INACTIVATION OF p53 IN NORMAL HUMAN CELLS INCREASES G 2 /M ARREST AND SENSITIVITY TO DNA-DAMAGING AGENTS Jocelyn CE ´ RALINE 1 , Gae ¨l DEPLANQUE 1 , Brigitte DUCLOS 1 , Jean-Marc LIMACHER 1 , Amor HAJRI 1 , Francine NOEL 1 , Christophe ORVAIN 1 , Thierry FRE ´ BOURG 2 , Claudine KLEIN-SOYER 3 and Jean-Pierre BERGERAT 1 * 1 Laboratoire d’Oncologie Mole ´culaire, Institut de Recherche contre les Cancers de l’Appareil Digestif, Ho ˆpitaux Universitaires de Strasbourg, Strasbourg, France 2 Unite ´ de Ge ´ne ´tique Mole ´culaire, Centre Hospitalier Universitaire de Rouen, Rouen, France 3 INSERM U.311, Etablissement de Transfusion Sanguine, Strasbourg, France p53 mutations are found in about 70%of human cancers. In order to evaluate the role of these mutations in response to chemotherapeutic agents, it is important to distinguish be- tween p53 response to DN A-damaging agents in normal and in tumour cells. H ere, using normal human fibroblasts(N H Fs), we show that cisplatin and UV radiation induce G 2 /M arrest which is temporally linked to p53-protein induction. T o study the contribution of p53 to this G 2 /M arrest, we inhibited p53 induction in NHFs using p53 anti-sense oligonucleotides. Following exposure of N H Fs to U V radiation, the inhibition of p53-protein induction leads to a greater accumulation of cells in the G 2 /M phase, but also to a decreased fraction of cells in the G 1 phase. W e propose that p53 does not induce G 2 /M arrest directly, and that the extent of this arrest may depend on the fraction of cells that do not stop at the G 1 phase following exposure to DN A-damaging agents. Furthermore, inhibition of p53-protein induction leadsto increased sensitiv- ity of NHFs to UV radiation. These results suggest that inhibition of p53 protein enhances sensitivity to DNA- damaging agents in normal human cells. Int. J. Cancer 75:432– 438, 1998.  1998 Wiley-Liss, Inc. Damaged bases and chromosome breaks may give rise to mutations during cell division. To maintain the integrity of their genome, higher eukaryotes have developed mechanisms to recog- nize and repair DNA damage prior to cell division. These occur at cell-cycle checkpoints, including G 1 /S and G 2 /M. Failure in one of these mechanisms could be a key step in tumour progression. The tumour-suppressor protein p53, which is most strongly implicated in human cancer (Skilling et al., 1996), participates in the cell response to DNA-damaging agents (Kastan et al., 1991). Loss or inactivation of the p53 tumour-suppressor protein correlates with genetic instability (Donehower et al., 1992). In normal cells, signals arising from damaged DNA lead to activation of the p53 response pathway, resulting in cell-cycle arrest, DNA repair and apoptosis of certain cells (reviewed by Harris, 1996). p53 protein induces G 1 /S arrest by the transcriptional activation of the p21 waf1/ cip1 gene, but its contribution to G 2 /M arrest is currently controver- sial. Some studies suggest that p53 could be involved in the G 2 /M restriction point (Stewart et al., 1995; Agarwal et al., 1995), while G 2 /M arrest following X-irradiation has been observed in p53- deficient cells (Han et al., 1995). It has also been shown that wild-type p53 is implicated in the G 2 exit (Guillouf et al., 1995). The consequences of p53 loss on cell survival also remain unclear. The link between p53 and sensitivity or resistance to DNA- damaging agents is still debatable. Loss of p53 function is linked to resistance in some cells (Lee and Bernstein, 1993) and to increased sensitivity in others (Fan et al., 1995; Hawkins et al., 1996; Wahl et al., 1996). To clearly evaluate the role of p53 in G 2 /M arrest and in cell sensitivity, a cell system lacking functional p53 is indispens- able. Most of the studies cited above were performed with p53-deficient or mutant tumour cells. Genetic abnormalities might have arisen from the loss of p53 in these cells, masking the effects of p53 inactivation. To overcome this difficulty we used an anti-sense oligonucleotide to inhibit p53-protein expression. We present evidence that, following genotoxic stresses, the inhibition of p53 in normal human cells leads to increased G 2 /M arrest and to greater sensitivity following exposure to DNA-damaging agents. MATERIAL AND METHODS Cell culture and exposure to DNA-damaging agents Primary NHFs were obtained from dermis explants of newborn foreskin. The p53 +/+ phenotype of these cells was established according to the technique described by Flaman et al. (1995). Cells were grown in Dulbecco’s minimum essential medium (DMEM) supplemented with 1% penicillin, 1% streptomycin and 10% FCS (GIBCO, Paisley, UK). Stock cultures of cells were harvested and aliquots of 5  10 5 cells were seeded on duplicate in 100-mm Petri dishes. All DNA-damaging treatments were performed after 48 hr incubation at 37°C with 5% CO 2 . HPLC-purified p53 anti-sense fully phosphorothioated oligonucleotide (AS1) with a length of 20 bases (Hirota et al., 1996) (nucleotides 772-791, GenBank, acces- sion number X01405, 5'-CCCTGCTCCCCCCTGGCTCC-3') was added at a final concentration of 1 μM, 12 hr before treatment with DNA-damaging agents. For control, HPLC-purified p53 anti-sense fully phosphorothioated oligonucleotide (AS2) (nucleotides 209-286, Gen- Bank, accession number X01405, 5'-CGGCTCCTCCATGGCAGT-3') and ERCC2 anti-sense oligonucleotide (nucleotides 1829-1849, Gen- Bank, number X52470, 5'-CGGTGATCTCCAGAGTATGC-3') were also added at a final concentration of 1 μM, 12 hr before DNA damage. All oligonucleotides were purchased from Eurogentec, Serain, Belgium. For UV irradiation, the medium was removed, cells were washed twice with PBS and exposed to a 254-nm UV lamp at doses of up to 20 J/m 2 . The energy of UV light was controlled by a radiometer (VLX-3W, 254 nm). Irradiated or non-irradiated cells were then incubated for various time periods. For the cisplatin treatment, cells were exposed to various doses of cisplatin (0–10 μg/ml) for 1 hr. The medium was then removed and the cells were washed twice with PBS and incubated in fresh medium for the indicated time periods. Enzyme-linked immunosorbent assays (ELISA) and Western-blot analysis The levels of p53 protein in NHFs following exposure to DNA-damaging agents and in NHFs in which p53-protein induc- tion was targeted by the anti-sense prior to genotoxic stress, were evaluated by quantitative ELISA (Pantropic p53, Oncogene Sci- Contract grant sponsors: Association pour la Recherche contre le Cancer (ARC); Contract grant number: 1316; Contract grant sponsors: Ligue De ´partementale Contre le Cancer du Haut-Rhin; Association pour la The ´rapie Ge ´nique des Cancers (ATGC). *Correspondence to: Laboratoire d’Oncologie Mole ´culaire, Institut de Recherche contre les Cancers de l’Appareil Digestif, Ho ˆpitaux Universi- taires de Strasbourg, BP 426, 67091 Strasbourg cedex, France. Fax: (33) 03 88 11 90 97. E-mail: Jean-Pierre.Bergerat@ircad.u-strasbg.fr Received 6 July 1997; Revised 15 September 1997 Int. J. Cancer: 75, 432–438 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer