ICANCERRESEARCH57. 1171—1179,March 15, 19971 The seminal observation that p53 mediates a G1 cell cycle arrest following y-irradiation was originally proposed to be analogous to the RAD9-mediated G2 DNA damage checkpoint in budding yeast (5). The RAD9 arrest pathway enhances cell survival by delaying pro gression into mitosis to allow for DNA repair (17). Based on this similarity, it was suggested that the p53-dependent G1 arrest is tran sient to allow time for DNA repair prior to entrance into S phase (5, 18). The model predicts that @53@ cells should have enhanced DNA repair, leading to decreased chromosomal aberrations and increased survival compared to pS3@ cells. Contrary to this prediction, this and other studies demonstrate that pS3@ cells have a reduced ability to form colonies after ‘y-irradiation relative to isogenic pS3@ cells (19— 26), although a few studies suggest that there is no correlation be tween p53 status and radiosensitivity (27). In addition, whereas the presence of an intact p53 pathway limits the long-term accumulation of chromosomal abnormalities in vivo, it does not appear to affect chromosome aberration frequency in the first postirradiation mitosis or spontaneous or induced mutation frequencies (25, 28—31). In contrast, some reports indicate that p53 may enhance survival and decrease chromosomal aberrations after UV irradiation, perhaps due to G1 delay and/or direct involvement in DNA repair (12, 32, 33), suggesting potential differences in the responses to ionizing versus Uv radiation.However,it has also been suggestedthat whereasthe efficiency of DNA repair is reduced by loss of p53, clonogenic survival is increased (10). The above observations are consistent with the alternative hy pothesis that the primary role of the p53-dependent @ arrest pathway in maintaining genetic stability after y-irradiation is through elimination of cells with DNA damage from the reproduc-@ tively viable population by permanent arrest or apoptosis, rather than through enhanced repair during temporary arrest (34). We observed previously that y-irradiated NDFs undergo a permanent, p53-dependent arrest resembling senescence (34, 35). Such cells exhibit prolonged induction of the cyclin-dependent kinase inhib itor @21WAF1/CIP1/SDI1 (34), which has been associated with senes cence (36). They remain viable as determined by trypan blue exclusion but are enlarged, irregularly shaped, and do not divide to form viable colonies for up to 10 weeks (21, 34, 37). These findings are consistent with other studies showing that large frac tions of normal cells irradiated in G0 remain in G0-G1 for a prolonged period after treatment (38, 39). A role for p53 in the irreversible G0-G1 arrest of NDF following ‘y-irradiationis more concordant with the higher radiosensitivity of p53k versus pS3@ cells. In this report, we determine the fate of several isogenic p53 @ and p53_ human fibroblast and epithelial strains following -y-irradiation in G0. It is well established in cell types that normally undergo apoptosis, such as thymocytes, that pS3@ strains are more radiosensitive than isogenic pS3@ strains, primarily because of the resistance of pS3@ cells to apoptosis (4, 6). Analogously, the data presented here indicate that in cells that do not normally undergo apoptosis, such as NDF, p53 @ strains are more radiosensitive than p53 strains because of induction of a senescent-like state over multiple cell cycles. 1171 p53Mediates Permanent ArrestoverMultipleCellCycles in Response to y-Irradiationt Steven P. Linke, Kristie C. Clarkin, and Geoffrey M. Wahl2 GeneExpressionLaboratory,The Salk Institutefor BiologicalStudies,La Jolla, California92037[S.P.L, K.C.C., G.M.WI, and Departmentof Biology,Universityof California-San Diego, La Jolla, California 92093 (S. P. U ABSTRACT A new technique that monitors cell cycle progression over multiple cycles was used to gain insight into how p53 limits the emergence of variants with structural chromosome alterations following y-irradiation. G0-synchronized, p53k (with a functional p53 pathway) normal human fibroblast and epithelial strains underwent a dose-dependent permanent arrest in the initial G0-G1 phase after irradiation. The dose-response curves indicate that a single event, such as an irreparable DNA break, may be sufficient to induce arrest. p53' cells that escaped the initial G0-G1 phase after irradiation entered S phase in at least two waves. However, many of these cells underwent long-term arrest in subsequent phases. In contrast, virtually all of the cells in isogenic p53 (with a nonfunctional p53 pathway) strains escaped from the first G0-G1 phase without delay, regardless of the dose. p53W @ were also eliminated in subsequent phases but at significantly lower frequencies. Consistent with these find ings, the reproductive viability of p53 cells was higher than p53@ cells. The nonclonogemc fraction appeared to be eliminated within three cycles for both cell types. In addition, artificial holding in G0 after Irradiation, which allows for the repair of potentially lethal damage, led to similar increases in survival in pS3@ and p53 cells. These data are inconsistent with the hypothesis that the primary function of p53-dependent G0-G1 arrest in response to 7-irradiation Is to allow additional time for DNA repair. Rather, they indicate that p53 helps maintain genetic stability by eliminating cells with damaged chromosomes from the reproductively viable population. INTRODUCTION The p53 tumor suppressor is an important factor for maintaining genetic stability through its participation in multiple cell cycle check points. Cells undergo a p53-dependent G0-G1 phase cell cycle arrest or apoptosis in response to a variety of environmental insults and cancer treatments, including y-irradiation, UV radiation, and some chemotherapeutic drugs (1—7).Radiation and DNA strand-breakage drugs induce p53-dependent arrest by directly damaging DNA (5), whereas drugs that deplete ribonucleotide pools can induce p53- dependent arrest in the absence of DNA damage (7). Because p533 cells progress into S phase with depleted nucleotide pools, they can undergo DNA breakage, which can lead to structural chromosome changes such as gene amplification (2, 3). Thus, p53 can both prevent cells with DNA damage from entering S phase and prevent cells from entering S phase under conditions favoring replication-associated damage. p53 is also involved in a checkpoint that prevents re-repli cation of DNA in the presence of mitotic spindle inhibitors (8). In addition, other studies suggest that p53 may also contribute to DNA repair, S-phase progression, a G2 checkpoint, and centrosome repli cation (9—16). Received 8/12/96; accepted 1128/97. Thecostsof publicationof thisarticleweredefrayedinpartbythepaymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by grants from the National Cancer Institute and the G. Harold and Leila Y. Mathers Charitable Foundation. 2 To whom requests for reprints should be addressed, at Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037. 3 The abbreviations used are: p53, nonfunctional p53 pathway; p53k, functional p53 pathway; NDF, normal human diploid fibroblast; BrdUrd or BI-dU,bromodeoxyuridine; PLD, potentially lethal damage; DSB, double-strand break; neo, neomycin. Research. on November 3, 2021. © 1997 American Association for Cancer cancerres.aacrjournals.org Downloaded from