Figures and data
Dysregulation of Polκ in cisplatin treated and resistant cancer cells.
(A) Western blot analyses of various cell-free lysates of cell lines without and treated with 10 μM cisplatin were carried out using Polη and Polκ antibodies. β-actin was used as a loading control. (B) Protein band intensities were estimated and fold change after β-actin normalization was shown in bar graph as mean ± SEM (n=2). Statistical significance determined by two-way ANOVA, Šídák’s multiple comparisons test (**p < 0.01, ***p < 0.001, ****p < 0.0001). (C) Schematic representation of generation of cisplatin resistant OSCC cell lines (H357-R and SCC9-R) from their respective cisplatin sensitive H357-S and SCC9-S cells. (D) RT-PCR analysis of various DNA polymerases in cisplatin resistant and sensitive OSCC cells. ACTIN was taken as a loading control. (E) Western blot analyses of lysates of OSCC cells using specific antibodies of various DNA polymerases or their subunits as mentioned. β-actin was detected as a loading control.
Absence of Polκ re-sensitizes OSCC resistant cells to cisplatin.
(A) H357-S, H357-R and H357-R POLK KO cells were treated with 5 μM cisplatin and analyzed for Polκ expression by Western blot analysis. β-actin was used as a loading control. (B) Colony-forming assay was performed using H357-S, H357-R and POLK KO cells without and with 5 μM cisplatin (i). Bar graph represents colony percentage as mean ± SEM (n=3) as determined by two-way ANOVA with Tukey’s multiple comparisons test (***p < 0.001, ****p < 0.0001, and ns- nonsignificant) (ii). (C) Cells were treated with cisplatin for 48 hours and then MTT assays were performed. Graph indicates the mean ± SEM (n=3), two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, ***p < 0.001, ****p < 0.0001, and ns- nonsignificant). Purple asterisks indicate comparison between H357-R to H357-S, pink asterisks indicate comparison between H357-R to H357-R POLK KO. (D) After treatment with 5 µM cisplatin for 48 hours, cells were stained with Annexin V/PI and analyzed by flow cytometry. Percentage of cells in each quadrant is as indicated (i). Bar graph represents percentage of total apoptosis (early and late apoptosis). Data were represented as mean ± SEM (n=3). Statistical significance determined by two-way ANOVA, Tukey’s multiple comparisons test, (***p < 0.001, ****p < 0.0001, and ns- nonsignificant) (ii).
Absence of Polκ enhances genomic instability and delays cell cycle progression in OSCC resistant cells.
(A) Representative alkaline comet images showing DNA tailing in cells following treatment with 5 μM of cisplatin for 48 hours (i). The bar graph represents percentage of tail DNA. Data represented as (mean ± SEM; n=3). Statistical significance determined by two-way ANOVA with Tukey’s multiple comparisons test (****p < 0.0001, and ns- nonsignificant) (ii). (B) Representative immunofluorescence images of H357-S, H357-R and H357-R POLK KO cells showing γH2AX (red) puncta and DAPI (blue) and corresponding merged images (i). Quantification of γH2AX foci numbers is indicated in a bar diagram as mean ± SEM (n=3) (ii). Statistical significance determined by Ordinary one-way ANOVA, Tukey’s multiple comparisons test, (****p < 0.0001, and ns- nonsignificant). Scale bars indicate 7.5 μm. (C) Western blot analyses of H357-S, H357-R and H357-R POLK KO lysate using γH2AX antibody was carried out. β-actin was used as a loading control. (D) Western blot analyses of H357-S, H357-R and H357-R POLK KO lysate probed with PARP1 antibody was carried out. β-actin was used as a loading control. (E) A representative cell cycle profiles of H357-S, H357-R and H357-R POLK KO cells at different time points after releasing from double-thymidine block were shown. G1, S, and G2M phases were demarcated (i). The percentage of cells in G1, S and G2-M phases at 12 h is estimated and plotted. Data was represented as mean ± SEM (n=2) (ii). Statistical significance determined by Ordinary one-way ANOVA, Tukey’s multiple comparisons test, (*p < 0.05, and ns- nonsignificant) (ii).
DNA replication in OSCC cisplatin resistant cells is governed by Polκ.
(A) H357-S, H357-R and H357-R POLK KO cells were treated with the indicated concentration of drugs HU (i), Aphidicolin (ii) and Camptothecin (iii) for 24 hours, and then an MTT assay was performed. These graphs indicate the mean ± SEM (n=3) and were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns- nonsignificant). Purple asterisks indicate comparison between H357-R to H357-S, pink asterisks indicate comparison between H357-R to H357-R POLK KO. (B) A schematic representation of the critical motifs of Polκ and their mutations involved in catalytic activity and PCNA interaction is shown. Knockout cells harboring GFP-tag of WT, PIP mutant and Catalytically Dead (CD) Polκ were analyzed by Western blotting. EV indicates cells possessing an empty vector. β-actin was taken as an internal control. (C) Transfected cells were treated with the indicated concentrations of HU (i), Aphidicolin (ii) and Camptothecin (iii) for 24 hours, and then MTT assays were performed. These graphs indicate the mean ± SEM (n=2) and statistically verified by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns-nonsignificant). Cyan, orange and violet asterisks indicate comparison between EV to GFP-WT, GFP-CD and GFP-PIP respectively. (D) A schematic representation of DNA fiber analysis in the presence of HU and representative images of various replication events in H357-S, H357-R and H357-R POLK KO cells were shown. The fork speeds were measured and the values of three independent experiments were pooled and plotted. Bar graph showing percentage of various replication events in H357-S, H357-R and H357-R POLK KO cells. Data were represented as mean ± SEM (n=3). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (**p < 0.01, ***p < 0.001, ****p < 0.0001, and ns-non significant). Scale bars indicate 10 μm. (E) Immunofluorescence co-localization analysis of H357-S and H357-R cells showing p12 and p50 subunits of Polδ (red), Polκ (green), DAPI (blue), and corresponding merged images. Merged images represent co-localization of Polδ with Polκ in the nucleus. Zoom-in merged (only green and red) view of dashed box area showed in upper right corner of corresponding image. Scatter plot of representative merged images is shown. Percentage of co-localization was shown in bar diagram. Scatter plot and co-localization percentage were obtained from confocal microscope Stellaris 5. Data were represented as mean ± SEM (n=3). Statistical significance was determined by t-test, (**p < 0.01, ***p < 0.001). Scale bars indicate 7.5 μm. (F) Co-immunoprecipitation was performed in H357-R whole cell extract using either Polκ or PCNA antibody. Interaction between Polκ with PCNA/p50/p12/Polκ and interaction between PCNA with Polκ/p50/p12/PCNA were analyzed by Western blotting. IgG was consider ed as a negative control. Representative best immunoblots from two independent experiments are shown.
Structural function of Polκ in the protection replication fork and restart post-cisplatin exposure.
(A) Western blot analysis shows ectopic expression of GFP-tagged WT, PIP mutant and Catalytically Dead (CD) mutants in H357-R POLK KO cells. EV indicates an empty vector. β-actin was considered as an internal control (i). Colony-forming assay was performed using H357-R and H357-R POLK KO transfected cells (ii). Bar graph represents colony percentage as mean ± SEM (n=4), statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, ****p < 0.0001, and ns- nonsignificant) (iii). (B) Cells were treated with the indicated concentrations of cisplatin for 48 hours and then an MTT assay was performed. These graphs indicate the mean ± SEM (n=3), two-way ANOVA, Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns- nonsignificant). Pink asterisks indicate comparison between H357-R to H357-R POLK KO EV. Cyan, orange and violet asterisks indicate comparison between EV to GFP-WT, GFP-CD and GFP-PIP respectively. (C) Schematic representation of DNA fiber analysis as carried out in the presence and absence of cisplatin (i). Representative replication event images were shown (ii). The fork speed were measured and the values of three independent experiments were pooled and plotted (iii). Bar graph showing percentage of various replication events in H357-S, H357-R and H357-R POLK KO cells with and without cisplatin (iv). Data were represented as mean ± SEM (n=3). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns-non significant). Scale bars indicate 10 μm. (D) Representative images of DNA fibers in H357-R and H357-R POLK KO cells transfected with EV and mutants (GFP-WT, GFP-PIP, GFP-CD) without or with treatment with 5 μM cisplatin for 24 hours before labelling are shown (i). The fork speeds were measured and the values of three independent experiments were pooled and plotted for H357-R and H357-R POLK KO transfected cells with or without cisplatin (ii). Bar graph shows the percentage of various replication events with and without cisplatin. Data were represented as mean ± SEM (n=3) (iii). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ****p < 0.0001, and ns-non significant). Scale bars indicate 10 μm.
Absence of Polκ dysregulate ATM -ATR signaling and DNA strand break repair pathways.
(A) Western blot analyses of cell-free lysates of H357-S, H357-R and H357-R POLK KO cells without and treated with 5 μM cisplatin were carried out, and were probed with the indicated antibodies against checkpoint protein. β-actin was detected as a loading control. (B) Similar Western blot analyses but probed with antibodies of proteins involved in HR pathway. (C) Similar Western blot analyses but probed with antibodies of proteins involved in NHEJ pathway. (D) Western blot analyses of nuclear extracts of H357-S, H357-R and H357-R POLK KO cell lines without and treated with 5 μM cisplatin were carried out and were probed with indicated specific antibodies. Histone H3 was used as a loading control.
Stabilization of proteins involved in checkpoint and DSB repair pathways form proteasomal degradation by Polκ.
(A) RT-PCR analysis of indicated genes was carried out in H357-R and H357-R POLK KO cells (i) and the mRNA expression levels were represented in bar graph (ii). Data were represented as mean ± SEM (n=2). Statistical significance determined by two-way ANOVA with Tukey’s multiple comparisons test (****p < 0.0001, and ns- nonsignificant). (B) Western blot analyses of cell lysates of H357-R POLK KO and H357-R POLK KO cells without and treated with 10 μM of MG132 for 6 hours was conducted and probed with indicated antibodies. β-actin was used as a loading control. Densitometric fold change relative to proteins in H357-R cells has been provided just below each band. (C) Representative immunofluorescence images of H357-R POLK KO and H357-R POLK KO cells treated with 10 μM of MG132 show DAPI (blue), Chk1 (green), Rad51 (green), Chk2 (red), BRCA2 (red), and Artemis (red), and corresponding merged images. Scale bars indicate 7.5 μm.
Dysregulation of proteasomal degradation and deubiquitinating systems in cisplatin resistant OSCC.
(A) A comparative transcriptomics data of H357-S and H357-R was retrieved and re-analyzed. String analysis revealed a total of 349 dysregulated genes of protein degradation pathways (i), out of which 13 genes are significantly up-regulated deubiquitination pathways (ii), 76 genes are down-regulated involved in ubiquitination pathways (iii), and 8 genes are down-regulated those are components of 26S proteasomal complex (iv). (B) Endpoint RT-PCR analysis of indicated genes was carried out in H357-S, H357-R and H357-R POLK KO cells (i) and qRT–PCR gene expression analysis of the indicated genes in H357-S, H357-R and H357-R POLK KO cells using SYBR Green. ACTIN was used as a control. ΔΔct values were calculated and compared for the H357-S, H357-R and H357-R POLK KO cells. Calculated fold change was plotted. Data were represented as mean ± SEM (n=3) (ii). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (**p < 0.01, ****p < 0.0001, and ns- nonsignificant). (C) Western blot analyses of H357-S, H357-R and H357-R POLK KO lysate were carried out and probed with indicated antibodies. β-actin was used as a loading control. (*) indicates a non-specific band for USP2 antibody. USP18 antibody detected two sizes of the protein. Fold change in protein expression was shown in a bar graph. Data represented as mean ± SEM (n=2). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, ****p < 0.0001, and ns- nonsignificant).
PCNA-Polκ-USP18 axis stabilizes DDR proteins.
(A) Co-immunoprecipitation was performed in H357-R whole cell extract using either Polκ (i) or USP2 and USP18 antibodies (ii). Similar interaction between PCNA with USP2 and USP18 was determined. IgG was considered as a negative control. Representative best immunoblots from two independent experiments are shown. (B) Immunofluorescence co-localization analysis of H357-R cells showed USP2 (red), USP18 (red), Polκ (green), DAPI (blue), and corresponding merged images. Merged images represent co-localization of USP2 and USP18 with Polκ in the nucleus. Zoom-in merged (only green and red) view of dashed box area showed in upper right corner of corresponding image. (C) Western blot analyses of cell lysates of H357-R, MG132 untreated and treated H357-R USP18 knockdown cells were carried out and expression levels of indicated proteins were determined. Densitometric fold change relative to β-actin has been provided just below each band. (D) Cells were treated with the indicated concentrations of cisplatin for 48 hours and then an MTT assay was performed. The graph indicates the mean ± SEM (n=3), and statistical significance was determined by two-way ANOVA, Šídák’s multiple comparisons test, (*p < 0.05, **p < 0.01, ***p < 0.001, and ns-nonsignificant). Pink asterisks indicate comparison between H357-R (si NTC) to H357-R (si USP18). (E) H357-S, H357-R and H357-R POLK KO cells were treated with the indicated concentrations of T2AA alone and with cisplatin were treated for 48 hours and then an MTT assay was performed. Graph indicates the mean ± SEM (n=3), two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns- nonsignificant). Purple asterisks indicate comparison between H357-R to H357-S, pink asterisks indicate comparison between H357-R to H357-R POLK KO, and green asterisks indicate comparison between H357-S to H357-R POLK KO.
Dual tripartite interactions mediated by Polκ govern uninterrupted DNA replication and genome stability in OSCC cisplatin resistant cells.
In the cells having low Polκ expression the bulky adducts generated by cisplatin could not be bypassed leading to more fork stalling and collapse, which ultimately leads to cell death. Whereas in cisplatin resistant cells where Polκ is overexpressed, it modulates two axes independent of its catalytic activity. The PCNA-Polκ-Polδ axis facilitates efficient replication and proliferation of cells, the other axis PCNA- Polκ-USP18 stabilizes DNA damage response proteins to activate checkpoint and HR/NHEJ repair pathways during cisplatin exposure.
List of oligonucleotides used in the study.
