptosis, flow cyto-metry was used to define the population of sub-G0/G1 cells after UV. The results from this assay are consistent with HPV oncogene-mediated inhibition of apoptosis as the percentage of sub-G0/G1 cells after UV was significantly increased by oncogene knockdown (Fig. 4B). As a control and to further confirm that the increase in the sub-G0/G1 po-pulation was due to BAX-induced apoptosis, BCL2, an anti-apoptotic BAX antagonist was transfected into HeLa cells. BCL2 expression pre-vented the accumulation of sub-G0/G1 cells after UV (Fig. 4B).
    3.3. Generation of UV- and cisplatin-resistant HPV+ cervical cancer cells
    The ability of HPV oncogenes to both sensitize cells to UV and protect them from UV-induced apoptosis portrays the complexity of their manipulation of the cellular damage response. To further probe this relationship, UV-resistant HeLa cell lines were generated by re-peated exposure to UV followed by a recovery period (Fig. 5A). This process was repeated at least 4 times and until individual resistant colonies could be isolated using trypsin soaked cloning rings. These colonies were then expanded and their sensitivity to UV was compared to parental HeLa cells via MTT assay. The LD50 was calculated for each resistant cell line and used to select highly resistant cells for further analysis (Fig. 5B and Table 1).