Fig 1.
HPV oncogenes expressing cells impair ICL repair and depend on FancD2 for cisplatin sensitivity.
(A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-D) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA (siFancD2 or siControl) and treated with indicated doses of cisplatin for 48 hr. (D) Survival curve of siControl cells treated with indicated doses of cisplatin for 48 hrs.
Fig 2.
HPV oncogenes impair the repair of ICLs.
(A) Outline of an experiment to evaluate repair of cisplatin-induced ICLs using the modified alkaline comet assay. Cells were treated with 1.5 μM cisplatin for 2 hr and then incubated in cisplatin-free medium. Triangles (Δ) represent the time points (0, 24, 48 and 72 hr after cisplatin release) when cells were harvested and frozen. Immediately before comet analysis, cells were thawed and irradiated with 12 Gy to deliver a fixed level of random DSBs. (B) Representative comet images of untreated and cisplatin treated+ irradiated cells at 0hr and 72 hr. The insets are the magnified comets. A mean tail moment from a representative assay is shown. (C) Quantification of % ICL present in LXSN and E6/E7 expressing HFK cells at different post-treatment incubation time. The percentage of ICLs remaining was determined as described in Methods. Data represent mean ± SEM of at least 3 independent biological replicates. * (p< 0.05), *** (p ≤ 0.001), **** (p< 0.0001) denote a statistically significant difference from LXSN at the same post incubation time. ‘ns’ denotes non-significant differences.
Fig 3.
HPV oncogenes increase chromatin-bound monoubiquitinated FancD2/ FancI.
(A) Immunoblot showing FancD2/ FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, total FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.
Fig 4.
HPV oncogenes increase FancD2 foci formation but impair colocalization of FancD2 with DSBs, and mislocalized FancD2 in E6 cells causes reduction in Rad51 recruitment to DSBs.
(A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.
Fig 5.
HPV E6 causes delayed de-ubiquitination of FancD2.
(A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in Fig 4A, following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. FancD2 Ub:non-Ub ratio in cells following cisplatin withdrawal and UVB exposure was plotted alongside Figure 5B and 5C. ** (p< 0.01) denotes a statistically significant difference from 0 hr cisplatin withdrawal. ‘ns’ denotes non-significant differences. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in Fig 6A. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 and total FancI in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.
Fig 6.
Cell-cycle specific process of FancD2 monoubiquitination/ de-ubiquitination is not abrogated in E6 cells.
(A) Outline of synchronization assay in HFK cells using double-thymidine block. Cells were synchronized by double-thymine block and released at various time points. Immunoblot for FancD2, FancI, cyclin A and vinculin of LXSN (B) and E6 cells (C). Cell-cycle phases at each time point were determined by flow cytometry of DNA content (D). Asynchronous (Asyn.) cells, which have not undergone thymidine block, were included for comparison.
Fig 7.
E6 mediated increased FancD2 monoubiquitination is p53 independent, but delayed FancD2 de-ubiquitination is dependent on p53 degradation.
(A) Immunoblot for p53 (upper panel) and RT-PCR analysis of 16E6 and GAPDH expression (lower panel) in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm2 UVB (D) and processed similarly as in Fig 5.
Fig 8.
E6 attenuates Palb2 expression and foci formation.
(A-C) HFKs were transduced with LXSN, E6 or E6/E7 (A) Immunoblot showing Palb2 expression. Actin serves as a loading control (left panel). Graph showing relative Palb2 protein expression from 3 independently derived E6/E7 cell lines (right panel). (B) Cells were untreated or treated with cisplatin (3 uM) for 24 hr and immunostained with Palb2, pH2AX, and DAPI. Cells with >5 Palb2 foci were counted and the percentage of positive cells is plotted (n = 3, mean ± SD). * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas ns indicates non-significant. (C) HFKs were transfected with siControl or siPalb2. Immunoblot confirming depletion of Palb2 (upper panel). Cisplatin survival assay for siRNA-transfected cells (lower panel).