Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14775–14780, December 1998 Cell Biology A p53-dependent S-phase checkpoint helps to protect cells from DNA damage in response to starvation for pyrimidine nucleotides (Li-Fraumeni cellsyREF52 cellsygene amplificationycell cycle arrestyG1 checkpoint) MUNNA L. AGARWAL,ARCHANA AGARWAL,WILLIAM R. TAYLOR,OLGA CHERNOVA,YOGESH SHARMA, AND GEORGE R. STARK* Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 Contributed by George R. Stark, October 9, 1998 ABSTRACT Normal mammalian cells arrest primarily in mechanisms that do not involve broken DNA (16). Paulson et G1 in response to N-(phosphonacetyl)-L-aspartate (PALA), al. (23) have shown that, in the absence of a functional p53 which starves them for pyrimidine nucleotides, and do not pathway, PALA treatment of tumor cells leads to DNA generate or tolerate amplification of the CAD gene, which damage and thus to CAD gene amplification. Furthermore, in confers resistance to PALA. Loss of p53, accompanied by loss some cells the induction of p53 and associated G1 arrest are of G1 arrest, permits CAD gene amplification and the conse- defective in response to PALA but normal in response to quent formation of PALA-resistant colonies. We have found g-radiation (24), suggesting that there are distinct and sepa- rat and human cell lines that retain wild-type p53 but have lost rable pathways of p53 activation in response to these different the ability to arrest in G1 in response to PALA. However, these stimuli. In this report, we describe the responses of several cell cells still fail to give PALA-resistant colonies and are pro- lines to pyrimidine nucleotide depletion or radiation-induced tected from DNA damage through the operation of a second DNA damage. All of these lines have functional, wild-type p53 checkpoint that arrests them reversibly within S-phase. This and are not permissive for gene amplification. However, S-phase arrest, unmasked in the absence of the G1 checkpoint, instead of accumulating in G1 after treatment with PALA, two is dependent on p53 and independent of p21ywaf1. of the cell lines arrest within S-phase. We suggest that activa- tion of p53 by pyrimidine starvation causes a stable and Normal mammalian cells maintain genomic integrity through reversible arrest in G1 and also within S-phase, thus regulating controls that regulate their ability to progress through the cell genomic stability at more than one point of the cell cycle. cycle when they encounter environmental stress (1, 2). When these controls are deranged, the result is genomic instability, MATERIALS AND METHODS a hallmark of neoplasia. The tumor suppressor protein p53, commonly inactivated in tumors, is a major regulator of cell Cell Lines and Culture Conditions. The MDAH041 post- cycle progression in response to DNA damage or arrest of crisis cell line, kindly provided by M. Tainsky (25), was derived DNA synthesis (3–5). The signals generated by these stresses from the fibroblasts of a patient with Li-Fraumeni syndrome. lead to an increase in the level of p53 protein and also stimulate There is a frame shift mutation in one p53 allele at codon 184, its ability to activate transcription, leading to cell cycle arrest and the normal p53 allele has been lost during in vitro y propagation (20). Normal human WI38 fibroblasts were ob- in both G1 (6–8) and G2 M (9, 10). An important transcrip- tional target of p53 is the cyclin-dependent kinase inhibitor tained from the American Type Culture Collection. TR9-7 p21ywaf1 (11), which helps to mediate cell cycle responses to cells, expressing wild-type p53 under the control of a tetracy- DNA damage (12–14). In primary human fibroblasts, the cline-regulated promoter, were derived from MDAH041 cells (17). Cell lines containing a neo marker were grown in the p53-mediated cell cycle arrest in response to DNA damage is m y irreversible (15). However, overexpression of p53 without presence of G418 (600 g ml). Early passage p53-null and y p21-null mouse embryo fibroblasts (MEFs) were provided by DNA damage leads to reversible arrest in both G1 and G2 M (16–18). L. Donehower and G. Hannon, respectively. Normal human By using p53-null mouse or human cells, or by using the E6 fibroblast (LF1) cells and their p21-null derivative (H07.2–1) protein of human papilloma virus 16 to inactivate p53, it was were gifts of J. Sedivy (26). Low passage primary rat embryo shown that cells become permissive for CAD gene amplifica- fibroblast (REF) cells were provided by Andrei Gudkov (University of Illinois, Chicago) and REF52 cells were as tion and thus N-(phosphonacetyl)-L-aspartate (PALA) resis- tance when p53 is absent (19–21). In contrast, cells with intact described by Franza et al. (27). All cells were grown in 10% CO2 in DMEM, supplemented with 10% fetal bovine serum. p53 arrest stably, predominantly in G1, and do not form resistant colonies in PALA (19, 20). These observations reveal PALA Selections. PALA (NSC224131), an inhibitor of the aspartate transcarbamylase activity of the multifunctional that PALA induces a p53-dependent cell cycle arrest in G1, thus suppressing gene amplification and genetic instability CAD enzyme, was obtained from the Drug Synthesis and (16). In rodent cells, CAD gene amplification is the only Chemistry Branch, Developmental Therapeutics Program, mechanism observed for PALA resistance, but human cells Division of Cancer Treatment, National Cancer Institute, become resistant to PALA through additional mechanisms Bethesda, MD. Treatments of cells with PALA were as described by Agarwal et al. (28). For each cell line or strain, the including but not limited to CAD gene amplification (22). ID was determined and selections of 2 3 105 cells per 10-cm In normal human fibroblasts deprived of pyrimidine nucle- 50 plate were done in the presence of 10% (volyvol) dialyzed fetal otides by PALA, wild-type p53 mediates G1 arrest through 3 calf serum, using PALA at 3 ID50 (29). The selective The publication costs of this article were defrayed in part by page charge Abbreviations: PALA, N-(phosphonacetyl)-L-aspartate; REF, rat em- payment. This article must therefore be hereby marked ‘‘advertisement’’ in bryo fibroblast; HPRT, hypoxanthine phosphoribosyl transferase; accordance with 18 U.S.C. §1734 solely to indicate this fact. MEF, mouse embryo fibroblast; 6TG, 6-thioguanine. © 1998 by The National Academy of Sciences 0027-8424y98y9514775-6$2.00y0 *To whom reprint requests should be addressed. e-mail: starkg@ PNAS is available online at www.pnas.org. cesmtp.ccf.org. 14775 Downloaded by guest on October 2, 2021 14776 Cell Biology: Agarwal et al. Proc. Natl. Acad. Sci. USA 95 (1998) medium was changed every 4–5 days until colonies of 100–200 cells were observed, typically in 3–4 weeks. Western and Northern Analyses. Total cellular protein was isolated by lysing cells in 20 mM TriszHCl, pH 7.5, 2% (wtyvol) SDS, 2 mM benzamidine, and 0.2 mM phenylmethanesulfonyl fluoride. Protein concentrations were determined by the Brad- ford method (Bio-Rad). Proteins (25 mgylane) were separated by SDS-10% PAGE and electroblotted to polyvinylidene difluoride membranes (Stratagene; ref. 30). After transfer, the gels were stained with Coomassie blue (Sigma) to verify equal loading. The membranes were probed with mAb DO-1 (Santa Cruz Biotech), directed against p53, and bound antibody was detected by enhanced chemiluminescence (Amersham). Northern analysis: Total RNA was extracted with the Trizol reagent (GIBCOyBRL) as specified by the manufacturer. Analysis was performed as described by Sambrook et al. (31). Cell Cycle Analysis. The DNA content of trypsinized cells was determined by staining with propidium iodide, using a Cycletest kit (Becton Dickinson). Cell cycle distribution was determined by using a FACScan instrument (Becton Dickin- FIG. 1. Induction of p53 and p21ywaf1 by PALA. (A) Cells were son), CELL FIT software, and an HP340 Series 9000 Worksta- treated for 4 days with 0.5 mM PALA, and p53 was detected by tion (Hewlett–Packard). Dead cells were gated out by using Western analysis, using the DO-1 antibody, which was detected by pulse processing. enhanced chemiluminescence. (B) Levels of p21 mRNA. Total RNA Measurement of DNA Synthesis. For tritium labeling, C11 was separated by electrophoresis in an agarose gel and transferred to and MDAH041 cells were grown in 6-well plates and treated a nylon membrane. p21 mRNA was detected with a specific human 3 m 3 with PALA at 3 ID50. After 3 days, 5 Ci of H-labeled cDNA probe. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) thymidine was added to each well, and the cells were incubated served as loading control. for 2 hr more. After a trichloracetic acid wash, 0.5 ml of 2 M perchloric acid was added to each well at 60°C for 30 min. The However, TR9-7 cells plus tetracycline and C11 cells retain resulting solution was transferred to scintillant and 3H incor- many other p53-dependent responses of normal human fibro- poration was determined. The residue was dissolved in alkali blast cell strains, in contrast with their p53-null MDAH041 parents. Similarly, the rat cell line REF52, which has wild-type for protein determination. For BrdUrd incorporation, cells 3 p53, retains many normal responses characteristic of REF cell grown on coverslips were treated with PALA (3 ID50) and strains. For example, none of these cell lines exhibit gene were incubated for an additional 4 hr in medium containing 10 m amplification in selective concentrations of PALA (refs. 29 and M BrdUrd. The incorporation of BrdUrd into DNA was 34 and data presented below), TR9-7 (plus tetracycline), and determined by immunostaining (Amersham). y REF52 cells arrest normally at the G1 S boundary in response to DNA damage (see below), and TR9-7 and C11 cells retain y RESULTS the normal p53-dependent arrest at G2 M in response to inhibition of DNA synthesis (5).