br Cisplatin cisplatin Sigma Aldrich catalog
Cisplatin: cisplatin (Sigma-Aldrich catalog # 479306-1G) is a pla-tinum based antineoplastic agent that forms cytotoxic adducts with the DNA dinucleotide d(pGpG) that induces intrastrand cross-links. Olaparib: olaparib (LC laboratories catalog # O-9201) is a selective inhibitor of PARP1/2 with IC50 of 5 nM/1 nM in cell-free assays.
3.1. HPV+ cervical cancer cell lines are sensitive to sub-erythemal doses of UV
Fig. 1. Sensitivity of cervical cancer to UV. A. This graph depicts the sensitivity of cervical cancer cell lines to ultraviolet radiation when compared to primary human foreskin keratinocytes (HFK) as measured by MTT. The black square points and solid line represent HFKs. The black circle points and dotted line represent HeLa cells, a cervical cancer cell line. B. This graph depicts the combined results of the colony formation assays. The solid black line and solid black squares represent HeLa and the dotted line and dot points are non-cancer HFK. C. This graph depicts the percentage of apoptotic (propidium iodide po-sitive) cells 48 h after increasing doses of UV. For all, n = 3, *p < 0.05 by unpaired t-test and error bars represent mean ± SD.
and inducing a highly coordinated signaling response. HPV oncogenes transform keratinocyte cells in part by disrupting both processes. HPV E7 degrades the master 130-40-5 regulator, RB. While both HPV E6 and E7 induce aberrant activation of DNA damage proteins. We hypothesize that the continued expression of HPV E6 and E7 in cervical cancer cell lines will make them less able to respond to UV and as a result more sensitive to UV exposure. This hypothesis was tested by defining the
Fig. 2. Confirmation of lentiviral transduction. A. Representative immunoblot showing E7 levels in HeLa cells transduced with the empty vector PLL, shRNA E6-1, or shRNA E6-2. Lysates were collected 2 days post-lentiviral infection (PI) or 4 days PI. Tubulin is the loading control. B. Representative immunoblot showing E7 levels in HPV18 HeLa cells transduced with empty vector PLL or a gradient of shRNA 18E6-1. C. Representative immunoblot showing E7 levels in HeLa cells transduced with the empty vector PLL or shRNA E6-1. Lysates were collected 1 and 2 weeks PI. Tubulin is the loading control. D. Graphs showing relative E7 protein determined by densitometry.
sensitivity of a HPV+ cervical cancer cell line (HeLa) to UV. As a control, untransformed primary keratinocytes derived from neonatal foreskins (HFKs) were also observed because HPV infects and trans-forms keratinocytes. HeLa cells were significantly more sensitive to a gradient of low dose UV (0–10 mJ/cm2) than HFKs when viability was measured by MTT assay (Fig. 1A).
As a complimentary measure of UV sensitivity, a colony formation
assay was used to measure the toxicity of the same range of UV doses. These results confirm the data obtained by the MTT assay. Indeed, HeLa cells were significantly more sensitive to all exposures of UV in the colony formation assay, with virtually no colonies visible after 1 mJ/ cm2 of exposure (Fig. 1B and Supplemental Fig. 1). Conversely, HFKs formed readily detectable colonies at all observed doses of UV. The sensitivity resulting from an inability to properly respond to UV
Fig. 3. Increased UV sensitivity in E6/E7 knockdown HeLa cells. A. This graph depicts UV sensitivity in HeLa cells transduced with the empty vector PLL or shRNA E6-1 as measured by MTT assay. B. This image depicts UV sensitivity in HeLa cells transduced with empty vector, a shRNA Control, or a shRNA 18E6E7 by colony formation assay (CFA). C. This graph depicts UV sensitivity in HeLa cells transduced with the empty vector PLL or shRNA 18e6-1 by cell counting. For all, n = 3, *p < 0.05 by unpaired t-test and error bars represent mean ± SD.
damage is likely manifest in elevated apoptosis. Unfortunately, neither a colony formation assay nor a MTT detect apoptosis directly and thus they cannot distinguish apoptosis from other types of cell death. To determine if HPV+ cervical cancers have an increased propensity to undergo apoptosis after UV exposure, we measured propidium iodide (PI) staining as a standard indicator of apoptosis (Fig. 1C). PI is a membrane impermeable dye that intercalates between DNA base pairs. We used a fluorescence-based assay to detect PI. Cell membranes be-come permeable during apoptosis allowing PI uptake measurable at 617 nm. Taking the ratio of cells that are stained with PI to the total cell count allows us to determine the percentage of apoptotic cells after UV exposure. We found HeLa cells had significantly higher levels of PI
staining compared to HFKs when both were exposed to either 2 or 4 mJ/cm2 of UV (Fig. 1C).