[CANCER RESEARCH 61, 88 –96, January 1, 2001] p53 Modulates Base Excision Repair Activity in a Cell Cycle-specific Manner after Genotoxic Stress 1 Hagai Offer, Irit Zurer, Gaspar Banfalvi, Marian Reha’k, Ayellet Falcovitz, Michael Milyavsky, Naomi Goldfinger, and Varda Rotter 2 Departments of Molecular Cell Biology [H. O., I. Z., A. F., M. M., N. G., V. R.] and Biological Chemistry [G. B., M. R.], Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT To elucidate the nature of the cross-talk between the p53 protein and the DNA repair machinery, we have investigated the relationship between the two throughout the cell cycle. Base excision repair (BER) was analyzed in cell cycle phase-enriched populations of lymphoid cells expressing wild-type p53. Our study yielded the following novel findings: (a) BER exhibited two distinct peaks of activity, one associated with the G 0 -G 1 checkpoint and the second with the G 2 -M checkpoint; (b) although the overall BER activity was reduced after exposure of cells to 400R, there was an augmentation of the G 0 -G 1 -associated BER activity and a reduc- tion in the G 2 -M-associated BER activity; and (c) modulations in these patterns of BER after genotoxic stress were found to be p53 regulated. p53 protein levels induced after -irradiation were distributed evenly in the various cell cycle populations (analyzed by the PAb-248 anti-p53 mono- clonal antibody). However, both the dephosphorylation of serine 376 of p53 (contained in the PAb-421 epitope) and the specific DNA binding activity, as well as apoptosis, were enhanced toward the G 2 -M popula- tions. Furthermore, inactivation of wild-type p53, mediated by mutant p53 expression, abolished the alterations in the BER pattern and showed no induction of a G 2 -M-associated apoptosis after -irradiation. These results suggest that after genotoxic stress, stabilized p53 enhances the G 0 -G 1 - associated BER activity, whereas it predominantly reduces BER activity at the G 2 -M-enriched populations and instead induces apoptosis. After genotoxic stress, p53 functions as a modulator that determines the pattern of BER activity and apoptosis in a cell cycle-specific manner. INTRODUCTION Genomic stability, central to the maintenance of the normal cellular life, is heavily dependent on the ability of the cell to sense and recognize damaged DNA and then to either repair or degrade it. Apoptosis after exposure to DNA-damaging agents can be regarded as a mechanism for preventing the propagation of genetically aberrant cells that have sustained a high level of DNA damage (1). Data accumulated over the years suggest that the tumor suppressor gene p53 plays a pivotal role at several molecular junctures in these pathways (2–5). Collectively, it suggests that activated p53, after genotoxic stress, may either trigger the onset of DNA repair, leading to the completion of the cell cycle, or alternatively, induce apoptosis and/or cellular differentiation, leading to exit from the cell cycle (reviewed in Refs. 6 and 7). The notion that p53 directly induces apoptosis has been shown unequivocally in many independent ways. However, less is known about the molecular mechanism(s) that un- derlies the role of p53 in the DNA repair machinery. The wild-type p53 protein was described to be associated with a number of known DNA repair pathways, including nucleotide excision repair (8 –10). In addition, we showed that p53 in its wild-type protein conformation, encoded by either human or murine temperature-sensitive mutant p53, directly induced BER 3 activity in vitro and in vivo (11). 4 Depletion of p53 from the nuclear extracts abolished this enhanced activity (11). The p53 COOH terminus is thought to be essential for “sensing” and detecting damaged DNA (12–14). p53 plays a central role in the G 0 -G 1 checkpoint, where it has been suggested to induce cell growth arrest and apoptosis, and in the G 2 -M checkpoint, where it is involved in the control of apoptosis and DNA repair induction (reviewed in Refs. 10, 15, and 16). The involvement of p53 in the DNA damage-dependent delay in the G 2 phase of the cell cycle and its contribution to DNA repair in this phase were described by several studies (17–20). Knockout of either p53 or p21/waf1 gene expression in human colorectal cancer cell lines caused premature exit from DNA damage-dependent G 2 arrest and failure of cytokinesis, resulting in endoreduplication of the tetraploid cells and formation of polyploid giant cells. High levels of p53 can modulate the G 2 growth arrest prior to mitotic chromosomal condensation (21). To further decipher the molecular cross-talk between the p53 protein and the DNA repair machinery, we studied their activities at specific phases in the cell cycle. In our work, we focused on the role of p53 in BER, a DNA repair pathway that is in charge of the removal of modified bases induced by endogenous and exogenous stress (11). We have chosen to examine these parameters in two different cell lines. One is the pre-B-lymphoid cell line 70Z/3, which expresses wild-type p53 and seems to exhibit a normal cell cycle pattern (22) and a derived cell line that expresses mutant p53 (70Z/3-M8). The other is the p53 null L12 early pre-B cells (23) and derived clones expressing the p53 temperature-sensitive mu- tant (ts; Ref. 24). Enrichment of well-defined cell populations representing the individual cell cycle phases was achieved by centrifugal elutriation (20). We found that the pattern of BER activity is modulated during the cell cycle. Cells exhibit two distinct peaks of BER, at similar levels of activity. One peak is associated with the G 0 -G 1 -enriched populations and the other with the G 2 -M-enriched populations. Exposure of cells to 400R significantly enhanced the G 0 -G 1 -associated peak of BER activity. Under these conditions, the G 2 -M-associated BER activity was reduced, and instead, cells underwent apoptosis. Our present data show that after genotoxic stress, p53 also functions as a modulator that determines the pattern of BER activity in a cell cycle-specific manner. MATERIALS AND METHODS Cell Lines. The murine pre-B-cell line 70Z/3 (25) and the murine early pre-B p53 null cell line L12 (23) were grown in suspension at 37°C in RPMI 1640 containing 10% FCS and 2  10 -5 M -mercaptoethanol. 70Z/3-M8 is a stable clone derived by transfection of pSVLM8p53, a p53 mutant cDNA with an alternatively spliced COOH terminus and the drug-resistant gpt gene (26). L12p53ts-derived clones express the LTRp53ts mutant plasmid. The Received 5/10/00; accepted 10/26/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants from the Israel-USA Binational Science Foundation and the German Israeli Foundation for Scientific Research and Development and the Israel Cancer Research Fund. V. R. is the incumbent of the Norman and Helen Asher Professorial Chair in Cancer Research at the Weizmann Institute. 2 To whom requests for reprints should be addressed, at Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-9344501; Fax: 972-8-9465265; E-mail: varda.rotter@weizmann.ac.il. 3 The abbreviations used are: BER, base excision repair; ts, temperature sensitive; FACS, fluorescence-activated cell sorter; PI, propidium iodide; AP, apurinic; EMS, electrophoretic mobility shift assay. 4 H. Offer, M. Milyavsky, N. Erez, and V. Rotter. p53 involvement in BER does not require its transcriptional activity, submitted for publication. 88 Research. on June 19, 2015. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from