Characterization of XP-C and Wild-Type (WT) fibroblasts used in this study.
XPC protein expression is lost in XP-C cells compared to WT cells
The expression of XPC protein was examined in both WT and XP-C cells immortalized from patient primary fibroblasts. Immuno-staining of both cells using an antibody against the XPC protein was carried out. In contrast to WT cells, XP-C cells showed a total absence of XPC protein. Remarkably, XPC was exclusively localized in the nuclei of WT cells (Fig. 1a).
Increased photosensitivity of XP-C cells in response to UVB irradiation compared to WT cells.
To examine the photosensitivity of XP-C cells relative to WT, both kinds of cell lines were seeded until 80% confluency then subjected to increasing doses of UVB. 24 hours after UV treatment, cell viability was recorded as a measurement of the cells’ reducing capacity (Prestoblue@ assay). Viability was normalized based on the calculation of the percentage of viability compared to the viability of control non-irradiated cells set as 100% of viability. Both cell lines showed a decrease in viability as a function of increased UVB doses. XP-C cells were more photosensitive compared to the WT ones showing a sharper significant decrease in viability at each UVB dose (p < 0.001). The UVB dose leading to 50% of mortality was determined for both XP-C and wild-type cells (LD50). XP-C cells showed a much lower LD50 (about 0.02j/cm2) compared to WT cells (about 0.19j/cm2) (Fig. 1b). These results confirm the strong sensitivity to UVB of XP-C cells used in this study as described in other model21.
Absence of XPC impairs DNA repair of UVB-induced lesions
XPC protein is essential for lesion recognition step of GGR22. For that, we aimed to determine the effect of XPC mutation on the repair of lesions. The two major photoproducts generated after UVB exposure are CPDs and 6-4PPs that are formed between pyrimidine dimers either TT, TC, CT, or CC. The cells were seeded until 80% confluency then irradiated at 0.02 j/cm2, corresponding to the previously determined XP-C LD50, and then collected post UVB treatment at different time points. In order to monitor DNA lesions, DNA was extracted and digested to be analyzed by LC-MS/MS. For CPDs, the four different lesion types were quantified. WT cells showed a decrease in different CPDs lesions’ amounts as a function of time to reach a minimum after 48h indicating efficient repair of DNA damage while in contrast, XP-C cells showed constant elevated amounts of lesions as a function of time (Fig. 1c). It should be noted that not all lesions were present in the DNA in the same quantities: the majority of CPD lesions were of TT-CPD nature followed by TC-CPD, CT-CPD while the least abundant lesion was CC-CPD in both kinds of cells. In addition, the repair kinetics in wild-type cells differed between lesion types with the fastest repair observed for the CT-CPD. Only the TT and TC lesions could be detected for the 6-4PPs, as the two remaining lesions were less frequent. 6 − 4 PPs were repaired faster than CPD with total repair 24h post UV in WT cells compared to the higher amount of lesions in XP-C cells. There were no discrepancies in the repair kinetics between the TT and TC 6-4PP lesions in normal cells (Fig. 1c). Taken together, our results show that wild-type but not XP cells efficiently repair photoproduct resulting from UVB irradiation.
Primary Screen identified 16 candidate compounds that increase XP-C cells viability post UVB irradiation
To identify compounds that would correct the photosensitive phenotype of XP-C cells, we did set up a robotic assay in 96-well microplate to monitor cell viability after UVB irradiation (see methods and supplementary Fig. 1). The robustness of the assay was calculated as a Z’ factor23 at various UVB doses on a Z’ plate including 48 bioactive controls mimicking the desired effect (here, non-irradiated protected XPC cells in DMSO) and 48 bioinactive controls (here, irradiated cells in DMSO). A library of 1280 FDA and EMA approved drugs (Prestwick@) was then screened on XP-C cells at 10µM for 24h in duplicates using this assay. Briefly, following drug treatment, the cells were UVB irradiated at 0.05j/cm2 based on Z’ Factor calculation (supplementary figure S1), then post-treated with the same drugs and incubated at 37°C for an additional 24h period of time before the measurement of cell viability. In this setup, the effect of drug treatment on cell viability can be either preventive or curative to the UVB-induced damage. The bioactivity of each tested compound, signifying the drugs’ beneficial effects on photo-resistance, was calculated by determining the fluorescence of each well with respect to the fluorescence of non-irradiated DMSO treated wells set as 100% (bioactive control) and irradiated DMSO treated wells set as 0% (bioinactive control). The robust Z score was also calculated per plate as an additional normalization technique23. The hit selection was based on two criteria: compounds that possess a bioactivity ≥ 25% and a robust Z score ≥ 2.5. Sixteen molecules were identified as primary hits on this basis and selected for further characterization of their bioactivity on XP cells (Fig. 2).
Selection of three bioactive drugs increasing XP-C cells viability after UVB irradiation
In order to select the most efficient compounds showing robust activity on XPC viability among the 16 primary hits, a secondary screen was carried out using the same assay as the initial primary screen. Additional conditions were included together with the initial screen’s conditions. The drugs were indeed screened at three different concentrations (1, 5 and 10µM). Moreover, the cells were irradiated at two doses of either 0.02j/cm2 or 0.05j/cm2. Finally, each drug was screened in triplicate in each of the different irradiation conditions and drug concentrations. The same viability readout was measured as for the primary screen. The acetohexamide drug was also added as a positive control as it was reported to increase XP-A cells’ viability in response to UV irradiation 24 where XPA is a protein involved in the damage verification process downstream damage recognition post irradiation25. For each drug, a dose-response curve of bioactivity as a function of drug concentration (in µM) was plotted for the different doses tested. The drugs that showed the highest bioactivity were isoconazole, clemizole hydrochloride, and bifonazole inducing 20 to 40% increase in XP-C cell viability depending on the UVB exposition and drug concentration. In contrast, the acetohexamide only mediated a 20% increase in viability of XPC cells and displayed no increased bioactivity when applying increasing concentrations to the cells (Fig. 3). This secondary screen defines three promising drugs protecting XPC cells from UVB toxicity
Identification of drugs that promote the repair of DNA damage
In order to determine the mechanisms of action of the 16 primary hits and acetohexamide, we tested whether they could promote DNA repair activity in XP-C cells. For that, DNA damage analysis was carried out after cells were treated with the 17 drugs and irradiated at a dose of 0.05j/cm2 using the same protocol as for the primary screen. After the measurement of bioactivity, the plates were fixed and immuno-stained with an anti-6-4PP antibody for the quantification of the amount of DNA damage following UVB irradiation in the presence of drug or not (supplementary figure S2). The positive control showing maximal DNA damage was the DMSO treated irradiated cells while the negative control with minimal damage is the non-irradiated DMSO treated cells. XP-C cells treated with either isoconazole or clemizole hydrochloride showed a significant decrease in the amount of 6 − 4 PP at the single-cell level compared to untreated controls suggesting enhanced repair activity due to drug treatment. This decrease in DNA damage was significant at a 5µM concentration for both drugs (p < 0.001) while not at 1µM that may be a too weak concentration or at 10 µM where the difference was not found to be significant (Fig. 4). These results thus identified isoconazole and clemizole hydrochloride as two drugs promoting DNA repair in response to UVB irradiation in XP-C cells.
Dose-response analysis of drug bioactivity as a function of UVB doses in XP-C and WT cells
To further characterize the effect of isoconazole and clemizole hydrochloride drugs on XP-C cells versus wild-type cell viability enhancement, UV dose-response experiments were carried out. In contrast to the previous experiments, the dose-response analysis here was performed on both the XP-C and WT cell lines and viability was normalized to that of non-irradiated cells (fixed at 100%). This task aims to decipher whether the effect of the two drugs on cell viability is specific to XP-C cells or also valid in WT cells. The two cell lines were treated with either isoconazole, clemizole hydrochloride or DMSO then subjected to increasing UVB doses. Both drugs enabled enhanced photo-resistance to the increasing UVB doses in both XP-C and WT compared to DMSO treated cells (Fig. 5a & 5b). Such photo-resistance was significant for both drugs in XP-C cells with a p-value < 0.05. However, in WT cells, the treatment with isoconazole showed significant photo-resistance against increased UV doses irradiation (p-value < 0.05), unlike clemizole hydrochloride whose increase in viability was not significant. In conclusion, this protective effect may not be specific to XP-C cells as a similar protective effect was also shown for the WT cells. This is consistent with their observed ability to increase DNA repair efficiency.
Investigation of pre- versus post- drug treatment efficacy
As mentioned above, the chemical treatment consisted of two phases, a pre-irradiation treatment to test the preventative effect of the drug and a post-irradiation treatment to examine its curative effect. In order to determine which treatment phase had a more essential role in the XP-C cells phenotypic normalization, the two phases of treatment were analyzed separately via the determination of drug bioactivity. It should be noted that bioactivity is defined by the ability of the drugs to impose an effect on living matter which in this case is its ability to increase photo-resistance of the cells between non-irradiated cells set at 100% and irradiated non-treated cells set at 0%. The effect of each of the pre- or the post- treatment was compared to that of the combined pre- plus post-treatment regimen. For isoconazole, the post-irradiation treatment was sufficient to induce similar protection as the combined treatment at both 5 and 10 µM (with no significant differences) while the pre-treatment had no effect on XP-C cells viability at none of the tested concentrations (Fig. 6a). At 10µM the difference in bioactivity was significant between pre and post-treatment as well as between the pre- and combined treatment with a p-value < 0.01, signifying that both pre-treatment and combined regimens share significant protection compared to pretreatment. In the case of clemizole hydrochloride, the pre-irradiation treatment showed slight bioactivity reaching 20% increased cell viability at the highest concentration of drug of 10 µM. The post-treatment however was again more effective than the pre-treatment on XP-C viability reaching an average of 50% increase of cell viability at 10µM. The combination of both pre- and post-irradiation treatment showed the highest bioactivity with a significant difference compared to the pretreatment at both 5 and 10µM p-value < 0.05, and no significant difference with the post-treatment only. Thus, despite a slight protective potential of clemizole hydrochloride, both drugs (isoconazole and clemizole hydrochloride) may essentially serve as curative remedy post-UVB irradiation.
Double Drug treatment has no synergistic nor additive effect
Both compounds isoconazole and clemizole hydrochloride have an azole ring in their structure (and may thus target a same molecular biological target or similar mechanisms). We, therefore, examined whether the combined treatment with both drugs at 10µM could improve further or not the acquired photo-resistance compared to single-drug treatment. Accordingly, XP-C cells were treated with either drug alone or with both and then subjected to irradiation at a dose of 0.02j/cm2. The double treatment had the same bioactivity as the single treatment with isoconazole with no benefit (Fig. 6b). Hence, no synergetic effect was obtained upon double drug treatment.
Isoconazole and clemizole hydrochloride do not affect cell proliferation
In a first step, and in order to analyze the proliferative state of the irradiated cells and whether the different treatments can modify this profile, cells were stained with Ki67 antibodies and analysis was carried out at the single-cell level. Ki67 antigen was expressed during all phases of the cell cycle (G1, S, G2, and M) but not in quiescent cells. Both XP-C and WT cell lines were positive to Ki67 signifying that the cells are not in quiescent state post UV. Cells treated with either DMSO, isoconazole, clemizole hydrochloride, or both drugs showed no difference in the Ki67 expression profile (supplementary figure S3).
Ki67 antibodies stain cells in various stages of the cell cycle, and it was previously reported that some non-proliferating cells tend to test positive for Ki67 due to antigen retention26. Moreover, bulky adducts generated by UV tend to block the progression of replication forks decreasing DNA replication. Therefore, in a second step, EDU incorporation assay was performed to clarify whether this positive staining was due to antigen retention or rather the cells were capable of recovering from the DNA replication blockade following different treatments. It should be noted that EDU insertion into DNA allows the identification of cells that have progressed through the S phase. XP-C cells were treated with either DMSO, isoconazole or clemizole hydrochloride in a pre- and post-treatment regime and then irradiated with UV to be finally cultured in the presence of EDU at different time points (2h. or 4h. post UV). Cells were then collected and stained to be analyzed by flow cytometry. When comparing the course of EDU incorporation in DMSO treated cells over the different time points post UV, it was clear that the intensity of EDU decreases as a function of time till becoming null at 24h post UV. This signifies that the cells were no longer able to undergo DNA replication at 24h. post UV. Isoconazole had a slight effect on increasing EDU positive population at 2h post UV, but both drugs failed to increase DNA replication beyond that showing similar effects to DMSO (Fig. 7). These results indicate that cell proliferation does not seem to be the major mechanism underlying the protective effect induced by these drugs.
Isoconazole and not clemizole hydrochloride decreases both apoptosis and necrosis
In an attempt to decipher the cellular mechanisms of photo-resistance of the drug treatment, different cell phenotypes were analyzed. Apoptosis was therefore quantified using Cell Event which allows the indirect measurement of caspase 3/7 activity, key downstream players in the apoptosis activation cascade. XP-C cells were therefore treated with either drug or DMSO followed by UVB irradiation then staining with Cell event and PI 24h post UV. The samples were analyzed by flow cytometry. Out of the two drugs, Isoconazole showed a decrease in cell event % cells reaching 23.35% as well as lower PI-positive population percentage (9.57%) compared to DMSO treated cells showing higher population percentages of 41.23% and 11.57%, respectively. On the other hand, clemizole hydrochloride did not show any decrease in both quantified parameters. These results suggest that unlike isoconazole, it is probably not via the reduction of apoptosis that the clemizole hydrochloride mediates its protective role (Fig. 8). Similar outputs were also detected in WT cells (supplementary figure S4).