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A Low UVB Dose, with the Potential to Trigger a Protective p53-Dependent Gene Program, Increases the Resilience of Keratinocytes against Future UVB Insults

2005; Elsevier BV; Volume: 125; Issue: 5 Linguagem: Inglês

10.1111/j.0022-202x.2005.23909.x

1523-1747

David Decraene, Katrien Smaers, Daniel Maes, Mary S. Matsui, L. Declercq, Marjan Garmyn,

Molecular Biology Techniques and Applications

One protein central in the response of human keratinocytes to ultraviolet B damage is p53. By transactivating genes involved in either cell cycle arrest or DNA repair, p53 has a leading role in the recovery from this damage. Considering this role, we wished to investigate whether the triggering of a p53-dependent gene program by repetitive ultraviolet B (UVB) exposure can induce an adaptive response in human skin cells. In particular, we examined two p53-target genes, p21/WAF1 and p53R2, with a crucial role in p53-induced cell cycle arrest and p53-induced DNA repair respectively. Exposure to a mild UVB dose was able to induce an adaptive response in human keratinocytes, leading to increased survival of cells that maintain their capacity to repair DNA damage upon exposure to apoptotic doses of UVB. Our study indicates that this adaptation response is only achieved if the interval between subsequent UVB insults allows sufficient time for the p53-induced protective gene program to be induced. Our results also demonstrate that small but quickly recurring UVB exposures are as harmful as one intense, continual exposure to UVB irradiation. Future research will be oriented toward investigating alternative ways to induce an adaptive response without pre-exposing the cells to UV. One protein central in the response of human keratinocytes to ultraviolet B damage is p53. By transactivating genes involved in either cell cycle arrest or DNA repair, p53 has a leading role in the recovery from this damage. Considering this role, we wished to investigate whether the triggering of a p53-dependent gene program by repetitive ultraviolet B (UVB) exposure can induce an adaptive response in human skin cells. In particular, we examined two p53-target genes, p21/WAF1 and p53R2, with a crucial role in p53-induced cell cycle arrest and p53-induced DNA repair respectively. Exposure to a mild UVB dose was able to induce an adaptive response in human keratinocytes, leading to increased survival of cells that maintain their capacity to repair DNA damage upon exposure to apoptotic doses of UVB. Our study indicates that this adaptation response is only achieved if the interval between subsequent UVB insults allows sufficient time for the p53-induced protective gene program to be induced. Our results also demonstrate that small but quickly recurring UVB exposures are as harmful as one intense, continual exposure to UVB irradiation. Future research will be oriented toward investigating alternative ways to induce an adaptive response without pre-exposing the cells to UV. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide poly(ADP-ribose) polymerase ultraviolet B p53, a nuclear phosphoprotein, functions as a transcription factor with a central role in the stress-response of keratinocytes (Decraene et al., 2004Decraene D. Smaers K. Gan D. et al.A synthetic superoxide dismutase/catalase mimetic (EUK-134) inhibits membrane-damage-induced activation of mitogen-activated protein kinase pathways and reduces p53 accumulation in ultraviolet b-exposed primary human keratinocytes.J Invest Dermatol. 2004; 122: 484-491Crossref PubMed Scopus (37) Google Scholar). In its wild-type form, the p53 protein becomes activated in response to a myriad of stress types, not in the least genotoxic stress (through either ultraviolet radiation, ionizing radiation, or chemical agents). The major aim of this activation is to safeguard the genomic stability of the cells, through transactivation of a plethora of genes with an active role in either cell cycle arrest and global genomic DNA repair or even apoptosis when damage is too substantial. In this respect, p53 is often called “guardian of the genome”. p21/WAF1, 14-3-3σ, GADD45, B99, and cyclin G have all been attributed a role in the p53-dependent G1- or G2-arrest following stress (Decraene et al., 2001Decraene D. Agostinis P. Pupe A. de Haes P. Garmyn M. Acute response of human skin to solar radiation: Regulation and function of the p53 protein.J Photochem Photobiol B. 2001; 63: 78-83Crossref PubMed Scopus (52) Google Scholar). At the same time p53 improves repair efficiency by transactivating repair-related genes, including DDB2 (XPE), XPC, PCNA, GADD45 and p53R2 (Hwang et al., 1999Hwang B.J. Ford J.M. Hanawalt P.C. Chu G. Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in global genomic repair.Proc Natl Acad Sci USA. 1999; 96: 424-428Crossref PubMed Scopus (489) Google Scholar; Tanaka et al., 2000Tanaka H. Arakawa H. Yamaguchi T. et al.A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage.Nature. 2000; 404: 42-49Crossref PubMed Scopus (721) Google Scholar; Decraene et al., 2001Decraene D. Agostinis P. Pupe A. de Haes P. Garmyn M. Acute response of human skin to solar radiation: Regulation and function of the p53 protein.J Photochem Photobiol B. 2001; 63: 78-83Crossref PubMed Scopus (52) Google Scholar; Adimoolam and Ford, 2002Adimoolam S. Ford J.M. p53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene.Proc Natl Acad Sci USA. 2002; 99: 12985-12990Crossref PubMed Scopus (246) Google Scholar). On the other hand, p53 can exert control over apoptosis by upregulating several apoptosis-related genes when damage is so substantial that it jeopardizes the genomic integrity of the cell. This includes Fas/APO1 and KILLER/DR5, BAX, BID, PUMA, NOXA, p53AIP, PIG3, PERP, and PRG3 (Miyashita et al., 1994Miyashita T. Krajewski S. Krajewska M. et al.Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo.Oncogene. 1994; 9: 1799-1805PubMed Google Scholar; Owen-Schaub et al., 1995Owen-Schaub L.B. Zhang W. Cusack J.C. et al.Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression.Mol Cell Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (681) Google Scholar; Polyak et al., 1997Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. A model for p53-induced apoptosis.Nature. 1997; 389: 300-305Crossref PubMed Scopus (2174) Google Scholar; Wu et al., 1997Wu G.S. Burns T.F. McDonald III, E.R. et al.KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene.Nat Genet. 1997; 17: 141-143Crossref PubMed Scopus (903) Google Scholar; Attardi et al., 2000Attardi L.D. Reczek E.E. Cosmas C. Demicco E.G. McCurrach M.E. Lowe S.W. Jacks T. PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family.Genes Dev. 2000; 14: 704-718PubMed Google Scholar; Oda et al., 2000Oda K. Arakawa H. Tanaka T. et al.p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53.Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar; Decraene et al., 2001Decraene D. Agostinis P. Pupe A. de Haes P. Garmyn M. Acute response of human skin to solar radiation: Regulation and function of the p53 protein.J Photochem Photobiol B. 2001; 63: 78-83Crossref PubMed Scopus (52) Google Scholar; Ohiro et al., 2002Ohiro Y. Garkavtsev I. Kobayashi S. et al.A novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis-inducing factor (AIF).FEBS Lett. 2002; 524: 163-171Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar; Sax et al., 2002Sax J.K. Fei P. Murphy M.E. Bernhard E. Korsmeyer S.J. El Deiry W.S. BID regulation by p53 contributes to chemosensitivity.Nat Cell Biol. 2002; 4: 842-849Crossref PubMed Scopus (332) Google Scholar). The ultraviolet B (UVB) fraction of sunlight damages DNA of skin cells, with the most important lesions being cyclobutane pyrimidine dimers (Decraene et al., 2002Decraene D. Agostinis P. Bouillon R. Degreef H. Garmyn M. Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis, providing more time for cyclobutane thymine dimer removal in primary human keratinocytes.J Biol Chem. 2002; 277: 32587-32595Crossref PubMed Scopus (55) Google Scholar). Insufficient repair of these lesions leads to photocarcinogenesis in the long run. To cope with these adverse effects, skin cells have adapted a typical UVB response that involves the activation of this p53-dependent protective gene response program. This study investigates whether a low UVB dose, with the potential to trigger a protective p53-dependent gene response, might increase the resilience of keratinocytes against upcoming UVB insults. In particular, we focused on two important mediators of the p53-protective response: p21/WAF1 and p53R2. p21/WAF1 mediates a G1 arrest, by preventing cyclin kinase-mediated phosphorylation of the retinoblastoma protein, an essential step for the G1/S transition to proceed. p53R2 has recently been identified as a target gene of p53, involved in DNA repair upon genotoxic stress, including UV (Lozano and Elledge, 2000Lozano G. Elledge S.J. p53 sends nucleotides to repair DNA.Nature. 2000; 404: 24-25Crossref PubMed Scopus (107) Google Scholar). p53R2 encodes a subunit of ribonucleotide reductase, a rate-limiting enzyme for DNA synthesis, which catalyzes the conversion of ribonucleoside diphosphates to deoxyribonucleotides (Tanaka et al., 2000Tanaka H. Arakawa H. Yamaguchi T. et al.A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage.Nature. 2000; 404: 42-49Crossref PubMed Scopus (721) Google Scholar). Through this pathway, p53R2 creates a supply of precursors, which specifically aid in DNA repair. We demonstrate that a low UVB dose indeed preconditions keratinocytes against a second damaging UVB dose, as indicated by an increase in cell survival and decrease in apoptosis. The present data suggest that the generation of this adaptive response takes time and occurs, at least partly, via a p53-dependent upregulation of the cell-cycle-arrest-associated gene p21/WAF1 and the repair-associated-gene p53R2. By mediating cell cycle arrest and DNA repair, p53 has a leading role in the recovery from UVB damage. We investigated the effect of UVB on the expression of p53 and two p53 target genes, p21 and p53R2, with a central role in, respectively, G1 arrest and DNA repair. Consistent with earlier observations (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar; Decraene et al., 2004Decraene D. Smaers K. Gan D. et al.A synthetic superoxide dismutase/catalase mimetic (EUK-134) inhibits membrane-damage-induced activation of mitogen-activated protein kinase pathways and reduces p53 accumulation in ultraviolet b-exposed primary human keratinocytes.J Invest Dermatol. 2004; 122: 484-491Crossref PubMed Scopus (37) Google Scholar), p53 protein levels accumulated in UVB-exposed cells. Phosphorylation of p53 at serine 15, which is required for p53 stabilization (She et al., 2000She Q.B. Chen N. Dong Z. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation.J Biol Chem. 2000; 275: 20444-20449Crossref PubMed Scopus (285) Google Scholar), was readily observable both after irradiation with a low (8 mJ per cm2) or a high (32 mJ per cm2) UVB dose. The time course was different depending on the dose, as keratinocytes irradiated with 8 mJ per cm2 UVB displayed peak levels of phosphorylated p53 (ser15) 3–6 h post-irradiation, whereas it took up to 18 h before peak levels of phosphorylated p53 (ser15) were observed in keratinocytes irradiated with a higher UVB dose (32 mJ per cm2). The time course of p53 accumulation closely followed the timecourse for serine 15 phosphorylation (Figure 1). But only the low UVB dose (8 mJ per cm2) resulted in increased levels of p53R2 and in increased levels of p21 at later time points. A significant increase of p53R2 expression is only observed from 18 h onward. Increased p21 expression can be observed from 6 h, and peak levels persist up to 18 h post-irradiation. We used the A235 keratinocyte cell line, which contains only one p53 allele and no longer expresses any p53 mRNA (A253 (p53-)), possibly due to mutations in the regulatory region of the remaining p53 allele (Reiss et al., 1992Reiss M. Brash D.E. Munoz-Antonia T. Simon J.A. Ziegler A. Velluci V.F. Zhou Z.L. Status of the p53 tomor suppressor gene in human squamous carcinoma cell lines.Oncol Res. 1992; 4: 349-357PubMed Google Scholar). We stably re-integrated wt-p53 cDNA into the genome of the A253 cell line by retroviral transfer (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar). Primary keratinocytes, (NHK), p53-negative (A253 p53(-)), and p53-positive (A253 p53(+)) cells were exposed to a low UVB dose (8 mJ per cm2). p53 and p53R2 levels were examined by western blot analysis. No p53 protein can be detected in A253 p53(-), whereas p53 expression is restored in the A253 p53(+) cells. Although p53 is re-expressed in the A253 p53(+) cells, irradiation with 8 mJ per cm2 UVB did not result in further induction of the p53 protein at 6 and 24 h post-irradiation. This lack of increase in protein level does not necessarily imply that p53 would not be activated under these conditions, since latent p53 can be activated by low-level UVB irradiation without a detectable increase in protein level (Hupp et al., 1995Hupp T.R. Sparks A. Lane D.P. Small peptides activate the latent sequence-specific DNA binding function of p53.Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (437) Google Scholar). Kinetics of p53R2 induction in NHK after a low UVB dose are identical to Figure 1. No p53R2 expression is observed in the A253 p53(-) cells. The A253 p53(+) cells show increase of p53R2, but only at later timepoints postirradiation. Thus, upon irradiation with 8 mJ per cm2, the kinetics of p53R2 induction in NHK, containing wt-p53, and A253 p53(+) cells, re-expressing wt-p53 are quite similar, whereas no p53R2 induction is seen in the A253 p53(-) cells, confirming the p53 dependence of the p53R2 induction after the low UVB dose used (Figure 2). We next investigated the effect of a pre-exposure to a low UVB dose on the response to a second, high UVB dose. We again examined the basal levels of p53R2 and p21 first. Keratinocytes were either sham irradiated or irradiated with 8 mJ per cm2 UVB. 24 h later, keratinocytes were exposed to the second, high UVB dose of 32 mJ per cm2. p53, p53R2, and p21 levels were examined by western blot analysis. Significantly increased basal levels of p21 and p53R2 can be observed when, 24 h before the second, high UVB-dose, cells are pre-exposed to a low UVB dose of 8 mJ per cm2 (Fig S1). Download .jpg (.04 MB) Help with files Figure S1Conditioning to a low UVB-dose increases basal levels of p53R2 and p21 Cultured keratinocytes were either sham-irradiated or exposed to a low UVB-dose (8 mJ per cm2). Since pre-exposure to a low UVB dose upregulates genes involved in the recovery of DNA damage, we wished to investigate whether pre-exposure to a low UVB dose protects keratinocytes against a second, high UVB dose. Cell survival was examined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survival assay. Keratinocytes were either sham-irradiated (gray bars) or pre-exposed to 8 mJ per cm2 UVB (striped bars). 24 h later, these cells were irradiated once more with a second UVB dose ranging from 0 to 32 mJ per cm2 as indicated. Viability was determined 24 h after the final UVB insult by MTT. Figure 3a demonstrates that pre-exposed keratinocytes (8 mJ per cm2) exhibit elevated survival rates when exposed to a second, high UVB insult (24–32 mJ per cm2) 24 h later. A possible reduction of apoptosis was determined by looking at caspase-3 induced poly(ADP-ribose) polymerase (PARP) cleavage through western blot analysis. Keratinocytes were either sham-irradiated or pre-exposed to 8 mJ per cm2 UVB. Twenty-four hours later, these cells were again irradiated with a second UVB dose ranging from 0 to 32 mJ per cm2 as indicated. Lysates were collected 24 h after the final UVB insult. In agreement with the observed increase of cell viability, less PARP cleavage is seen in keratinocytes irradiated with a high UVB-dose (24–32 mJ per cm2), if these cells are first pre-exposed to a low UVB dose (8 mJ per cm2; Figure 3b). Next, we investigated the effect of dose fractionation on the response of human keratinocytes to UVB, and in particular, on the efficiency of DNA repair and on the induction of apoptosis. The ability of keratinocytes to repair UVB-induced cyclobutane thymine dimers was examined by Southwestern dotblot analysis. Cells were irradiated once with either 8 mJ per cm2 or 24 mJ per cm2 UVB or were irradiated with 24 mJ per cm2 UVB fractionated into three low doses of 8 mJ per cm2. The time interval between these consecutive irradiations was either 3 or 24 h. Both the initial amount of lesions, present after the final UVB insult, as the amount of lesions remaining 24 h after the final irradiation were examined. This residual damage is expressed as a percentage of the initial damage (mean±SEM, n=6). In 24 h, keratinocytes are able to repair a large amount of cyclobutane thymine dimers (mean residual damage of 24%), if the initial UVB dose is low (8 mJ per cm2; Figure 4a). At higher levels of UVB damage (24 mJ per cm2), keratinocytes do not show significant repair of damaged DNA (Figure 4a). Fractionation of this high dose into three low doses of UVB irradiation (3 × 8 mJ per cm2) does not stimulate the repair capacity if the interval between the consecutive doses is small (3 h; Figure 4a), since in those conditions no significant repair occurred. The repair efficiency is restored (mean residual damage of 25%) when the interval between the consecutive UV doses is long enough (24 h; Figure 4a). We also studied the effect of dose fractionation on apoptosis. Again, cells were irradiated either once with 8 or 24 mJ per cm2 UVB or were irradiated with 24 mJ per cm2 UVB fractionated into three low doses of 8 mJ per cm2. The time interval between these consecutive irradiations now ranged from 30 min to 24 h. Apoptosis, monitored by caspase-3 dependent PARP cleavage, is reduced when a lethal dose is fractionated into three low doses of UVB, but only when interval between the subsequent UVB insults is long enough (24 h), which is consistent with the timing required for p53-induced expression (Figure 4b). There is ample evidence that evolution has provided many adaptive protective mechanisms from the intracellular to tissue to the whole organism level. One intracellular adaptive protective mechanism that has been highly conserved is the induction of a superfamily of proteins, called heat-shock proteins (Jantschitsch and Trautinger, 2003Jantschitsch C. Trautinger F. Heat shock, UV-B-induced DNA damage and mutagenesis in skin.Photochem Photobiol Sci. 2003; 2: 1-5Crossref PubMed Scopus (2) Google Scholar). At the tissue level, UV-induced melanin pigmentation and thickening of the epidermis are adaptive mechanisms that protect the skin against further damage after subsequent UV exposures (Gilchrest and Eller, 1999Gilchrest B.A. Eller M.S. DNA photodamage stimulates melanogenesis and other photoprotective responses.J Investig Dermatol Symp Proc. 1999; 4: 35-40Abstract Full Text PDF PubMed Scopus (154) Google Scholar). At the intracellular level, keratinocytes are known to survive from UV exposure without permanent (mutagenic) DNA damage by inducing a sophisticated protective UV response program, in which temporary growth arrest and repair of DNA damage are key steps to guarantee a successful outcome. After a low UVB dose cells arrest in G1, before the S phase (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar). During this arrest, time is provided for the cell to repair its damage. UVB predominantly induces cyclobutane thymine dimers and these bulky DNA lesions are repaired by the nucleotide excision repair process. The p53 protein is involved in this protective response at least partly through transactivation of protective target genes. We demonstrate here that, although p53 protein phosphorylation at the serine 15 residue and the accompanying p53 stabilization increases both after a low and high UVB dose, only a low UVB dose, which leads to cell survival, results in upregulation of the protective genes p21 and p53R2. This upregulation takes time, a time lag that is in agreement with the protective role of p53 through its function as a transcription factor. It takes several h before increases in p21 or p53R2 protein levels are noticeable, and peaks in p21 and p53R2 protein expression are best observable, respectively, 6–18 and 18–24 h post-irradiation (Figure 1). The involvement of cdk inhibitor p21 in a G1 arrest upon UV has been demonstrated before (Petrocelli et al., 1996Petrocelli T. Poon R. Drucker D.J. Slingerland J.M. Rosen C.F. UVB radiation induces p21Cip1/WAF1 and mediates G1 and S phase checkpoints.Oncogene. 1996; 12: 1387-1396PubMed Google Scholar), including a study of our own (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar) where we demonstrate a p53-dependent p21 induction mediating a G1 arrest after the same low UVB dose of 8 mJ per cm2. p53R2 is a recently identified p53 target gene involved in DNA repair (Tanaka et al., 2000Tanaka H. Arakawa H. Yamaguchi T. et al.A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage.Nature. 2000; 404: 42-49Crossref PubMed Scopus (721) Google Scholar). Precursors for DNA repair are not supplied from the dNTP pool. It is p53R2 that supplies the urgent precursors for DNA synthesis at the actual site of DNA repair, within the damaged nuclei of arrested cells. To our knowledge, this is a demonstration of p53R2 upregulation in normal human keratinocytes by low doses of UVB and not by high doses. This observation is in agreement with the role of p53R2 in DNA repair, and the absence of repair but switch to apoptosis when doses are too high and repair becomes impossible. Furthermore, by using p53-positive and p53-negative cells, we could confirm that the induction of p53R2 is p53-dependent since its induction was restricted to the p53-positive cells (Figure 2). It is less clear, however, whether a low UVB dose that triggers a protective p53-dependent gene response leading to cell survival, through the induction of growth arrest and repair, would also increase the resilience/resistance of skin cells against an upcoming UVB insult. Therefore, we investigated whether experimental conditions that induced p21 and p53R2 would protect the cell against a second damaging UVB dose. In agreement with the protective function of these p53 target genes, pre-exposure to a low dose protected the cells against a second high dose, administered 24 h later. Indeed, as compared with non-pre-exposed cells, cells pre-exposed to 8 mJ per cm2 demonstrated increased survival rates and a decrease in apoptosis, when exposed to a second high UVB dose of 32 mJ per cm2 (Figure 3). Furthermore, dose fractionation of 24 mJ per cm2 into 3 low doses of 8 mJ per cm2 resulted in a reduction of apoptosis, but only when the time interval in between was long enough (24 h). With a decreasing time interval between fractionated doses (3 h or less), no reduction in apoptosis can be seen. Additionally, complete recovery of DNA repair is only observed when the time interval in between is long enough (24 h). With a decreasing time interval (3 h), no significant removal of cyclobutane thymine dimers is observed (Figure 4). These observations emphasize that a minimum time is required for the cell to activate its repair capacity. But, once a cell has been allowed time for recovery and p53-dependent gene expression, it becomes more resilient against subsequent UVB insults. We cannot totally exclude that, besides the observed gene program, other p53-dependent (Decraene et al., 2001Decraene D. Agostinis P. Pupe A. de Haes P. Garmyn M. Acute response of human skin to solar radiation: Regulation and function of the p53 protein.J Photochem Photobiol B. 2001; 63: 78-83Crossref PubMed Scopus (52) Google Scholar, Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPa is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar) and p53-independent checkpoint signaling events (Shackleford et al., 1999Shackleford R.E. Kaufmann W.K. Paules R.S. Cell cycle control, checkpoint mechanisms, and genotoxis stress.Environ Health Perspect. 1999; 107: 5-24Crossref PubMed Scopus (219) Google Scholar) could contribute to the observed adaptive response. A G1 arrest, mediated by p53-dependent p21 induction (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar) and a p53-dependent induction of repair-associated gene p53R2 (Figure 2), however, have been confirmed in our experimental conditions. These observations indicate that the observed upregulation of p53 and its target genes p21 and p53R2 after a low UVB dose contributes at least partly to the increased resilience of keratinocytes against further UVB insults. Provided there is enough time to recover, a gradual exposure to mild levels of UVB can increase the resistance of skin cells via the upregulation of p53 target genes, involved in growth arrest and repair. Together with other adaptation mechanisms at the cellular level, such as heat-shock protein induction, and at the tissue level, such as tanning and epidermal thickening, which also require time, the skin may become more resistant against acute damaging effects of UV. Further research, however, is warranted in order to explore alternative ways to activate a protective adaptive response without causing the damage that induces it in the first place. Hence, this p53-dependent gene program could become a potential therapeutic target to test actives that increase the own resilience of skin cells against UVB damage. Thus, in addition to sun-avoidance and sunscreen use, activation of pathways that protect genetic material of the cell, like this p53-dependent gene program, would mean an absolute surplus value in the protection of skin against UVB damage. In this way it would not only become possible to protect the skin better against UV damage but also to remove the damage more efficiently by stimulating temporary growth arrest and repair. Primary human keratinocytes were isolated from foreskins of young donors (less than 6 y) as described (Gilchrest, 1983Gilchrest B.A. In vitro assessment of keratinocyte aging.J Invest Dermatol. 1983; 81: 184s-189sCrossref PubMed Scopus (124) Google Scholar). Third to fifth passage cells were used in experiments. The procedure has been approved by the ethical committee of the university of Leuven. Experiments performed adhered to the Declaration of Helsinki guidelines. Keratinocytes were grown in serum-free and growth-factor-containing medium (Keratinocyte-SFM; Invitrogen, Merelbeke, Belgium; supplemented with bovine pituitary extract (50 μg per mL) and human recombinant epidermal growth factor (5 ng per mL)). The human SCC A253 cell line was chosen for its lack of endogenous p53 expression (American Type Culture Collection). Stable retroviral-mediated infection of A253 cells with full-length human wt-p53 was performed as described previously (Courtois et al., 1997Courtois S.J. Woodworth C.D. Degreef H. Garmyn M. Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.Exp Cell Res. 1997; 233: 135-144Crossref PubMed Scopus (27) Google Scholar). Prior to UVB irradiation, cells were washed twice with phosphate-buffered saline (PBS), then irradiated through a thin film of PBS, and refed with their own medium. Cells were exposed through the cover of the dish, which filters out residual UVC (Brown et al., 2001Brown D.B. Peritz A.E. Uitto J. Gasparro F.P. Ultraviolet-filtering properties of commonly used tissue cell culture plasticware.Photodermatol Photoimmunol Photomed. 2001; 17: 126-129Crossref PubMed Scopus (11) Google Scholar). The UVB source was a parallel bank of three Philips TL 20W12 tubes with a peak output around 310 nm (Philips, Haasrode, Belgium). Output was measured through the cover of a tissue culture dish with an IL700 radiometer (International light, Newburyport, Massachusetts). At the indicated time points, cells were scraped in lysis buffer and proteins were isolated and separated by SDS PAGE, as described (Decraene et al., 2004Decraene D. Smaers K. Gan D. et al.A synthetic superoxide dismutase/catalase mimetic (EUK-134) inhibits membrane-damage-induced activation of mitogen-activated protein kinase pathways and reduces p53 accumulation in ultraviolet b-exposed primary human keratinocytes.J Invest Dermatol. 2004; 122: 484-491Crossref PubMed Scopus (37) Google Scholar) Monoclonal anti-human PARP (poly(ADP-ribose) polymerase, monoclonal anti-human p53 (Clone DO1) were purchased from Pharmingen (BD Biosciences, Erembodegem, Belgium). Polyclonal anti-p53R2 was purchased from ProSci Inc (Poway, California) and mouse monoclonal anti-p21 was purchased from oncogene. Polyclonal phospho-p53 (Ser15) was obtained from Cell Signaling (Beverly, Massachusetts). Cell viability was assessed using the tetrazolium salt MTT (Sigma-Aldrich, Bornem, Belgium). Cells were seeded in 96-well plates. 24 h after UV irradiation, cells were incubated with MTT (1 mg per mL) in PBS for 1 h. Cleavage of MTT by dehydrogenase enzymes of metabolically active cells yields a blue formazan product. The formazan product was dissolved in DMSO and the absorbance at 550 nm was measured by spectrophotometry. At the indicated time points, total cellular DNA was extracted using the QIAamp DNA Mini Kit (Qiagen Benelux B.V., Venlo, the Netherlands), and DNA concentration was determined with a spectrophotometer. 300 ng of DNA was loaded onto a nitrocellulose membrane using a dotblot manifold and fixed for 3 h at 80°C. The membrane was blocked, incubated with the primary antibody, and subsequently with a biotinylated secondary antibody (Amersham Pharmacia, Roosendaal, the Netherlands), as described previously (Decraene et al., 2002Decraene D. Agostinis P. Bouillon R. Degreef H. Garmyn M. Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis, providing more time for cyclobutane thymine dimer removal in primary human keratinocytes.J Biol Chem. 2002; 277: 32587-32595Crossref PubMed Scopus (55) Google Scholar). Following a final rinse, DNA damage was visualized using enhanced chemiluminescence as described by the supplier (Amersham Pharmacia, Roosendaal, Netherlands) and quantified by densitometric analysis. The primary antibody used is monoclonal anti-thymine dimers (clone H3, Affitech, Norway) directed against cyclobutane thymine dimers. This work was supported in part by Grant OT/00/33 from the University of Leuven and Grant 0211.99 from the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen”, Belgium. The following material is available online for this article. Figure S1 Conditioning to a low UVB-dose increases basal levels of p53R2 and p21 Cultured keratinocytes were either sham-irradiated or exposed to a low UVB-dose (8 mJ per cm2).