A p53-dependent S-phase checkpoint helps to protect cells from DNA damage in response to starvation for pyrimidine nucleotides

Abstract

Normal mammalian cells arrest primarily in G1 in response to N-(phosphonacetyl)-L-aspartate (PALA), which starves them for pyrimidine nucleotides, and do not generate or tolerate amplification of the CAD gene, which confers resistance to PALA. Loss of p53, accompanied by loss of G1 arrest, permits CAD gene amplification and the consequent formation of PALA-resistant colonies. We have found rat and human cell lines that retain wild-type p53 but have lost the ability to arrest in G1 in response to PALA. However, these cells still fail to give PALA-resistant colonies and are protected from DNA damage through the operation of a second checkpoint that arrests them reversibly within S-phase. This S-phase arrest, unmasked in the absence of the G1 checkpoint, is dependent on p53 and independent of p21/waf1.

Figure 1

Induction of p53 and p21/waf1 by PALA. (A) Cells were treated for 4 days with 0.5 mM PALA, and p53 was detected by Western analysis, using the DO-1 antibody, which was detected by enhanced chemiluminescence. (B) Levels of p21 mRNA. Total RNA was separated by electrophoresis in an agarose gel and transferred to a nylon membrane. p21 mRNA was detected with a specific human cDNA probe. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) served as loading control.

Figure 2

Cell cycle distributions of human (A) and rat (B) cells. After treatment with PALA (3 × ID₅₀) or γ-radiation (5 Gy), the cells were sorted after staining with propidium iodide. The percentages of cells in G₁, S, and G₂/M are indicated. (C) Cell cycle distributions as a function of time of treatment with PALA.

Figure 3

DNA synthesis in PALA-treated cells. (A) BrdUrd labeling of PALA-treated MDAH041 and C11 cells. Cells grown on coverslips were treated with PALA (3 × ID₅₀) for 3 days and pulse-labeled for 4 hr. After staining with anti-BrdUrd antibody, the cells were photographed (×57). (B) [³H]thymidine labeling of PALA-treated C11 and MDAH041 cells. Cells were treated with PALA (3 × ID₅₀) for 3 days and pulse-labeled for 2 hr. The incorporation of ³H was normalized to the total amount of protein in each well.

Figure 4

Cell cycle distributions of p21/waf1-null human cells. (A) Three days after treatment with PALA (50 μM, 3 × ID₅₀), the cells were sorted after staining with propidium iodide. The percentages of cells in G₁, S, and G₂/M are indicated. (B) G₁/S ratios for untreated and PALA-treated normal and p21-null human cells.

Figure 5

p53-Dependent effects of PALA-induced pyrimidine nucleotide starvation.