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Gene Therapy (2008) 15, 576–590 r 2008 Nature Publishing Group All rights reserved 0929-1903/08 $30.00 www.nature.com/cgt

ORIGINAL ARTICLE A dual role of p21waf1/cip1 gene in of HEp-2 treated with or methotrexate S Kraljevic Pavelic1, T Cacev2 and M Kralj3 1Laboratory of systems biomedicine, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia; 2Laboratory of molecular oncology, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia and 3Laboratory of functional genomics, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia

We investigated the effects of p21waf1/cip1 gene overexpression in human laryngeal squamous carcinoma cells HEp-2 lacking expression on apoptosis induction upon the treatment with two commonly used chemotherapeutic agents, cisplatin and methotrexate. For that purpose, we employed cDNA arrays and qPCR to monitor upon treatment with AdCMV- alone or in combination with the chemotherapeutic compounds. We found that p21waf1/cip1 gene overexpression provoked apoptosis of HEp-2 through the induction of the TNFRSF9 gene and activation of 7. In addition, we have proved that p21waf1/cip1 can assume a dual role in apoptosis in the same cell system depending on the chemotherapeutic agent: its overexpression enhances apoptosis in cisplatin-treated cells and attenuates apoptotic signals in methotrexate-treated cells. The observed dual role of p21waf1/cip1 was in direct correlation with the modulation of 3 and 7 activation and changes in the expression of GADD45a gene. The results presented herein encourage future use of targeted p21waf1/cip1 gene therapy in cancer treatment in a well-defined therapeutic and genetic context. Cancer Gene Therapy (2008) 15, 576–590; doi:10.1038/cgt.2008.28; published online 16 May 2008 Keywords: apoptosis; GADD45a; p21waf1/cip1; HEp-2 cells; cisplatin; methotrexate

Introduction dependent inhibitors (CKIs) that bind to cyc/Cdk pairs and negatively regulate , are crucial in It has now been widely accepted that cancer is a polygenic biological processes such as development, differentiation 2 defined by the imbalance between cell prolifera- and genotoxic stress. One of the best-described and tion and cell . Typical features of cancer cells first-discovered CKIs is the p21 protein encoded by the waf1/cip1 primarily include a disregard of signals to stop prolifera- respective growth-inhibitory gene p21 , which can tion and differentiation, capacity for sustained prolifera- exert its role in different phases of the cell cycle. The tion, evasion of apoptosis, invasion and angiogenesis. The protein p21 belongs to the cip/kip family along with p27 majority of these processes involve control over the cell and p57. They all have the ability to bind and inhibit cyc/ 2 cycle.1 In cancer cells proliferation is disrupted in many Cdk complexes, preferentially those containing Cdk2. different ways, and understanding which molecular However, it seems that p21 is the only CKI capable of 3 players are deregulated and which are the mechanisms interacting with almost all cyc/Cdk complexes, thus behind their deregulation could pave the way for target important in the cell growth arrest induced upon discovery, new diagnosis procedures as well as for genotoxic stress. When overexpressed, p21 exerts its 4 improved cancer treatment. Normally, cell-cycle progres- cytostatic role through G1,G2 or S-phase arrest. sion is regulated by sequential activation of (cyc)/ However, the final outcome of its binding to cyc/Cdk cyclin-dependent kinase (Cdk) pairs, whereby cyclin- complexes could be sometimes stimulation of these complexes, which depends on the p21 stoichiometry and cellular localization.5 Besides its ability to bind cyc/Cdk Correspondence: Dr S Kraljevic Pavelic, Laboratory for systems complexes, p21 can interact with other cellular biomedicine, Division of Molecular Medicine, Rudjer Boskovic involved in the growth, DNA synthesis or DNA repair Institute, Bijenicka cesta 54, Zagreb 10000, Croatia. (for example, growth arrest and DNA damage-inducible E-mail: [email protected] and Dr M Kralj, Laboratory for functional genomics, Division of Molecular Medicine, Rudjer protein GADD45 and proliferating cell nuclear ) and in transcriptional regulation (for example, transcrip- Boskovic Institute, Bijenicka cesta 54, Zagreb 10000, Croatia. 4 E-mail: [email protected] tion factor ). It has been well established that waf1/cip1 Received 30 October 2007; revised 21 January 2008; accepted 21 p21 is a direct transcriptional target of the key February 2008; published online 16 May 2008 tumor-suppressor gene p53, which is induced upon DNA Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 577 damage in the cells expressing the wild-type p53. Never- through the induction of TNFRSF9 gene and activation theless, p21waf1/cip1 is not necessary for the p53-induced of . We have proved that p21 can assume a dual apoptosis. In addition, p21waf1/cip1 can be activated by role in apoptosis in the same cell system and thus different, p53-independent mechanisms that include provided new grounds for optimizing therapy in the transcriptional activation by E2Fs, STATs, AP2, treatment of cancer cells lacking p53 expression. C/EBPa, C/EBPb and activation by tumor-suppressors BRCA1, Wnt-1 and transforming -b.4 The p21 induction seems to be transient, thus suggesting its Materials and methods role in the early stages of the cell-cycle arrest rather than in the permanent cell-cycle arrest, as was demonstrated to Cell lines be the case in the senescent cells.3 Because of all these Tumor cell lines HEp-2 (ATCC no. CCL-23), CAL 27 characteristics, p21 has been considered a tumor-suppres- (ATCC no. CRL-2095) and Detroit 562 (ATCC no. CCL- sor protein for many years. However, this well-established 138) were cultured as monolayers and maintained in the role has been contested by recent studies revealing its 10% DMEM (Dulbecco’s modified Eagle’s medium— engagement in the cancer survival. For example, it has DMEM supplemented with 10% fetal calf serum (FCS) been established that p21 acts as a negative regulator of (Gibco, Carlsbad, CA, USA), 2 mML-glutamine, À1 À1 both, p53-dependent and p53-independent apoptosis in 100 U ml penicillin and 100 mgml streptomycin) in a 2 cancer cells, especially in those subjected to different humidified atmosphere with 5% CO2 at 37 1C. HEK-293 stress stimuli, for example, during which cells (ATCC no. CRL-1573) were maintained in high p21-dependent growth arrest permits repair or even glucose DMEM. prevents DNA damage.4,5 Antiapoptotic role of p21 may also be the consequence of its binding to initiator Adenoviruses and pro-caspases, whereby it inhibits their cleavage (activa- Replication-defective adenoviral recombinant Ad5CMV- tion). For example, inhibition of pro-caspase 3 cleavage p21 (Introgen Therapeutics Inc., Houston, TX, USA) was by p21 results in abrogation of Fas-mediated .6 used for the introduction of the gene p21waf1/cip1 into cells. In addition to direct inhibition of different apoptotic The vectors contain the cytomegalovirus (CMV) promo- pathways in cancer cells, it seems that p21waf1/cip1 exerts tor, p21 cDNA, SV 40 polyadenylation signal in a paracrine mitogenic effects, whereby it stimulates cell minigene cassette inserted into the E1-deleted region of growth by inducing secreted antiapoptotic and mitogenic modified Ad5. As a control vector dl 312 was used. Viral factors.7 As p21waf1/cip1 can protect cells from chemotherapy- vectors were propagated and titrated in HEK-293 cells. induced apoptosis, small molecules could be used to Cells were harvested 36–40 h after infection, pelleted, attenuate its activity and increase the sensitivity of tumor resuspended in phosphate-buffered saline (PBS) and cells to anticancer drugs. Therapeutic possibilities of lysed; cell debris was removed by subjecting the cells to p21waf1/cip1 targeting in cancer cells with attenuating small CsCl gradient purification. Concentrated was molecules are even more accentuated in light of the fact dialyzed, aliquoted and stored at À80 1C. that p21 overexpression is an early event in some development.8 Immunocytochemistry However, p21waf1/cip1 is important as a mediator of the The cells were seeded on eight-well glass chamber slides growth arrest response induced by some anticancer drugs, (Nunc, Roskilde, Denmark) in 10% DMEM at 1 Â 105 such as (Iressa) in head and neck squamous cells per well for HEp-2, 1.5 Â 105 cells per well for CAL- carcinoma cells,9 and can even assume a proapoptotic role 27 and 3 Â 105 cells per well for Detroit 562. At 24 or 72 h under certain conditions in specific biological systems, after the infection with Ad-CMV-p21 or Ad-dl 312 at 20, that usually lack functional p53.2,4 In such systems p21 30 and 50 MOIs the cells were washed in the PBS and waf1/cip1 overexpression induced either upon p21 gene fixed in methanol with 1.5% hydrogen peroxide (H2O2) introduction10,11 or upon activation by tumor (Kemika, Zagreb, Croatia). After washing with PBS, factor (TNF) family receptors,12 provokes blocking buffer (Dako, Glostrup, Denmark) was added apoptosis or increases the rate of apoptosis. Nevertheless, and cells were incubated for further 20 min at room the exact mechanisms by which p21 promotes apoptosis in temperature. Primary monoclonal against p21 cancer cells are not disclosed yet and further studies are (anti-mouse p21 immunoglobulin G (IgG); Pharmingen, needed to make it exploitable in cancer therapy. San Diego, CA, USA), at concentration 2.5 mgmlÀ1 were In this study, we investigated the effects of p21waf1/cip1 allowed to bind overnight at 4 1C in humid atmosphere. gene overexpression in human laryngeal squamous The cells were washed with PBS and the secondary carcinoma cells lacking p53 expression HEp-2 on polyclonal against mouse IgG (Amersham apoptosis induction upon the treatment with two NA931V, Uppsala, Sweden) diluted in PBS at concentra- common chemotherapeutic agents, cisplatin and metho- tion 4% was added and incubated for a further 45 min trexate. We observed the opposite roles of p21 in at room temperature. The cells were then incubated apoptosis depending on the chemotherapeutic agent, with peroxidase–antiperoxidase complex (Dako) (1:100) which was directly correlated with the expression of two for 30 min. Finally the cells were stained with 0.025% genes: BIK and GADD45a. Finally, we found that p21 diaminobenzydyn tetracloride solution (DAB, Sigma, overexpression provoked apoptosis of HEp-2 cells Taufkirchen, Germany) containing 3% H2O2 for 5 min

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 578 and counter-stained with hematoxylin (Dako). Slides were point was performed in duplicate in three individual mounted in glycerol medium (Glycergel Dako) and experiments. The percentage of the cells in each cell-cycle analyzed by light microscope. The p21-positive cells phase was based on the obtained DNA histograms and (brownish colored) were counted and the percentage determined using the ModFit LT program. Statistical was expressed as a number of positive-infected cells analysis was performed in Microsoft Excel by using the compared to mock-infected cells. At least 100 cells were ANOVA at Po0.05. counted. V test Antiproliferative assays Detection and quantification of apoptotic cells at single Antiproliferative effect of cisplatin (PLIVA 10, PLIVA, cell level was performed using Annexin V-FLUOS Zagreb, Croatia), methotrexate (methotrexate; Pharmacia kit (Roche, Basel, Switzerland), according to & Upjohn, London, UK) and Ad-CMV-p21 were tested. the manufacturer’s recommendations. The cells were The cells were inoculated onto standard 96-well microtiter seeded in six-well plates (3 Â 105 cells per well) and plates on day 0 at concentrations 1 Â 103 cells per well for treated according to the required schedule. After the HEp-2, 1.5 Â 103 cells per well for CAL-27 and 3 Â 103 desired length of time, both floating and attached cells cells per well for Detroit 562. Test agents in five 10-fold were collected. The cells were then washed with PBS with dilutions (10À8–10À4 mol lÀ1), or viral dilutions (10–50 the addition of 2% FCS, pelleted and resuspended in two MOI (multiplicity of infection) were then added and staining solutions prepared in the 4-(2-hydroxyethyl)-1- incubated for a further 24, 48 and 72 h. Working dilutions piperazineethanesulfonic acid buffer containing either were freshly prepared on the day of testing. After Annexin V-fluorescein labeling reagent, PI or their incubation, the cell growth rate was evaluated by combination. Camptotecyn (20 mM; Sigma) was used as performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- the control apoptosis-inducing agent. The cells were then diphenyltetrazolium bromide) , as previously de- analyzed with Becton Dickinson FACSCalibur flow scribed.13 Each test point was performed in quadruplicate cytometer (10 000 counts were measured). Fluorescence in three individual experiments. Each result is a mean compensation and analysis was performed with Summit value from three separate experiments. V3.1 software (Cytomation Inc., Glostrup, Denmark). Similarly, for determination and comparison of the Annexin V-labeled cells were determined to be early adenovectors effect alone or in combination with cisplatin apoptotic and Annexin V and PI cells were determined to or methotrexate, the modified MTT test as described be late apoptotic/necrotic cells. Each test point was below was used. Viral dilutions at 40 and 50 MOI were performed in duplicate in two individual experiments. prepared and the cells were either infected with adeno- The results are shown as mean percentages from three , or mock-infected (10% DMEM). After 1.5 h of separate experiments for all groups and were further infection cisplatin dilutions (0.25, 0.5, 0.75, 1, 1.25, 5, 10, statistically analyzed in Microsoft Excel by using the 15, 20, 25 mgmlÀ1), methotrexate (5 Â 10À9,10À8, ANOVA at Po0.05. 5 Â 10À7,1Â 10À7,1Â 10À6,1Â 10À5 and 1 Â 10À4)or 10% DMEM were then added and incubated for 24, 48 RNA isolation and 72 h. After incubation, the cell growth rate was The cells (1 Â 106) were seeded in 10 mm dishes (Falcon evaluated by performing the MTT assay as described San Jose, CA, USA). Total RNA was isolated in two above. The percentages of growth (PGs) were calculated technical replicates in three biological experiments from as ratios of mean absorbance values for each treatment treated and untreated cells by using TRIZOL (Invitrogen, with its respective control multiplied with 100. Carlsbad, CA, USA) according to the manufacturer’s In addition, the growth rate of cells was measured by recommendations. RNA concentrations were determined counting the numbers of viable cells by Trypan blue spectrophotometrically at 260 nm and the RNA quality exclusion. The experiments was repeated three times and was checked routinely by gel . statistic analysis was performed in Microsoft Excel by using the analysis of variance (ANOVA) at Po0.05. DNA-chip analyses For the DNA-chip analyses, total RNA was obtained in Cell-cycle analysis three biological experiments each performed in two A total of 2 Â 105 cells per well were seeded in six-well technical replicates from treated and untreated cells. plates. The cells were infected with Ad-CMV-p21 at 40 Commercial arrays containing highly specific 96 bp MOI and/or were treated with cisplatin and methotrexate fragments of apoptosis-related genes (Apoptosis cDNK at concentrations 0.5 mgmlÀ1 and 1 Â 10À7 mol lÀ1, res- GEArray, Q Series HS-002 from SuperArray, Frederick, pectively. After the desired length of time the attached MD, USA, http://www.superarray.com/) were used ac- cells were trypsinized, combined with floating cells, cording to manufacturer’s recommendations. Briefly, a washed with PBS and fixed with 70% ethanol. Immedi- total of 3 mg of the obtained RNA per sample was ately before the analysis, the cells were washed with PBS transcribed to cDNA (probe) with the reverse transcrip- and stained with 1 mgmlÀ1 of propidium iodide (PI) with tion cocktail containing specific apoptosis-related pri- the addition of 0.2 mgmlÀ1 of RNAse A. The stained cells mers, reverse transcriptase (200 U; MMLV reverse were then analyzed with Becton Dickinson FACSCalibur transcriptase, Promega, Madison, WI, USA), deoxy- flow cytometer (20 000 counts were measured). Each test nucleotides at concentrations 5 mM dATP, 5 mM dCTP,

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 579 5mM dGTP, 0.5 mM dTTP (Eppendorf, Hamburg, analysis Germany), 1 M dithiothreitol, and the RNAse inhibitor Treated and untreated cells were lysed with the buffer (40 U; RNase inhibitor; Promega). The AmpoLabeling- containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethane- LPR Kit L-03 (SuperArray) was used for signal ampli- sulfonic acid, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2 mM fication and biotin (1 mM, Biotin-16-dUTP; Roche) was ethylene glycol tetraacetic acid, 10% glycerol, 1% Triton used for cDNA labeling. Pre-hybridization of arrays was X-100 and inhibitor cocktail (Roche, performed in cylinders obtained from the manufacturer Switzerland). Total proteins (40 mg) were resolved on 9 with previously denatured salmon sperm DNA (Shared or 12% SDS–polyacrylamide gel depending on the salmon sperm; Invitrogen) at final concentration studied protein at constant 100 V and subsequently 100 mgmlÀ1 at 60 1C in a hybridization oven. After 2 h transferred to nitrocellulose membrane (Bio-Rad, of pre-hybridization, the labeled probe was added onto Hercules, CA, USA) at constant 200 mA using Mini- arrays and hybridized for 12 h in the hybridization oven at PROTEAN Cell Hercules, CA, (Bio-Rad). Membranes 60 1C at 5–10 g. The hybridized arrays were washed with were blocked for 1 h at room temperature with 4% nonfat the solution I (2 Â SSC (20 Â SSC; 3 M NaCl, 0.3 M dry milk in Tris-buffered saline Tween-20 (TBST; 50 mM Na-Citrate, pH 7.0), 1% SDS) and II (0.1 Â SSC, 0.5% Tris base, 150 mM NaCl, 0.1% Tween-20, pH 7.5). SDS). Blocking and chemiluminescent detection was Subsequently, membranes were incubated overnight at performed with the kit components according to the 4 1C in 3% nonfat dry milk in TBST supplemented with manufacturer’s recommendations. The signal was cap- primary antibodies against p21 (monoclonal anti-p21 tured with chemiluminiscent films (Lumi-Film Chemilu- mouse IgG, diluted 1:200; Santa Cruz Biotechnology, minescent Detection Film; Roche). The films were Santa Cruz, CA, USA), caspase 7 (monoclonal anti- developed automatically by using the Thermal Imaging procaspase 7, diluted 1:1000; BD Pharmingen, San Diego, System FPI-500; Amersham Pharmacia. The scanned CA, USA) and caspase 3 (monoclonal anti-procaspase 3 films were analyzed by using the manufacturer’s com- mouse IgG, diluted 1:200; Santa Cruz Biotechnology). mercial software GEArray Expression Analysis Suite. The membranes were then washed with TBST and Housekeeping genes (GAPDH and PPIA) were used for incubated for 1 h at room temperature in TBST contain- normalization and minimal value was used for back- ing a secondary anti-mouse antibody linked to horse- ground correction. Normalized values were statistically radish peroxidase. The signal was detected by the Western analyzed among all groups with SPSS (SPSS for Lightening Chemiluminiscence Reagent Plus kit (Perkin Windows) by ANOVA (at Po0.05, Tukey’s honestly Elmer, Waltham, MA, USA) and visualized on the significantly different (HSD) for post-hoc comparisons). VersaDoc Imaging System 4000 (Bio-Rad). Signal in- tensities of the particular bands were measured and Quantitative RT-PCR analyzed by the Quantity One software (Bio-Rad). Anti- For qPCR, total RNA was isolated in three biological a-tubulin (monoclonal anti- a-tubulin mouse IgG, diluted experiments each performed in two technical replicates 1:1000; Sigma) was used as a loading control. from treated and untreated cells. High-capacity cDNK Archive Kit (Applied Biosystems, Foster City, CA, USA) was used for reverse of total RNAs obtained from three biological replicates into the cDNAs. The cDNAs were used in the reverse transcription (RT)–PCR Results reaction for quantitative analyses (qPCR) using ABI Antiproliferative effect of AdCMV-p21, methotrexate PRISM 7000 Sequence Detection System (Applied and cisplatin on head and neck squamous cell Biosystems). In the predeveloped TaqMan assay reagents carcinoma cell lines for genes: p21waf1/cip1 (Hs00355782_m1), TNFRSF9 The responses of HEp-2, CAL 27 and Detroit 562 cells to (Hs00155512_m1) BIK (Hs00154189_m1), CASP7 the individual therapeutic agents (cisplatin, metotrexate (Hs00169152_m1), DFFB (Hs00237077_m1), GADD45A and AdCMV-p21) or the combination of the chemothera- (Hs00169255_m1), 18sRNA (Hs99999901_s1), RPLP0 peutics and adenovirally mediated p21waf1/cip1 gene (Hs99999902_m1), PPIA (Hs99999904_m1) and GAPDH expression were analyzed by determining the relative (Hs99999905_m1) (Applied Biosystems) were used. The viability of the treated cells. In addition, the gene transfer PCR reactions were carried out according to the efficiency was determined by immunocytochemical detec- manufacturer’s protocol. tion of the cellular p21 protein expression. The endogen- Gene expression monitored by qPCR relative to the ous expression of the p21 protein was detected in CAL 27 control condition (untreated cells and/or cells treated with cells (70% of total cell number) and Detroit 562 cells methotrexate or cisplatin for comparisons of combined (30% of total cell number). The p21 protein expression treatments involving both AdCMV-p21 and the cyto- levels increased in all cell lines upon AdCMV-p21 stiatic compounds) was shown as fold change. Data were treatment, depending on the MOI. In HEp-2 cells, no statistically analyzed by using The Relative Expression endogenous p21 protein expression was detected, so Software Tool (REST-MCS) using the randomization increased p21 expression is attributed solely to the tests14,15 and normalization with three reference genes adenovirally mediated p21waf1/cip1 gene transduction (18S RNA; RPL0 and PPIA) having stable expression in (Table 1). As expected, the empty vector AdCMV-dl tested treatments. 312 did not cause the induction of p21 protein expression.

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 580 Table 1 The percentage of HEp-2 cells expressing the p21 protein, 24 and 72 h after the infection with AdCMV-p21 Treatment Percentage of p21-positive cells (%)

24 h 72 h

Control 0 0 Ad-CMV-p21 MOI 20 31 30 Figure 2 Giant and apoptotic cells 72 h after infection of HEp-2 (a) Ad-CMV-p21 MOI 30 36 35 and CAL 27 (b) cells with AdCMV-p21 at 50 MOI (multiplicity of Ad-CMV-p21 MOI 50 50 55 infection). Red arrows point to giant cells (several fold increase in the cell volume) and blue arrows point to cells undergoing apoptosis (condensed nuclei). The cells were immunocytochemically stained, whereby the p21 protein expression was observed as brown staining.

42% at an MOI of 50. Interestingly, even for the most sensitive cell line (HEp-2), substantial growth inhibition was observed only after 6 days of treatment with MOIs of 40 and 50. Therefore, these vector concentrations were used for further experiments. Morphological changes were observed both in HEp-2 and CAL 27 cells after the 24- and 72 h treatments. The cells were significantly enlarged after 24 h, which was indicative for the cell-cycle arrest induced by AdCMV- p21. After 72 h, a giant cell formation was observed both in HEp-2 and CAL 27 cells, as well as morphological changes pointing to the induction of apoptosis, mainly in Hep-2 (Figure 2). As expected, cisplatin exerted a strong and similar antiproliferative effect on all tested cell lines (data not À6 shown) (IC50 values were about 3 Â 10 M), which corresponds to 0.5 mgmlÀ1, whereas methotrexate differen- tially inhibited the growth of all tested cell lines, being somewhat less active on Detroit 562 (IC50 values were À8 about 4.9 Â 10 M), and slightly cytotoxic to HEp-2 cells. These results were in accordance with the previously published results.16,17 The results of treatments with AdCMV-p21, chemo- therapeutics and combination treatments are shown in Figure 3. The combined treatment with AdCMV-p21 at an MOI of 40 and cisplatin (0.25–5 mgmlÀ1) or metho- À4 À9 trexate (1 Â 10 –5 Â 10 M) differed among the tested cell lines. The p21waf1/cip1 gene overexpression had no effect on the CAL-27 cell growth inhibition induced by cisplatin (data not shown). But, a trend was observed Figure 1 Effect of AdCMV-p21 at 10–50 MOI (multiplicity of toward a dose-dependent reduction of viability (up to infection) on the growth of HEp-2, CAL 27 and Detroit 562 tumor 16%) of HEp-2 cells treated with AdCMV-p21 and cells. The percentages of growth (PGs) were assessed by the MTT cisplatin in comparison with cells treated with cisplatin (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay alone. Interestingly, completely opposite trend was after a 6-day treatment in three biological replicates. Statistically observed in the combined treatment of Detroit 562 cells relevant results (Po0.05) are marked with the asterisk symbol. The with AdCMV-p21 at an MOI of 50 and cisplatin where empty vector AdCMV-dl 312 was used as control. the percentage of viable cells was 10–20% higher in the combined treatment in comparison with the cisplatin treatment alone. Moreover, AdCMV-p21 had no effect on AdCMV-p21 induced a time- and dose-dependent the cell growth inhibition of CAL-27 and Detroit 562 cells antiproliferative effect on HEp-2 and CAL 27 cells, induced with methotrexate (data not shown). However, whereas Detroit 562 cells were completely resistant to the an enhanced surviving fraction of HEp-2 cells (up to treatment (Figure 1). The strongest antiproliferative effect 20%) treated with methotrexate and AdCMV-p21 was of AdCMV-p21 was observed for HEp-2 (29–71%) observed, pointing to a possible promotion of cell survival whereas the growth inhibition of CAL 27 cells reached by p21waf1/cip1 gene overexpression.

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 581

Figure 3 The effect of AdCMV-p21 at 50 MOI (multiplicity of infection) on the growth of cells treated with cisplatin or methotrexate after the 72 h treatment of HEp-2 and Detroit 562. The dose–response curves were obtained by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. The results are shown as percentages of growth (%) for each treatment point±standard deviation. Statistically significant changes are marked with the asterisk symbol (Po0.05).

This differential effect of p21waf1/cip1 gene overexpres- confirmed by a set of experiments with low concentrations sion induced by AdCMV-p21 on the growth of HEp-2 of cytostatic compounds (0.5 mgmlÀ1 for cisplatin and À7 treated with cisplatin or methotrexate was additionally 1 Â 10 M for methotrexate), whereby the cells were

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 582 Table 2 The results of the HEp-2 cell-cycle analysis

Treatment Cell-cycle phase (%)

SubG0/G1 G0/G1 SG2/M

24 h Control 6.6±2.9 43.1±0.5 42±3.6 14.9±4.1 AdCMV-p21 40 MOI 9.6±1.1 67.1±1.4 * 25.1±2.6* 7.8±1.2* Cisplatin 0.5 mgml-1 5.1±0.5 49.7±1.9 * 34.5±6.4 15.4±5.1 Cisplatin+AdCMV-p21 6.2±0.1 40.5±2.2 38.9±5.6 20.5±3.4 À7 Methotrexate 10 M 8.8±0.2 55±0.9* 44.8±0.7 0.6±0.4* Methotrexate+AdCMV-p21 10±0.8 47.2±1.4 * 48.9±1.5 3.9±0.1*

48 h Control 10±4 52.3±1.9 37.4±2 10.2±0.1 AdCMV-p21 40 MOI 11±3.7 35.4±0.9* 38.5±2.1 26±1.1* Cisplatin 0.5 mgml-1 21.2±1.9 50.4±9.6 36.4±2.9 12.8±6 Cisplatin+AdCMV-p21 20.7±1.8 41.5±3* 43.3±6.9 15.3±3.9 À7 Methotrexate 10 M 14.7±0.7 65.9±3.3* 27.1±10 7.1±6.9* Methotrexate+AdCMV-p21 10.8±2.1 51.1±0.5 41.6±0.1 7.3±0.4*

72 h Control 14.9±0.5 50.5±0.4 31.8±2.8 17.7±2.4 AdCMV-p21 40 MOI 25.4±3.6* 32.4±3.4* 49.6±8.7 18±5.3 Cisplatin 0.5 mgml-1 44.1±2.4* 13.1±0.4* 25.5±0.7 61.3±0.5* Cisplatin+AdCMV-p21 77.7±2.9* 39.5±9.2 58.1±11.2 3.1±2.7* À7 Methotrexate 10 M 29.4±1.1* 82.2±3.2* 13.5±2.8* 0* Methotrexate+AdCMV-p21 25.3±2.4* 69.9±2.1* 0.9±0.8* 29.2±1.2*

-1 À7 The cells were treated with AdCMV-p21 at 40 MOI, cisplatin at 0.5 mgml , methotrexate at 1 Â 10 M and with their combinations. The results are shown as cell percentages (%) in a particular cell-cycle phase±standard deviations. Statistically significant results are marked with asterisk (*) (Po0.05).

manually counted using Trypan blue staining after the arrested in the G0/G1 cell-cycle phase (31.7%) at all time 72 h treatment. There was a statistically significant points. Besides, both cytostatic compounds caused an decrease of viable cells (10–33%) in the combined increase in subG0/G1 cell proportion after the 72 h treatment of HEp-2 with AdCMV-p21 and cisplatin when treatment. The cell-cycle perturbations induced by chemo- compared with cells treated with cisplatin alone. Simi- therapeutics were similar to those induced by the larly, a constant increase of viable HEp-2 cells (13–21%) combined treatment with AdCMV-p21. However, there in the combined treatment with AdCMV-p21 and was a significant increase in the percentage of cells in the methotrexate compared with methotrexate treatment subG0/G1 (33.6%) treated with the combination of alone was also confirmed (data not shown). AdCMV-p21 and cisplatin in comparison to those cells treated with cisplatin alone after the 72 h treatment. In Cell-cycle perturbations in HEp-2 cells induced by addition, an increase of cells in the G2/M phase was AdCMV-p21, cisplatin and methotrexate alone or by observed in the combined treatment with methotrexate their combinations and AdCMV-p21 in comparison to the treatment with The previous results showed that overexpression of the methotrexate alone after 72 h. p21waf1/cip1 gene in HEp-2 cells treated either with AdCMV-p21, cisplatin, methotrexate, or their combina- Effects of AdCMV-p21 on the cisplatin or methotrexate tion caused a differential effect on the cell growth. apoptosis induction in HEp-2 cells Therefore, we have further investigated this phenomenon According to the obtained results, it is likely that by studying the cell-cycle perturbations caused upon the apoptosis occurs upon treatments of HEp-2 cells with introduction of the p21waf1/cip1 gene in HEp-2 treated with AdCMV-p21, cisplatin and methotrexate, however more cisplatin or methotrexate using . (Table 2). precise method should be used to confirm this observa- The overexpression of p21 gene caused a significant tion. Therefore, we performed the Annexin V test by G0/G1 cell-cycle arrest (24%) 24 h after the AdCMV-p21 using flow cytometric analyses (Figure 4). We were infection, whereas it induced a G2/M arrest after the 48h especially interested in the investigation of the potential treatment (16%). In addition, an increase of cells in pro- and antiapoptotic effects of p21waf1/cip1 gene over- subG0/G1 (10.5%), which is indicative for cells under- expression observed after the combined treatment with going apoptosis, was observed after the 72 h treatment. AdCMV-p21 and the chemotherapeutic compounds. We Initially, both cisplatin and methotrexate induced the have not been able to detect statistically significant cell-cycle arrest in the G0/G1 (7–12%). However, after the changes among the treatments and control after the 24 h 72 h treatment cisplatin caused an increase of cells in treatments. However, an increase in the number of cells G2/M phase (43.6%) at the expense of cells in G1 and S in different phases of apoptosis was detected after 72 h. phase, whereas the cells treated with methotrexate were AdCMV-p21 alone caused a 28% increase in the

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 583

Figure 4 The results of the annexin V test performed with HEp-2 cells after 72 h treatments with AdCMV-p21 at 40 MOI (multiplicity of infection), À1 À7 cisplatin at 0.5 mgml , methotrexate at 1 Â 10 M, alone or with their combinations. The results are shown as percentages of viable cells (R7 quadrant), percentages of early apoptotic cells (R8 quadrant) and percentages of late apoptotic cells (R6 quadrant)±standard deviation. Statistically significant changes for treatments in comparison to respective controls are marked with the asterisk symbol (Po0.05). apoptotic cell population. Cisplatin caused a 25% combined treatment of methotrexate and AdCMV-p21 increase of apoptotic cells, whereas methotrexate in- in comparison to cells treated with methotrexate alone, creased the number of apoptotic cells by 40%. pointing thus again to p21 pro-survival signaling. On the other hand, when both cisplatin and AdCMV- p21 treatments were administered, 20% more late- Changes in the expression of apoptosis-related genes in apoptotic cells were detected compared to the cisplatin HEp-2 cells treated with AdCMV-p21, cisplatin and treatment alone, pointing again to the overexpression of methotrexate or their combination p21waf1/cip1 gene’s influence on the induction of cell death. In order to identify the mechanism underlying the above- By contrast, there were 4% more viable cells and 13% mentioned phenomena, possibly related to the changes in more cells in the early phases of apoptosis in the the apoptosis signaling pathways, the expression of 96

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 584 À7 Table 3 Changes of apoptosis-relevant gene expression in HEp-2 cells treated with Ad-CMV-p21 at 40 MOI, methotrexate (1 Â 10 M), cisplatin (0.5 mgml-1) or with their combinations after the 24 h treatment by cDNA microarrays Treatment AdCMV-p21 Methotrexate AdCMV- Cisplatin AdCMV- À7 À1 (40 MOI) (1 Â 10 M) p21+methotrexate 0.5 mgml p21+cisplatin Gene Gene name

TNFRSF9 m 2.3 m 2.3 m 2.1 1.3 m 2.2 superfamily, member 9 DFFB DNA fragmentation factor, 40 kDa k 2.2 0.5 k 1.3 0.4 1.6 b-polypeptide GADD45a Growth arrest and DNA damage- 0.9 m 1.8 m 1.0 1.1 m 1.9 inducible-a CASP6 , apoptosis-related 1.0 k 0.6 k 0.5 0.8 0.7 cysteine peptidase CASP7 Caspase 7, apoptosis-related 1.0 1.0 m 0.9 m 1.8 m 1.8 cysteine peptidase BIK Bcl-2-interacting killer (apoptosis 1.2 1.3 k 0.5 0.7 1.0 inducing) MYD88 Myeloid differentiation primary 1.1 0.8 k 2.1 1.0 m 1.9 response gene (88) BNIP3 Bcl-2/adenovirus E1B 19 kDa 1.1 0.7 k 2.4 0.8 0.9 interacting protein 3 Upregulated genes are marked with the sign m and downregulated genes are marked with the sign k. Statistically relevant changes (Po0.05) in comparison to control are shown in bold.

Table 4 Gene expression monitored by quantitative PCR relative to the control condition presented in fold change±standard error Treatment/Gene AdCMV-p21 Methotrexate AdCMV- Cisplatin AdCMV- À7 -1 (40 MOI) (1 Â 10 M) p21 +methotrexate (0.5 mgml ) p21 +cisplatin

BIK 3.8±0.8* 2.6±0.5* À1.1±0.2 2.4±1.1 2.9±1.4 CASP7 1.5±0.1* 1.2±0.2 1.08±0.1 2.3±0.36** 2±0.2+ DFFB À2.7±0.04*** À1.5±0.7 À2.2±0.03* 2.1±0.3** 2.4±0.22+ GADD45a 3.1±0.41* 69.8±14.6* 5±2.9** 243±64*** 2±0.07** TNFRSF9 22.8±2.6*** 4.4±0.7*** 10.4±1.9* 1.9±0.32* 8.7±1.7* p21waf1/cip1 175.9±23.8* À1.2±0.1 163.9±14.1*** 2±0.24 723±84* *P ¼ 0.001; **P ¼ 0.015; ***P ¼ 0.003; +P ¼ 0.05.

genes involved in the regulation of apoptosis were trends. Only GADD45a and BIK genes were differentially analyzed, using cDNA microarray method (see ‘Materials expressed (Po0.05) between cells treated with the and methods’). Several differentially expressed genes combination of AdCMV-p21 and methotrexate and among all treatments when compared to the controls methotrexate alone, whereby both genes were down- were detected after the 24 h treatment (Table 3). The regulated to control levels in the combined treatment. expression of TNFRSF9 gene increased (2.3-fold change), When compared to control cells, the combined treatment whereas the expression of DFFB decreased (2.2-fold with AdCMV-p21 and methotrexate, exerted upregula- change) 24 h after the treatment with AdCMV-p21 at 40 tion of TNFRSF9 gene (2.1-fold change), along with MOI. The GADD45a and TNFRSF9 genes were upregu- downregulation of CASP6 (2-fold change), MYD88 (2.1- lated for 1.8- and 2.3-fold, respectively, by the treatment fold change) and BNIP3 (2.4-fold change) genes. À7 with methotrexate at 1 Â 10 M. Surprisingly, the treat- ment with cisplatin caused only upregulation in the qPCR confirmation of cDNA analyzes expression of CASP7 gene (1.8-fold change). However, The expression changes of the most interesting differen- when cisplatin was combined with AdCMV-p21 we tially expressed gene (BIK, CASP7, DFFB, GADD45a observed significant upregulation in the TNFRSF9 (2.2- and TNFRSF9) were additionally confirmed by qPCR fold change), GADD45a (1.9-fold change), CASP7 (1.8- (Table 4). The expression of the p21waf1/cip1 gene was used fold change) and MYD88 (1.9-fold change) expression. as experimental control. Although these results were in We were especially interested whether the p21waf1/cip1 concordance with the results obtained with cDNA gene overexpression combined with the respective che- microarrays, qPCR method as a far more sensitive motherapeutic agent, induced differential regulation of method, allowed for precise detection of fold changes apoptosis-related genes as a basis of the above-mentioned with high statistical significance for those genes found to

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 585 be differentially expressed in cDNA experiments. For Similarly, the procaspase 3 was cleaved in the treatment example, the BIK gene was significantly overexpressed in with cisplatin alone (1.5-fold) and in the combined the treatments with AdCMV-p21 (3.8±0.8) and metho- treatment cisplatin-AdCMV-p21 (1.4-fold) after the 24 h trexate (2.6±0.5), whereas its expression was not altered treatment. The changes in procaspase 3 status was more in the combined treatment with AdCMV-p21 and dramatic after the 48 h treatment, whereby it was cleaved methotrexate, neither it was altered in the treatments in the treatment with cisplatin alone (1.5-fold), in the with cisplatin, or cisplatin and AdCMV-p21 in comparison methotrexate treatment (1.3-fold) and completely in the to control. Similarly, the overexpression of GADD45a combined treatment cisplatin-AdCMV-p21, confirming was detected in all treatments. However, its expression again that the p21waf1/cip1 gene overexpression enhances levels were significantly lower when cisplatin or metho- apoptosis activated by cisplatin. trexate was used in combination with AdCMV-p21. It is known that HEp-2 cells constitutively express the Moreover, increased expression levels for CASP7 were E6 protein of the papilloma virus type 18 that is detected in treatments with AdCMV-p21 (1.5±0.1), responsible for the inactivation of the pro-apoptotic cisplatin (2.3±0.36) and in the combined treatment of tumor-suppressor protein p53. Therefore, we tested cisplatin and AdCMV-p21 (2.0±0.2). Overexpression of another p53-independent apoptotic protein that might the DFFB gene was found only in the treatment with ultimately lead to caspase activation in HEp-2, the c-Jun cisplatin, whereas its underexpression was observed in N-terminal (JNK) known to be activated treatments with AdCMV-p21 and in the combined (phosphorylated) upon different extracellular stimuli. We treatment with AdCMV-p21 and methotrexate. The observed a twofold rise in the level of phosphorylated TNFRSF9 gene expression was significantly changed in JNK (p-JNK) only in cells treated with a combination of all treatments. However, the highest expression values cisplatin and AdCMV-21 after the 24 h treatment, and a were observed in the treatment of HEp-2 with AdCMV- somewhat diminished level in treatments with methotrex- p21. In addition, its expression was higher in the ate (1.4-fold) and methotrexate and AdCMV-p21 (1.2- combined treatments with chemotherapeutics and fold). Moreover, we observed a threefold rise in the level AdCMV-p21 (8.7- to 10.4-fold change) when compared of p-JNK in the treatment with AdCMV-p21 and about to the respective chemotherapeutic alone (1.9- to 4.4-fold threefold rise in all other treatment regimes after 48 h in change). comparison to control, whereby it was somewhat reduced in the combined treatment with methotrexate and Western blot analyses AdCMV-p21, comparing to methotrexate alone. In order to measure the p21 protein levels upon AdCMV- At last, we verified the status of the Bik protein p21 infection of HEp-2 cells, western blot analyses were expression throughout all the treatments but were not performed 24 and 48 h after infection (Figure 5). The able to detect any changes in its expression regardless of results were consistent with immunocitochemistry and the treatment duration (data not shown). both methods confirmed that the transferred gene was overexpressed (also detected by qPCR), as well as translated into active protein. The p21 protein expression Discussion was observed exclusively in cells treated with AdCMV- p21, whereby it was significantly lower in the combination The CKI p21waf1/cip1 has been known as a universal treatment with cisplatin and AdCMV-p21, which was not inhibitor of the (cyc/cdk) that mediates the cell prolifera- the case at the gene expression level. tion arrest induced either by p53-dependent or -indepen- Moreover, we wanted to verify the status of main dent mechanisms.11 Because of this antiproliferative role, apoptosis effectors molecules at the protein level, namely it has been tested as a ‘gene drug’ for treatment of the caspases 3 and 7. Caspases are regulated at a post- malignant cells both in vitro and in vivo, whereby the translational level, ensuring they can be rapidly activated. exogenous introduction of p21waf1/cip1 caused either They are first synthesized as inactive procaspases, which apoptosis or the cell-cycle arrest.10,18–20 The aim of this are activated upon cell death stimuli by proteolytic study was to investigate the effects of p21 protein cleavage. Therefore, we performed expression analyses overexpression induced upon adenoviral introduction of of the procaspases 7 and 3 protein expression levels. Both the p21waf1/cip1 gene by AdCMV-p21, on the growth of procaspases 3 and 7 were cleaved in some treatments that squamous head and neck cancer cell lines HEp-2, CAL 27 was indicative for caspase activation and HEp-2 apopto- and Detroit 562. In addition, we tried to elucidate how sis. The procaspase 7 was cleaved in the combined p21waf1/cip1 modulates cell response to two common treatments of cisplatin and AdCMV-p21 (1.8-fold change) chemotherapeutics, cisplatin and methotrexate. Our and methotrexate and AdCMV-p21 (1.7-fold change) results are in concordance with previously published data after the 24 h treatment. In addition, it was cleaved in the showing strong antiproliferative effect of p21waf1/cip1 treatments with AdCMV-p21 (2.0-fold change), in the overexpression on HEp-2 and CAL 27 at higher tested combined treatment with cisplatin and AdCMV-p21 (2.7- vector concentrations (40 and 50 MOI). However, the fold change) and in the treatment with methotrexate (1.4- most pronounced cytostatic effects of all treatment combi- fold change) after 48 h. Interestingly, the procaspase 7 nations were seen in HEp-2, which was the reason we chose expression was not altered in the combined treatment with this particular cell line for detailed mechanistic studies on methotrexate and AdCMV-p21 after the 48 h treatment. p21waf1/cip1 role in the chemotherapeutic response.

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 586

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 587 Our results showed a several fold increase in the cell protein with a BH-3 domain crucial for its proapoptotic volume of HEp-2 upon AdCMV-p21 introduction, which activity.27 BH3-only proteins are activated upon cytotoxic can be ascribed to the G0/G1 arrest. This phenomenon has signals and prime cells for apoptosis by activation of the already been described by some authors as ‘giant cells’ intrinsic apoptotic pathway.28 Finally, p21waf1/cip1 gene formation characterized by impairment in introduction in HEp-2 resulted in the GADD45a gene followed by cell death resulting from ectopically induced overexpression known to be ubiquitously activated upon p21 protein overexpression.10,21 However, our study DNA damage independently of the p53 status.29 indicates that apart from formation of giant cells, a The role of GADD45a has been implicated in the portion of HEp-2 cells treated with AdCMV-p21 directly control of the cell-cycle G2/M checkpoint, induction of underwent apoptosis, as confirmed by a rise in transcript cell death and DNA-repair process induced upon DNA levels of the gene CASP7 accompanied by the activation damaging agents and growth arrest signals.29 Cisplatin of the caspase 7 protein. Apoptosis induction upon and methotrexate treatments have in common significant exogenously induced p21 protein overexpression was raise of GADD45a transcripts levels and activation of already observed for some cervical carcinoma cell lines JNK after a prolonged treatment. The question however with inactivated p53 protein11,20 as well as for some cells remains whether the observed overexpression of with mutated p53.4,22 As HEp-2 cells contain inactivated GADD45a gene in HEp-2 lies upstream or downstream p53 protein, it is possible that exogenously induced p21 of activated MAPK/SAPK-JNK pathway. According to protein overexpression might compensate for p53 defi- the literature data, both possibilities are likely to occur ciency and induce p53-independent apoptotic pathways. and should be further tested.30 But, according to our Apoptosis could be driven both by distinct extrinsic/death results it seems plausible to assume JNK activation receptor and intrinsic/mitochondrial pathways, which are downstream of GADD45a. mutually intertwined.23 Indeed, our transcriptomic results Cisplatin and methotrexate appear to activate different reveal that both apoptotic mechanisms are triggered at apoptotic pathways as partially expected from their the transcription level by AdCMV-p21 in HEp-2. Hereby, diverse mechanisms of action. Although cisplatin directly the involvement of extrinsic pathway is witnessed by a binds to DNA resulting in formation of intra- and strong overexpression of the TNFRSF9 gene encoding for interstrand adducts as well as in cross-linking DNA to the cell receptor TNFRSF9. TNFRSF9 gene is a member proteins,31 methotrexate mechanism of action is linked of tumor necrosis factor receptor (TNFR) superfamily to its antimetabolic activity and block of the DNA and encodes for a B39 kDa glycoprotein induced on the synthesis.32,33 Both, cisplatin and metotrexate treatments surface of T and natural killer cells during their induce G0/G1 arrest followed by apoptosis. This is in activation24 with a potential in cancer .25 concordance with previously published data showing that However, its exact role in apoptosis induction of cancer methotrexate induces the G0/G1 cell-cycle arrest in cells cells has not been studied in details. It was shown that its with inactivated p53.29 However, low concentration of aggregation could activate the mitogen-activated protein cisplatin used in these experiments was not completely (MAPK) cascades and stress-activated protein cytotoxic and a fraction of cells survived the treatment 25 kinases (SAPK)/Jun N-terminal kinases (JNK) in T cells and remained arrested at G2/M phase, suggesting that or nuclear factor-kB signaling cascade in 293 cells26 that GADD45a might be involved in this phenomenon. poses the question whether this receptor exerts a similar Generally, the cell growth inhibition of HEp-2 treated role in HEp-2 as well. Indeed, we detected raised levels of with cisplatin was dose dependent, whereas the growth phosphorylated JNK upon AdCMV-p21 introduction, inhibition of cells treated with methotrexate was expo- À6 which raises the possibility that TNFRSF9 mediates cell nentially increasing up to the concentration of 1 Â 10 M death through the MAPK/JNK signaling. The proapop- and after this value, it reached a plateau. Further cell totic role of this signaling pathway has been implicated in death studies highlighted differential effects of these two many studies proving the evidence for the connection chemotherapeutics on apoptosis regulation. Accordingly, between MAPK/JNK signaling and both, inactivation of we established that methotrexate treatment strongly the antiapoptotic Bcl-2 and Bcl-xL proteins and activa- activated caspase 7, whereas cisplatin treatment signifi- tion of the proapoptotic BH3-only proteins.23 In fact, we cantly induced caspase 3 activation. detected overexpression of the BIK gene that encodes for Interestingly, when the p21waf1/cip1 gene was introduced the BH3-only protein, which additionally speaks in the into HEp-2 treated with either cisplatin or methotrexate, favor of apoptosis induction by p21 through the MAPK/ the fate of cells varied. The number of apoptotic cells JNK signaling in HEp-2. The BIK gene is a proapoptotic treated with a combination of cisplatin and AdCMV-p21 member of the Bcl-2 family and encodes for a 18 kDa was higher in comparison with those treated with cisplatin

À1 À7 Figure 5 Effect of AdCMV-p21 (40 MOI), cisplatin (0.5 mgml ), methotrexate 1 Â 10 M) and their combinations on the expression of the p21 protein (a), caspase 3 (b), caspase 7 (d) and on c-Jun N-terminal protein kinase (JNK) phosphorylation (c), after the 24 and 48 h treatments. The expression of a-tubulin was used as the loading control. The results are representative of three independent experiments: line 1—control; line 2—AdCMV-p21; line 3—cisplatin; line 4—cisplatin þ AdCMV-p21; line 5—methotrexate; line 6—methotrexate þ AdCMV-p21. Right panels represent the densitometric analysis of each band, normalized with the intensity of the loading control (a-tubulin).

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 588 alone. Contrary to this, the number of apoptotic cells On the other side, in methotrexate-induced apoptosis, treated with a combination of methotrexate and AdCMV- GADD45a gene seems to play somewhat different p21 was lower in comparison with those cells treated with role, probably as a player in the MAPK/SAPK-JNK methotrexate alone. We believe that such a dual role of pathway, which seems to be one of the main apoptotic exogenously induced p21 protein overexpression is linked pathways triggered by methotrexate in HEp-2. Our results to diverse apoptotic pathways triggered by cisplatin and showing JNK being phosphorylated both in treatments methotrexate in HEp-2. Although it has been reported with cisplatin and methotrexate as well as in the combined that p21 can assume a tumor-suppressor activity,4,10,34 treatments of these cytostatics with AdCMV-p21 raised many authors proved that p21 can assume an antiapop- the question how can the same pathway be involved in the totic role as well in different biological systems.3,4,35,36 opposite cell fate outcome. It has been documented that Similarly, its ectopically induced expression can either the proapoptotic function ascribed to MAPK/SAPK- enhance,19,37,38 or prevent apoptosis induced upon JNK pathway, especially in the genotoxin-induced chemotherapy treatment.39,40 A dual role in apoptosis apoptosis (for example, cisplatin), lies in the expression observed in our study is probably a consequence of p21 of Fas that promotes Fas-induced apoptosis as well protein interaction with particular protein(s) involved in as in MAPK/SAPK-JNK involvement in the activity of apoptosis triggered by cisplatin and methotrexate.41 Bax/Bcl proteins.45 On the other side, protective effects Previous investigations have shown that both the were ascribed for the MAPK/SAPK-JNK pathway, extrinsic and intrinsic apoptotic pathways are triggered probably due to the promotion of DNA repair mechan- by cisplatin in cells lacking p53 expression, which is also isms.45 It is thus possible that late phosphorylation of in concordance with our results.31 One of the signaling JNK in methotrexate-treated cells underlie the cell repair pathways triggered by cisplatin is the cell receptor process. If the damage is too severe, cells undergo (Fas/TRAIL)-mediated apoptotic pathway that leads to apoptosis. Indeed, we have observed elevated levels of initiator caspase activation, for example, caspases 8 or 10 the BIK transcript that diminished to the control level in and downstream to caspase 3 activation. In addition, the the combined treatment with methotrexate and AdCMV- mitochondria-mediated apoptotic pathway can also be p21. This suggests additional activation of the mitochon- activated upon cisplatin treatment by the proapoptotic drial pathway in the methotrexate-treated cells, probably BH3-only proteins, for example, Bid, as a consequence of through the involvement of the Bik protein whose gene initiator caspase activation, which ultimately leads to expression was attenuated upon AdCMV-p21 introduc- caspase 3 activation.42 Indeed, we detected caspase 3 tion. This speculation could be substantiated by a recent activation in HEp-2 treated with cisplatin as well as finding showing that Bim, also being a BH3-only protein, raised levels of CASP7 and DFFB transcripts, both genes triggers p53-independent apoptotic pathways, whereby it being involved in executive phases of apoptosis.23 It has could be activated through cytoskeletal sequestration.46 been established that activated caspase 3 consecutively For example, inducible expression of GADD45a in HeLa mediates the cleavage of p21 protein,43 thus reducing its cells resulted in growth suppression and apoptosis causing possible apoptosis-protective role. Indeed, we detected destabilization of the and translocation of lower p21 expression only at the protein level. Moreover, Bim into mitochondria.46 It seems that similar pheno- we found that p21, both alone, or in combination with menon observed in our study led to decreased levels of cisplatin, simultaneously activates caspase 7 and DFFB transcripts in the combined treatment with JNK, which along with the observed induction of methotrexate and AdCMV-p21. This hypothesis was TNFRSF9, substantiates the activation of apoptosis. On additionally confirmed by inactivation of caspases 3 and the contrary, activation of caspase 3 by methothrexate 7 in the combined treatment highlighting an apoptosis was not observed, which allows p21 to exert apoptosis- inhibition. One possible assumption for the p21-abro- preventing activity. gated apoptosis in HEp-2 could be explained through According to our results a good candidate for the downregulation of the GADD45a gene by exogenously observed pro- and antiapoptotic effect might be also the overexpressed p21waf1/cip1 gene. However, a study of GADD45a gene, because its transcript levels measured in apoptosis induction in HEp-2 by 5-fluorouracil, a widely combined treatments with AdCMV-p21 and both the used drug, showed that GADD45a protein tested cytostatic compounds decreased almost to the does not modulate apoptosis induced by this com- control level when compared to the treatments with pound.44 Moreover, JNK phosphorylation was observed cytostatic compounds alone. Our assumption is that in both in methotrexate- and combined methotrexate- HEp-2 cells treated with cisplatin activation of GADD45a AdCMV-p21 treatments, after a prolonged period. gene probably plays a role in DNA damage repair Although after 48 h p21 slightly reduced the pJNK processes and that p21 interferes with its transcription expression, we do not believe that this phenomenon is blocking the repair process linked to cell growth arrest, substantially important for its dual role, but lack of thus enhancing apoptotic pathways activated indepen- caspase 3 activation allows p21 to exert its apoptosis- dently of GADD45a. Accordingly, a recent study of protective role, whereby late-activated JNK promote apoptosis induction by carboplatin in human papilloma- DNA repair mechanism. virus-positive cell lines HeLa and HEp-2 has proven that In conclusion, the overexpression of p21waf1/cip1 gene in GADD45a protein itself does not modulate cisplatin- HEp-2 cells is likely to trigger the extrinsic apoptotic induced apoptosis in these cells.44 pathway through the upregulation of the TNFRSF9 gene

Cancer Gene Therapy Overexpressed p21 mediates HEp-2 apoptosis S Kraljevic Pavelic et al 589 role of genes/proteins transduction on the signaling pathways of transformed cells’ (0098093) and JEZGRE- TEST ‘Centre for integrative genomics, molecular diag- nostic, cell and gene therapy’ (14M09800). We thank Marko Marjanovic for the assistance in adenoviral vectors preparation and Dr Mirela Baus-Loncar for useful suggestions in qPCR experimental design and help with qPCR data analysis.

Disclosure/Conflicts of interest The authors declare no conflict of interest.

References

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