p53 and p21(Waf1) are recruited to distinct PML-containing nuclear foci in irradiated and Nutlin-3a-treated U2OS cells

Abstract

Promyelocytic leukemia nuclear bodies (PML-NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML-NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53-dependent growth inhibition. ALT-associated PML bodies (APBs) are specialized PML-NBs that include telomere-repeat binding-factor TRF1 and are exclusively in telomerase-negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell-cycle and p53 responses in ALT-positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin-3a. Both IR and Nutlin-3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin-3a. p53 was recruited to PML-NBs 3-4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML-NBs marked sites of apparently unrepaired DNA double-strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin-3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin-3a-treated cells. These data indicate (1) p53 is recruited to PML-NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53-p21 pathway activation increases the percentage of APB-positive cells, (3) p21 and p53 are recruited to ALT-associated PML-NBs after Nutlin-3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance.

Figure 1. Flow Cytometry analysis of U2OS cells treated with IR or Nutlin

U2OS cells (asynchronously growing and ~50% confluent) were either untreated (NT), treated with irradiation (IR, 10Gy) or treated with Nutlin (Nut; 5μmol/L). The cells were harvested at the indicated time points. The culture medium was not changed during the course of the experiment. Fixed cells were stained with propidium iodide (25 μg/mL) and subjected to flow cytometry analysis. A. Representative DNA profile histograms were analyzed using FlowJo (cell count versus propidium iodide/DNA content). The position of 2N and 4N cells is indicated. B. Cell cycle distribution was determined from DNA profiles using FlowJo. The numbers represent averages from three independent experiments. (mean ± SE; n = 3)

Figure 2. P53 and p21 induction in irradiated and Nutlin treated U2OS cells

U2OS cells were either untreated (NT), treated with irradiation (IR, 10Gy) or treated with Nutlin (Nut; 5μmol/L), as in Figure 1. Cell lysates were harvested at indicated time points and analyzed by Immunoblotting with indicated antibodies. Tubulin (Tub) was loaded as loading control.

Figure 3. p53 colocalized with PML in irradiated U2OS cells

U2OS cells on glass coverslips were either untreated (NT, A) or treated with irradiation (IR, 10Gy, B,C). Cells were fixed with 4% formaldehyde at indicated time points. P53, p21 antibodies were detected using an Alexa-488 conjugated goat anti rabbit IgG. PML antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. Cell nuclei were counterstained with DAPI (blue). Images were captured at 100× magnification. Representative images were shown. Representative foci with p53/PML colocalization were pointed with white arrows. Representative confocal images were shown in C.

Figure 4. Quantification of p53/PML colocalization in irradiated U2OS cells

Percentage of cells with p53/PML colocalization was determined at indicated time points after treatment. Percentage represents Mean ± SEM of three independent experiments with 200 cells counted per experiment.

Figure 5. p53 colocalized with γH2AX in irradiated U2OS cells

U2OS cells on glass coverslips were treated as described in Figure 2. A) Percentage of cells with >10 γH2AX foci was determined at indicated time points after treatment. Percentage represents Mean ± SEM of three independent experiments with 200 cells counted per experiment. B) γH2AX antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. P53, PML antibody were detected using an Alexa-488 conjugated goat anti rabbit IgG. Representative foci with p53/γH2AX or γH2AX/PML colocalization were pointed with white arrows.

Figure 6. p21 colocalized with PML in Nutlin treated U2OS cells

U2OS cells on glass coverslips were continuously treated with Nutlin (5 μM). Cells were fixed with 4% formaldehyde at indicated time points. A) P53, p21 antibodies were detected using an Alexa-488 conjugated goat anti rabbit IgG. PML antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. Representative foci with p21/PML colocalization were pointed with white arrows. B) Percentage of cells with p53/PML, p21/PML, p53/p21/PML colocalization was determined at indicated time points after treatment. Percentage represents Mean ± SEM of three independent experiments with 200 cells counted per experiment. C) Representative confocal images were shown.

Figure 7. p21 colocalized with γH2AX in Nutlin treated U2OS cells

U2OS cells on glass coverslips were treated as described in Figure 4. A) Percentage of cells with >10 γH2AX foci was determined at indicated time points after treatment. Percentage represents Mean ± SEM of three independent experiments with 200 cells counted per experiment. B) p21, PML antibodies were detected using an Alexa-488 conjugated goat anti rabbit IgG. γH2AX antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. Representative colocalization foci were pointed with white arrows.

Figure 8. p21 and p53 colocalized with TRF1 in PML-NB in Nutlin treated U2OS cells

U2OS cells on glass coverslips were either untreated (NT), treated with irradiation (IR, 10Gy) or treated with Nutlin (Nut; 5μmol/L). P53, p21, PML antibodies were detected using an Alexa-488 conjugated goat anti rabbit IgG. TRF1 antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. Representative colocalization foci were pointed with white arrows. Representative confocal images were shown in B.

Figure 9. APB positive cells increased in irradiated or Nutlin treated U2OS cells

APB positive cells were identified as those cells in which TRF1 and PML colocalized in large nuclear foci. PML antibodies were detected using an Alexa-488 conjugated goat anti rabbit IgG. TRF1 antibody were detected using a rhodamine-x conjugated goat anti mouse IgG. A) Percentage of APB positive cells was determined at indicated time points after treatment. Percentage represents Mean ± SEM of three independent experiments with 200 cells counted per experiment. B) U2OS cells were either transfected with siControl (siC), sip53, or sip21. At 24hrs after transfection, the cells were untreated (NT) or treated with Nutlin (Nut; 5μmol/L). Cells were fixed with 4% formaldehyde 2 days after treatment and subjected to immunofluoresence staining.