Differential requirement for p19ARF in the p53-dependent arrest induced by DNA damage, microtubule disruption, and ribonucleotide depletion - PubMed (original) (raw)

Differential requirement for p19ARF in the p53-dependent arrest induced by DNA damage, microtubule disruption, and ribonucleotide depletion

S H Khan et al. Proc Natl Acad Sci U S A. 2000.

Abstract

p19ARF has been implicated as a key regulator of p53 stability and activation. While numerous stresses activate the p53 growth arrest pathway, those requiring p19ARF remain to be elucidated. We used p19ARF knockout mouse embryo fibroblasts to show that DNA damage and microtubule disruption require p19ARF to induce p53 responses, whereas ribonucleotide depletion and inhibition of RNA synthesis by low doses of actinomycin D do not. The data provide evidence that the arrest pathway activated by ribonucleotide depletion involves some different signal transducers than those activated by DNA damage or microtubule disruption. We also present biochemical analyses that provide insights into the mechanism by which p53 and p19ARF cooperate in normal cells to induce cell cycle arrest.

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Figures

Figure 1

p19ARF-null MEFs become arrested during ribonucleotide depletion. Asynchronous populations of wild-type (WT), p53+/−, p53−/−, and p19ARF−/− MEFs were untreated or treated with 100 μM PALA for 24, 48, or 72 h. Cells were pulse labeled with BrdUrd and analyzed by flow cytometry. Cells that incorporated BrdUrd are shown above the diagonal line. The percentages of BrdUrd-positive cells are shown in the upper right corner of each plot. Representative dot plots for untreated or PALA-treated MEFs are shown in A (WT), B (p53+/−), C (p53−/−), and D (p19ARF−/−). (E) Western blot analysis of p21 and actin after 48-h PALA treatment of p53+/− and p19ARF−/− MEFs. Relative band intensities were quantified in all Western analyses with actin as an internal control.

Figure 2

Increased polyploidy in p19ARF-null fibroblasts after nocodazole treatment. Asynchronous cultures of p53+/−, p53−/−, p19ARF−/−, and p16−/−p19ARF−/− MEFs were treated with or without 0.05 μg/ml nocodazole for 48 h, fixed, and stained with propidium iodide for FACS analysis. Values in the upper right corner of each plot represent the percentage of cells with >4_N_ DNA content. Histogram plots of untreated or nocodazole-treated MEFs are shown in A (p53+/−), B (p53−/−), C (p19ARF−/−), and D (p16−/−p19ARF−/−).

Figure 3

Defective DNA-damage response in p19ARF-null MEFs. Asynchronous cultures of p53+/−, p53−/−, and p19ARF−/− MEFs were exposed to 6 Gy of γ-radiation. After 24 h, cells were pulse labeled with BrdUrd and analyzed by FACS. Cells above the diagonal line are BrdUrd positive. The values shown in the upper right represent the percentage of irradiated cells in S phase relative to untreated (% S irradiated/% S untreated). Representative plots are shown for p53+/− (A), p53−/− (B), and p19ARF−/− (C).

Figure 4

Involvement of p19ARF in the sustained induction of p21 after IR. Asynchronous populations of wild-type (WT) and p19ARF−/− MEFs were treated with 6 Gy of γ-radiation and harvested after 2, 4, 6, 8, 10, 24, or 48 h. For p53 and p21 analysis, 50 μg of protein was resolved on an SDS/10% polyacrylamide gel. For analysis of p19ARF, 100 μg of protein was resolved on a 12% polyacrylamide gel. (A) p53, p21, and actin protein levels in WT and p19ARF−/− MEFs 2 h (T2), 4 h (T4), 6 h (T6), 8 h (T8), 10 h (T10), 24 h (T24), and 48 h (T48) after IR compared with untreated (Unt). (B) p19ARF and actin protein levels in WT MEFs at the indicated times after IR compared with untreated (Unt). p53−/− and p19ARF−/− MEFs are also shown as positive and negative controls for p19ARF protein detection.

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Differential requirement for p19ARF in the p53-dependent arrest induced by DNA damage, microtubule disruption, and ribonucleotide depletion - PubMed (original) (raw)