Protein kinase C-activating tumor promoters modulate the DNA damage response in UVC-irradiated TK6 cells

Toxicology Letters 229 (2014) 210–219

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Protein kinase C-activating tumor promoters modulate the DNA damage response in UVC-irradiated TK6 cells Kyle P. Glover a,b , Lauren K. Markell a , E. Maria Donner a , Xing Han a, * a b

DuPont Haskell Global Centers for Health & Environmental Sciences, P.O. Box 30, 1090 Elkton Road, Newark, DE, USA University of the Sciences, Department of Biological Sciences, Cell and Molecular Biology Graduate Program, Philadelphia, PA, USA

H I G H L I G H T S







PKC-activating tumor promoters increase apoptosis after UVC-irradiation. Sustained UVC-induced gH2AX formation is observed in tumor promoter treated cells. The synergistic increase in apoptosis was p53-dependent in TPA + UVC treated cells. TPA modulates the expression of p53-target genes.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 May 2014 Received in revised form 17 June 2014 Accepted 18 June 2014 Available online 21 June 2014

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a non-genotoxic tumor promoter that dysregulates the protein kinase C (PKC) pathway and causes variable cellular responses to DNA damage in different experimental models. In the present study, we pretreated human lymphoblastoid TK6 cells (wild-type p53) for 72 h with TPA, and five other PKC-activating tumor promoters, to determine how sustained exposure to these chemicals modulates key DNA damage response (DDR) endpoints induced by UVCirradiation. Here we show that pre-treatment with PKC-activating tumor promoters augmented the sensitivity of TK6 cells to UVC-irradiation characterized by a synergistic increase in apoptosis compared to that induced by either stress alone. In addition, high residual levels of the DNA damage repair signal gH2AX was observed in tumor promoter treated cells indicating a delayed DDR recovery. NH32 (p53null, isogenic to TK6) cells were resistant to the synergistic effects on apoptosis implicating p53 as a central mediator of the DDR modulating effects. In addition, analysis of p53 target genes in TPA-pretreated TK6 cells revealed a significant modulation of UVC-induced gene expression that supported a shift toward a pro-apoptotic phenotype. Therefore, sustained exposure to tumor promoting agents modulates the UVC-induced DDR in TK6 cells, which may represent important synergistic interactions that occur during tumor promotion. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: TPA DNA damage response Tumor promotion p53

1. Introduction The DNA damage response (DDR) protects genomic integrity by sensing damaged DNA and transducing the signal to downstream effectors leading to cell cycle arrest and repair (Zhou and Elledge, 2000). Excessive damage leads to more extreme processes such as apoptosis, terminal differentiation and/or senescence to prevent the transmission of mutations to daughter cells. The DDR is therefore considered a central tumor suppressing pathway for

* Corresponding author. Tel.: +1 302 451 5808; fax: +1 302 451 3568. E-mail address: [email protected] (X. Han). http://dx.doi.org/10.1016/j.toxlet.2014.06.030 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

preventing neoplastic transformation and key DDR effectors, such as ATM/ATR, gH2AX, CHK2 and p53, are activated in premalignant tumors (Bartkova et al., 2005; Gorgoulis et al., 2005; Harper and Elledge, 2007). In contrast, defective DDR signaling is observed in late-stage carcinomas leading to genomic instability and malignancy (Hanahan and Weinberg, 2011). Selective pressure against cells with a functional DDR explains the high frequency of mutations in the p53 gene observed in human cancers (Vogelstein et al., 2000). p53 dictates the cellular response to DNA damage primarily through transcriptional activation of target genes involved in cell cycle arrest, DNA repair and apoptosis (Prives and Hall, 1999). In response to DNA damage, upstream kinases such as ATM/ATR or

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CHK1/2 phosphorylate p53 thus releasing it from the inhibitory E3 ubiquitin ligase MDM2. p53 levels then accumulate and translocate to the nucleus to mediate transcription. The decision to undergo repair or apoptosis is influenced by many factors including the overall levels of p53 accumulation, interaction with other transcription factors and selective promoter binding (Vousden and Prives, 2009). Given the importance of the DDR and p53 in tumor suppression, it is important to understand how tumor promoting stimuli interfere with this pathway. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a tumor promoting phorbol ester derived from croton resin that dramatically increases tumor formation in mouse skin previously treated with a mutagenic polyaromatic hydrocarbon (DiGiovanni, 1992). The mechanism of TPA-driven tumor promotion is linked to its strong affinity for protein kinase C (PKC), a characteristic shared among other phorbol ester tumor promoters as well as non-structurally related promoters such as indolactam-V and mezerein (DiGiovanni, 1992; Geiges et al., 1997). PKC signaling is integral to many cellular processes important in carcinogenesis including cell cycle regulation, apoptosis, differentiation, metastasis and angiogenesis (Griner and Kazanietz, 2007). Given the pleiotropic nature of PKC signaling, it is not surprising to find that PKC-activating tumor promoters, such as TPA, alter the cellular response to DNA damage. For instance, TPA exacerbates the apoptotic response to a variety of different DNA damaging agents including UV, arsenic trioxide, cadmium chloride, cisplatin and histone deacetylase inhibitors

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(Fernandez et al., 2004; Isonishi et al., 2000; Kimura et al., 1999; Rahmani et al., 2002; Wang et al., 2000). However, TPA can also inhibit the apoptotic response to DNA damage and attenuate p53 indicating that experimental conditions such as cell type, source of DNA damage and TPA exposure conditions may be critical factors that determine the interaction between TPA and DNA damage induced pathways (Magnelli et al., 1995; Mukherjee and Sikka, 2006; Schwartz and Jordan, 1997; Skouv et al., 1994). TPA dysregulates PKC in two ways, both of which are important in tumor promotion. First, activation of PKC by TPA leads to enhanced stimulation of PKC-mediated signal transduction. Second, the sustained activation of PKC by TPA leads to its degradation thereby altering downstream processes (Fournier and Murray, 1987). Therefore, TPA may affect cells differentially depending on the exposure time used in the experimental model which could account for the controversial findings described above. In the present study, we preconditioned human lymphoblastoid TK6 cells for 72 h with TPA, or other PKC-activating tumor promoters, to observe the effects of extended exposure to these agents. The preconditioned cells were then challenged with short-wavelength ultraviolet light (UVC, 254 nM) to induce a DDR. UVC-irradiation induces bulky cyclobutane dimers and 6-4 photoproducts in DNA leading to replication stress and double strand breaks (Sinha and Hader, 2002). We hypothesized that the altered response to UVC-induced DNA damage in tumor promoter altered cells would represent synergistic interactions that occur

Fig. 1. TPA inhibited cell growth and altered growth morphology of TK6 cells. (A) TK6 cells treated with TPA for 72 h had a dose-dependent reduction in cell growth (filled circles) without a significant loss in viability (trypan blue exclusion, open circles) compared to the untreated (DMSO solvent control) cells. (B) Treatment with TPA (1 nM) caused TK6 cells to grow in large aggregates of cells compared to a uniform suspension when treated with the solvent control, DMSO.

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in the tumor microenvironment enhanced by this class of compounds. To address this, we analyzed the apoptotic response to UVC-irradiation in tumor promoter-pretreated TK6 cells to determine the synergistic effects of exposure to both agents compared to either stress alone. The kinetics of gH2AX formation and recovery after UVC-irradiation were investigated to determine if DDR signaling was modulated in tumor promoter pretreated cells. Finally, we analyzed how p53-null cells respond in this model and if p53 target gene expression was altered by TPA-pretreatment. 2. Materials and methods

supplemented with calcium and magnesium (DPBS) and resuspended to a concentration of 0.4  106 cells/mL. Cells were then added to 60 mm untreated culture dishes and exposed to a germicidal UV lamp (254 nM) (UVP, Upland, CA) at 100–200 mW/ cm2 which was determined with a radiometer (UVX-25 Sensor, 254 nm, UVP). All exposures were conducted in a Chromato-Vue Mini Viewing Cabinet (Model C-10, UVP). After UVC-irradiation the cell suspensions were transferred to 15 mL centrifuge tubes and kept on ice until all samples were exposed. Non-irradiated cells were handled exactly like the UVC-irradiated samples only without exposure to the UV lamp. The cells were then centrifuged and resuspended in RPMI medium supplemented with a PKC-activating tumor promoter or DMSO and returned to the incubator.

2.1. Materials TPA (CAS 16561-29-8, Sigma–Aldrich, St. Louis, MO), 4aphorbol 12-myristate 13-acetate (4a-TPA) (CAS 63597-44-4, Sigma–Aldrich), phorbol-12,13-dibutyrate (PDBu) (CAS 3755816-0, Sigma–Aldrich), sapintoxin D (CAS 80998-07-8, Sigma– Aldrich), mezerein (CAS 34807-41-5, Santa Cruz Biotechnology, Dallas, Texas), ()-indolactam V (()-ind V) (CAS 90365-57-4, Santa Cruz Biotechnology) and resiniferonol 9,13,14-ortho-phenylacetate (ROPA) (CAS 57852-42-3, Santa Cruz Biotechnology) were diluted in sterile Hybri-MaxTM dimethyl sulfoxide (DMSO) (CAS 67-68-5, Sigma–Aldrich) 1,000 fold and stored frozen. 2.2. Cell lines and treatments The human lymphoblastoid TK6 cell line was purchased from the American Type Culture Collection (ATCC, CRL-8015, Manassa, VA) and propagated in RPMI medium (Mediatech, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (Mediatech) and penicillin/streptomycin (Mediatech). NH32 cells were received as a generous gift from Dr. Harold Liber (Colorado State University, Fort Collins, CO). Both TK6 and NH32 were subcultured every 2–3 days to maintain a culture density of less than 1.2  106 cells/mL. Cell viability and density were determined with a coulter counter (ViCell XR, Beckman Coulter, Indianapolis, IN). Cell growth inhibition was calculated by comparing the viable cell density of TPA treated cells with the untreated (DMSO solvent control) after 72 h treatment. The cells were treated with PKC-activating tumor promoters (TPA, PDBu, sapintoxin D, mezerin, ()-ind V or ROPA), the inactive isomer of TPA (4a-TPA) or DMSO (solvent control, 0.1%) for 72 h prior to UVC-irradiation. Following pretreatment, cells were washed with cold Dulbecco’s phosphate-buffered saline

2.3. Annexin-V assay At 48 h post UVC-irradiation, tumor promoter or vehicle treated cells were collected and washed in cold DPBS. Apoptotic cells were labeled with Alexa Fluor1 488 conjugated annexin-V (20x dilution) and counter stained with propidium iodide (PI, 20 mg/ mL) (Dead Cell Apoptosis Kit Cat# V-13245, Life Technologies, Carlsbad, CA). Ten thousand cells were analyzed per sample on a FACSCalibur with CellQuest Pro software (BD Biosciences, San José, CA). The percentage of early apoptotic cells was calculated from the PI-negative population to exclude late apoptotic/necrotic cells. The data were averaged from three to five experiments conducted in duplicate. 2.4. g H2AX assay At various time points post UVC-irradiation, tumor promoter or vehicle treated cells were washed twice in cold DPBS, fixed in 70% ethanol and stored frozen. On the day of the assay, cells were removed from the freezer, washed twice and blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline for 30 min at room temperature. The cells were then labeled with 100 mL of 1:100 anti-phospho-histone H2A.X (Ser139) (clone JBW301; Millipore, Billerica, MA) for 2 h at room temperature. Following incubation, the cells were washed twice with 1% BSA and resuspended with 100 mL of 1:30 fluorescein isothiocyanate (FITC)-conjugated F(ab0 )2 Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Labs; West Grove, PA) for 30 min at room temperature. Cells were washed twice following incubation and resuspended in 500 mL DNA staining solution (5 mg/mL PI with 1:320 RNase) for 20 min at 37  C and 30 min at room temperature

Fig. 2. TPA increased apoptosis and delayed recovery after UVC-irradiation in a synergistic manner. (A) TPA-pretreated (1 nM, 72 h) TK6 cells were exposed to UVC-irradiation (TPA + UVC) ranging from 5 to 20 J/m2 and analyzed for apoptosis (annexin V) by flow cytometry at 48 h post exposure. Synergistic effects were most pronounced after 10 J/m2 and saturated at 20 J/m2 compared to UVC alone (UVC). (B) TPA-pretreatment increased the apoptotic response to 10 J/m2 UVC (TPA + UVC) in a dose-dependent synergistic manner from 0.1 to 1 nM compared to TPA-treatment alone (TPA). (C) Compared to untreated control cells, TPA treatment alone (1 nM) reduced the cell growth rate slightly while UVC exposure at 10 J/m2 caused growth arrest to 24 h but recovered to normal growth rates by 48 h. TPA-pretreated cells also exposed to UVC-irradiation (TPA + UVC), in contrast, had significantly delayed recovery compared to untreated (DMSO solvent control) cells.

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prior to analysis. Ten thousand cells were analyzed per sample on a FACSCalibur with CellQuest Pro software. Cells were gated and separated on the FL3 channel to isolate the single cell population. gH2AX positive cells were visualized on the FL1 channel. The data were averaged from three experiments conducted in duplicate. 2.5. RNA isolation and QC At predesigned time-points after UVC-irradiation the cells were removed from the incubator and washed twice with cold DPBS. The cells were then flash frozen on dry ice and stored at 80  C until further processing. RNA was isolated using an RNeasy Kit (Qiagen, Valencia, CA) and the concentration and quality of the samples were verified on a Nanodropã spectrophotometer (NanoDrop, Wilmington, DE). cDNA was generated with the SuperScript1

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VILOTM cDNA Synthesis Kit (Life Technologies) with an gradient thermocycler (Eppendorf, Hamburg, Germany). 2.6. Quantitative real-time PCR (Q-PCR) Q-PCR was run on an ABI 7500 Real-Time PCR System using ABI master mix and TaqMan1 primers (Life Technologies). Gene expression levels were calculated using a standard curve for relative quantification with 3 biological replicates per sample. Three different house-keeping genes were initially analyzed including 18S, GAPDH and HPRT, however, only 18S was found to be acceptable for normalization purpose due to decreased expression of GAPDH and HPRT in UVC-irradiated samples. Differential expression was determined by comparing against the vehicle treated, non-irradiated sample at each time point.

Fig. 3. Other PKC-activating tumor promoting compounds inhibited cell growth and increased apoptosis after UVC exposure. (A) TK6 cells were pretreated with PKCactivating tumor promoters at concentrations ranging from 0.1 to 10,000 nM. 4a-TPA, a non-tumor promoting phorbol ester, was also evaluated. (B) Tumor promoter pretreatment at TPA-equivalent doses (70% inhibition of cell growth as observed in panel A) increased apoptosis (annexin V) 48 h after UVC-irradiation (10 J/m2) which was synergistic compared to that induced by UVC alone (*p < 0.01). In contrast, 4a-TPA pretreated cells had a similar response compared to the solvent control.

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2.7. Statistical analysis Statistical comparisons were conducted with a one way ANOVA with a post-hoc Tukey test for individual comparisons using Origin version 9 software (OriginLab Northampton, MA). 3. Results 3.1. Synergistic enhancement of UVC-induced apoptosis TK6 cells had a cytostatic response to TPA after a 72 h treatment that was dose dependent from 0.1 to 1 nM (Fig. 1A). At 1 nM, a 70% growth inhibition was observed without substantial loss in cell viability. In addition, TK6 cells grew in large aggregates in the presence of 1 nM TPA instead of a single cell suspension observed in the DMSO-treated solvent control (Fig. 1B). TPApretreated cells were slower growing but not quiescent as the percentage of cells in G0/G1 or S-phase was not altered after 72 h treatment (data not shown). We then exposed the cells to UVCirradiation to determine if TPA-treatment altered the apoptotic sensitivity to DNA damage. TPA-pretreated cells had a lower tolerance for UVC-stress and were significantly more apoptotic (Fig. 2A). The effect was most pronounced at 10 J/m2 UVC where TPA-pretreated cells were 55.0% apoptotic compared to 15.6% induced by UVC alone or 7.8% induced by TPA alone. This synergistic effect on apoptosis was dependent on the concentration of TPA which increased from 12.3% to 47.3% in cells treated across a dose range of 0.1–1 nM (Fig. 2B). Compared to the normal growth rate of TK6 cells, we observed a 24 h growth arrest followed by recovery after 10 J/m2 UVC exposure. In contrast, TPApretreated cells did not show recovery for 72 h after 10 J/m2 exposure (Fig. 2C). We then examined if other PKC-activating tumor promoters enhanced the apoptotic response to UVC-irradiation in a similar synergistic manner. In order to normalize the dose levels of this class of tumor promoting compounds, we determined a TPAequivalent concentration for each tumor promoter based on their respective growth inhibiting potential. Each PKC-activating tumor promoter reduced cell growth in the dose-dependent manner similar to TPA, albeit at log-fold higher concentrations (Fig. 3A). The dose–response curves show a cytostatic reduction in cell growth that plateaued at approximately 30% of the control without

significantly affecting cell viability. The TPA equivalent concentrations were 100 nM for PDBu, mezerein and sapintoxin D and 1 mM for ()-ind V and ROPA. At these concentrations the cells displayed a similar growth morphology as that induced by TPA displayed in Fig. 1B (data not shown). When the tumor promoter pretreated cells were exposed to UVC-irradiation at their TPAequivalent concentration we observed 40–50% of the cells to be apoptotic compared to 13.8% induced by UVC alone (Fig. 3B). 4aTPA, a non-tumor promoting stereoisomer of TPA with weak affinity to PKC (DiGiovanni, 1992), caused inhibition of growth at the highest concentration tested, 10 mM, but no changes in growth morphology were observed. In contrast to other PKC-activating tumor promoters, pretreatment with 4a-TPA did not enhance UVC-induced apoptosis (Fig. 3B). 3.2. Sustained H2AX phosphorylation (g H2AX) after UVC-irradiation Phosphorylation of H2AX at serine 139 (gH2AX) is an integral component of DDR signaling and a marker of replication stress induced by UVC (Fernandez-Capetillo et al., 2004; Ward and Chen, 2001). To determine if the lower apoptotic threshold of TPApretreated cells was associated with altered DDR signaling patterns, we analyzed the kinetics of gH2AX formation and clearance with and without UVC-irradiation. In our model we observed significant gH2AX formation 2 h after UVC-irradiation (Fig. 4). The effect was indicative of replication stress as the signal was primarily found in the S-phase population (Fig. 4B). gH2AX levels quickly declined in a time-dependent manner from 56.6% at 2 h to 5.7% by 24 h (solid line, Fig. 5A). TPA-pretreatment did not affect gH2AX induction as levels were similar to the UVC control at 2 and 4 h post-irradiation. However, by 8 h post-irradiation, TPApretreatment caused more cells to retain the gH2AX signal compared to the UVC control (32.0% versus 20.0%, respectively) and this trend was maintained through 24 h (18.1% versus 5.7%) (dashed line, Fig. 5A). TPA-pretreatment did not significantly induce gH2AX in the absence of UVC-irradiation indicating this effect was not due to increased background levels of gH2AX induced by TPA alone. We then compared the effect of other PKCactivating tumor promoters at 8 h post-irradiation and found a similar increase in gH2AX levels across this chemical class (Fig. 5B). In contrast, the gH2AX response in cells pretreated with 4a-TPA was similar to the DMSO control.

Fig. 4. UVC-irradiation caused gH2AX formation in S-phase cells. TK6 cells were exposed to UVC-irradiation (10 J/m2) and fixed in 70% ethanol 2 h post exposure. Cells were analyzed for gH2AX and DNA content with flow cytometry. (A) Baseline gH2AX levels were low in untreated TK6 cells while (B) 2 h after 10 J/m2 UVC, gH2AX was significantly increased in S-phase cells.

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DNA damage leads to increased expression of key DDR effectors involved in p53 regulation (MDM2), cell cycle arrest (p21, GADD45A), nucleotide excision repair (XPC, DDB2) and apoptosis (PUMA, NOXA, BAX) (Vousden and Prives, 2009). We analyzed the expression of these genes at 2, 4, 8, 12 and 24 h post UVCirradiation to determine the most sensitive time-point to analyze the effects of TPA (Fig. 7A). At 8-h post-UVC exposure each of these target genes were found to be 2 fold increased above the untreated control except BAX. BAX expression was not significantly increased by UVC at any time-point, however, the anti-apoptotic BCL2 gene was significantly inhibited at 8 h which indicated a proapoptotic shift in the BAX/BCL2 ratio (Basu and Haldar, 1998). Each gene, except MDM2, decreased from the 8 h expression level by 24 h which indicated gene expression recovery by this time point. MDM2 expression was maintained at the 8 h levels (4 fold increase over the control) through the 24 h time point. We then determined if induction and recovery of UVC-induced gene expression was significantly altered in TPA-pretreated TK6 cells by analyzing mRNA levels normalized to the untreated controls at 8 and 24 h post UVC-irradiation (Fig. 7B). TPA-pretreatment caused a statistical significant increase (p < 0.01) in the expression of the cell cycle regulatory genes p21 and GADD45A at both 8 and 24 h post-UVC. The two nucleotide excision repair genes, XPC and DDB2, were lower in the TPA-pretreated cells compared to the UVC control at both 8 and 24 h, although the difference for DDB2 at 8 h was not statistically significant. The average expression level of

Fig. 5. TPA-pretreated cells had sustained gH2AX formation after UVC-irradiation. (A) TK6 cells were analyzed for gH2AX formation after UVC-irradiation (10 J/m2) which peaks after 2 h and declines over time up to 24 h post UVC exposure. TPApretreated cells have similar induction of gH2AX but maintain higher gH2AX levels 8 h post exposure indicating delayed recovery. Untreated (DMSO solvent control) cells (filled square) or TPA-treatment alone (open square) were not significantly different at the 8-h time point and exhibited low background levels of gH2AX. (B) Pretreatment with other PKC-activating tumor promoters caused a similar increase in gH2AX at 8 h post UVC exposure (*p < 0.01).

3.3. Resistance of p53-null cells and altered transcription of p53 target genes p53 activation can lead to life or death decisions in cells following DNA damage through transactivation of target genes involved in cell cycle arrest or apoptosis. Since TPA-pretreated cells had a lower threshold for DNA damage induced apoptosis, we investigated if p53 was a key driver of this response by analyzing how NH32 cells, a p53-null cell line isogenic to TK6, respond to similar conditions (Chuang et al., 1999). NH32 cells responded to TPA-pretreatment similarly to TK6 with a cytostatic reduction in cell growth (Fig. 6A) and altered growth morphology (data not shown). However, TPA-pretreated NH32 cells were resistant to the synergistic increase in apoptosis following UVC-irradiation (Fig. 6B). This suggested that p53 and its regulated pathways played a key role in modulating the synergistic increase in apoptosis associated with the combined exposure to TPA and UVC compared to that induced by either stress alone. We therefore analyzed how p53 target gene expression was modulated by TPA after UVC-irradiation.

Fig. 6. NH32 cells (p53-null) were resistant to the synergistic effects of TPAtreatment combined with UVC-irradiation. (A) Treatment with 1 nM TPA was inhibitory to both TK6 and NH32 cell growth after 72 h treatment compared to the level of cell growth observed in the non-TPA treated control for each cell type. (B) In contrast to TK6, TPA-pretreated NH32 cells were resistant to the synergistic increase in apoptosis after 10 J/m2 UVC-irradiation (*p < 0.01). However, NH32 cells exposed to UVC alone underwent similar levels of apoptosis compared to TK6.

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Fig. 7. Transcription of p53 target genes in TK6 cells after UVC-irradiation was time-dependent and modulated by TPA. (A) mRNA was collected at 2–24 h post UVC-irradiation and analyzed by Q-PCR. The gene expression profile of p53 target genes was time-dependent after UVC-irradiation with maximal sensitivity observed at 8 h post exposure. Expression data is based on relative fold-change versus untreated (DMSO solvent control) cells. (B) TPA-pretreatment led altered gene expression patterns after UVCirradiation at 8 and 24 h (*p < 0.01). Relative levels for BAX were compared to BCL2 to determine the BAX/BCL2 expression ratio.

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NOXA was increased >2 fold compared to UVC alone but due to high variability between the replicate cultures, was not statistically significant. PUMA expression was increased by TPA-treatment alone but not significantly increased above the UVC control after UVC-irradiation. The BAX/BCL2 ratio was not significantly modulated by TPA indicating the synergistic increase in apoptosis is not attributed to the altered expression of these genes. The wellknown negative regulator and transcriptional target of p53, MDM2, was induced by TPA alone at 8-h (1.8-fold) but not further increased in cells also exposed to UVC-irradiation. By comparison, UVC-irradiation alone induced a 3.4 fold increase in MDM2. Therefore, MDM2 expression was inhibited after UVC-irradiation in TPA-pretreated cells compared to the UVC-irradiation alone at 8 h. However, by 24 h MDM2 expression levels were significantly higher in the TPA-pretreated cells after UVC-irradiation compared to UVC-irradiation alone. These results indicate that there was a selective modulation of p53-target genes in TPA-pretreated cells that may represent a shift toward a pro-apoptotic phenotype and reduced repair. Interestingly, TPA-pretreated cells had increased expression of MDM2, p21, GADD45A, DDB2, and PUMA at 8 h in the absence of UVC-irradiation signifying a background perturbation of p53 target gene expression. 4. Discussion During multistage carcinogenesis, the DDR becomes constitutively activated in early tumors primarily driven by oncogenic stress (Bartkova et al., 2005; Gorgoulis et al., 2005). Consequently, there is selective pressure against this tumor suppressive pathway as cancer progresses toward malignancy. In the multistage mouse skin model, repeated application of tumor promoting compounds, such as the PKC-activating phorbol esters, drives the clonal expansion of RAS transformed cells into early tumors that eventually lose p53 functionality and progress to carcinomas (DiGiovanni, 1992; Kemp, 2005). The primary mode-of-action of phorbol esters, and other PKCactivating tumor promoters, is the sustained activation followed by degradation of PKC isozymes. Deregulation of PKC signaling has a pleiotropic effect on cells as many downstream effectors (e.g., ERK, JNK, p38) of PKC cross-talk with other cellular pathways such as the DDR (Poehlmann and Roessner, 2010). Therefore, it is important to characterize how tumor promoting PKC-activating compounds interfere with DDR signaling. In our model, TK6 cells were grown in the presence of TPA, or other PKC-activating tumor promoters, for 3 days in order to sustain the pathway dysregulating effects of these compounds. When tumor promoter treated cells were challenged with UVCirradiation, we observed a significant increase in apoptosis compared to non-tumor promoter treated cells exposed under the same conditions. The decision to undergo apoptosis is cellular context dependent and pretreatment with PKC-activating tumor promoters shifts the cells toward a pro-apoptotic phenotype upon receiving DNA damage signals. This finding was in significant contrast to previous studies where apoptosis was attenuated in TPA-treated TK6 cells exposed to ionizing radiation (Schafer et al., 2002; Schwartz and Jordan, 1997). However, in these earlier studies TK6 cells were pretreated for 2 h prior to radiation exposure compared to 72 h used in this experiment. In a preliminary study, we were able to show that short-term TPA pretreatment could inhibit UVC-induced apoptosis, but only after 10 fold higher levels of UVC exposure than what was used in this experiment (100 J/m2 versus 10 J/m2). As pretreatment time increased from 24 to 72 h, we observed a synergistic increase in apoptosis similar to that observed in this experiment (Supplementary Fig. 1). TPA causes different effects after a short versus long treatment time as PKC isozymes are down-regulated in response to sustained activation (Fournier and Murray, 1987; Hansen et al., 1990). PKC down-

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regulation is associated with growth inhibition and increased cell death in multiple cell types (Buganim et al., 2006; Hsu et al., 1998; Whelan and Parker, 1998). In addition, HeLa cells treated with TPA undergo a synergistic increase in apoptosis in response to the DNA damaging agent cisplatin (Wang et al., 2000) which may be due to PKCa depletion and a reduction in glutathione (Isonishi et al., 2000). PKC down-regulation is also observed in mouse skin during TPA induced tumor promotion (Hansen et al., 1990). Therefore, it is possible that cells pretreated with PKC-activating tumor promoters are ultrasensitive to UVC induced DNA damage through mechanisms involving the dysregulation of PKC isozymes. Because apoptosis is not a DDR specific endpoint, we then determined if tumor promoter pretreated cells have altered DDR signaling and repair by measuring the formation of gH2AX after UVC-irradiation. Phosphorylation of H2AX at serine 139 (gH2AX) by ATR occurs early in the DDR around sites of replication fork stalling and double strand breaks (Ward and Chen, 2001). To allow for checkpoint recovery and DDR resolution gH2AX is dephosphorylated by WIP-1 or PP2A phosphatases (Cha et al., 2010; Chowdhury et al., 2005). Following UVC-irradiation, we observed a significant increase in the percentage of gH2AX positive cells that were primarily in S-phase. TK6 cells pretreated with TPA had similar levels of gH2AX induction at 2 and 4 h post-irradiation indicating that TPA does not interfere with DDR processes upstream of gH2AX formation, such as activation of ATM or ATR. However, the effects of TPA on gH2AX were apparent beginning 8 h post-irradiation where more cells retained the gH2AX signal. This effect was also induced by other PKC-activating tumor promoters at TPA-equivalent concentrations demonstrating that pretreatment with PKC-activating tumor promoters delayed recovery from UVC-induced DNA damage. Because sustained gH2AX signaling correlates with an increased sensitivity to DNA damage and cell death, we propose that PKC-activating tumor promoters lower the apoptotic threshold by prolonging the DNA damage signal (Banath et al., 2010; Chowdhury et al., 2005). A consequence of prolonged DDR signaling is the sustained accumulation of p53 thereby shifting the cellular microenvironment toward apoptosis (Latonen and Laiho, 2005; Vousden and Lu, 2002). Here we show that NH32 cells (p53-null) were resistant to the synergistic apoptotic effect of TPA. However, NH32 cells were still responsive to TPA-induced growth inhibition and UVC-induced apoptosis indicating a role for p53 in the synergistic response specific to the combined exposure toTPA and UVC. Although previous studies have shown p53 inhibition by TPA, these effects are likely specific to cell type and experimental design (Skouv et al.,1994). The role of p53 in tumor promotion is enigmatic as p53 was shown to actually be required for TPA induced promotion in p53 null mice (Kemp et al., 1993). However, the tumors formed in these mice were much more prone to malignant conversion which demonstrated the importance of p53 as a tumor suppressor primarily in the progression stage of carcinogenesis. Therefore, the role of p53 in tumor promotion is complex and the interaction between tumor promoting compounds and the DDR may funnel through the p53 transcriptional axis. We then investigated if UVC-induced transcription of p53 target genes was modulated in TPA-pretreated cells. We observed increased expression of cell cycle regulatory genes p21 and GADD45A, inhibition of nucleotide excision repair genes DDB2 and XPC, and a slight increase in pro-apoptotic genes PUMA and NOXA (although not statistically significant). This transcriptional profile likely represents a shift toward a pro-apoptotic phenotype in TPA-pretreated cells. Transcriptional activation of pro-apoptotic BH3-only proteins, such as PUMA or NOXA, shift the balance of proversus anti-apoptotic BCL-2 family proteins leading to mitochondrial membrane depolarization and initiation of the apoptotic cascade (Villunger et al., 2003). While cell cycle regulatory genes p21 and GADD45A are typically associated with cell cycle arrest and

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repair, they have also been shown to promote cell death in certain contexts. For instance, transient overexpression of p21 has been shown to increase the sensitivity to UV or cisplatin (Fotedar et al., 1999; Lincet et al., 2000). Also, GADD45a null mice are resistance to UV-irradiation induced apoptosis (Hildesheim et al., 2002). The expression profile of MDM2 also supports a pro-apoptotic phenotype in that UVC-induced MDM2 expression was inhibited in TPA-pretreated cells at 8 h but increased by 24 h. Inhibition of MDM2 occurs after high doses of UVC where p53 levels are rapidly increased and p21 is strongly induced at early time-points (Wu and Levine, 1997). Therefore, we hypothesize that TPA-pretreated cells have gene expression patterns indicative of higher levels of DNA damage than what was actually incurred. Increased MDM2 expression at 24 h is likely to be associated with delayed DDR recovery and prolonged p53 activation (Wu and Levine, 1997). Interestingly, TPA-pretreated cells did not alter the BAX/BCL2 ratio which is a standard marker for mitochondrial apoptosis indicating that extrinsic mechanisms of apoptosis may also be involved. Both nucleotide excision repair genes, XPC and DDB2, were down-regulated by TPA after UVC-irradiation. This effect correlates with our observation of delayed gH2AX signal resolution that may be indicative of DNA repair inhibition. Following high levels of DNA damage, DNA repair function decreases as p53 promotes apoptosis instead of repair (Offer et al., 2002). In this case, the inhibition of DNA repair gene expression may represent a shift in p53 transcriptional activity towards cell death. In addition, TPAtreatment alone induced MDM2 and p21, both well-established p53 target genes. This indicates that TPA stimulates a higher level of baseline p53 activity that, when combined with p53 induction by UVC-irradiation, might promote p53-dependent apoptosis instead of repair. However, it is possible that p53-independent processes are involved as TPA can also induce p21 expression in p53-compromised cells types through mechanisms involving MAPK pathways (Akashi et al., 1999; Das et al., 2000; Han et al., 2012). Therefore, compensatory mechanisms may be activated in p53-compromised cell types in response to TPAaltered signaling programs that converge on DDR effectors. 5. Conclusion In the present study, we show that TK6 cells pretreated with PKC-activating tumor promoters have sustained gH2AX signaling, altered expression of p53 target genes and a synergistic apoptotic response to UVC-irradiation. Given the essential role of the DDR in protecting against neoplastic transformation and the observation that constitutive activation of DDR markers occurs in early tumors, it is important to understand how chemicals interfere with this pathway. Although PKC-activating tumor promoters are nongenotoxic compounds, the signal transduction pathways altered by these compounds is extensive and interference with DDR signaling may represent a contributing mechanism toward tumor promotion. Considering the importance of tumor promotion in carcinogenesis, it is essential to further characterize the synergistic effects of stress response pathways activated in the tumor microenvironment to design better biomarkers and prevention strategies for cancer. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.

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