The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence

Abstract

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.

FIG 1

Irradiation-induced senescence in WI-38 fibroblasts. (A) WI-38 fibroblasts were gamma irradiated with 2 Gy or 15 Gy, and whole-cell lysates were analyzed by immunoblotting to detect the indicated proteins. d, days. (B) Representative images of SA–β-Gal staining of WI-38 cells 6 days after γ-IR. (C to E) Quantification of SA–β-Gal staining (C), living cells (D), and dead cells (E) within 6 days after irradiation. At least 50 to 100 cells per time point and irradiation dose were monitored. All experiments were done in triplicate. Mean values ± standard errors of the mean (SEM) are depicted.

FIG 2

PML NBs nonrandomly associate with persistent DNA damage foci. (A) Indirect immunofluorescence staining of WI-38 fibroblasts after γ-IR. Unirradiated (0 Gy) and irradiated cells (2 Gy or 15 Gy) were fixed at the indicated time points and stained with antibodies against PML and γH2AX. Scale bar, 5 μm. The inset in the bottom right image is an enlargement of the boxed area. (B to F) Quantification of γH2AX foci (B), association events between PML NBs and γH2AX (C), percentage of γH2AX foci associated with PML NBs (D), PML NBs (E), and theoretical probability of one random association between PML NBs and γH2AX foci (F). Quantifications of foci and potential associations were carried out using ImageJ software and customized macros. Mean values ± SEM are depicted.

FIG 3

PML NBs associate with UVA microirradiation-induced foci. WI-38 fibroblasts were irradiated with different doses of a UVA microbeam, fixed at the indicated time points, and immunostained to detect PML (green) and γH2AX (red). The regions within nuclei marked by white boxes are shown as magnified views (separated into single color channels shown as grayscale images) on the right of each overview image. (D) Note that in the images marked by asterisks, the signals for γH2AX were contrast stretched to visualize residual fluorescence. Scale bars = 5 μm.

FIG 4

PML NBs associate with DNA damage foci in senescent human and murine cells. (A) WI-38 fibroblasts at different PDs were subjected to Western blot analysis using antibodies against the indicated proteins (top). The number of SA–β-Gal-positive cells during serial passaging of these cells was also monitored (bottom). α-tub, α-tubulin. (B to G) Representative midnucleus confocal images of young WI-38 fibroblasts (B), replicative senescent WI-38 fibroblasts (C), unirradiated primary MEFs (pMEFs) (D), 15-Gy-irradiated MEFs after 6 days (E), MEFs treated with BrdU and DMA for 6 days (F), and replicative senescent MEFs (passage 6; PD 12) (G). The cells were fixed and immunostained to detect PML (green) and γH2AX (red). The percentages of of SA–β-Gal-positive cells are indicated (means ± SD). (H) Quantification of γH2AX foci (gray bars) and percentages of γH2AX foci associated with PML NBs (blue bars) for the indicated cells. Freshly isolated primary MEFs (passage 1) were used for irradiation and drug treatment, and replicative senescent MEFs were at passage 6 (PD 12). Experiments were done in triplicate. Mean values ± SEM are depicted.

FIG 5

PML NBs associate with DNA damage foci in human skin cells. Cryosections of human skin were stained with antibodies against PML (green) and phosphorylated ATM (red). (A and B) Representative confocal images of the epidermis (A) and the dermis (B). The insets are enlargements of the boxed areas. (C) The percentage of pATM-positive cells, as well as the association between pATM foci and PML NBs, was quantified in five skin sections from different donors. Mean values and SEM are depicted. (D) Antibodies against γH2AX (red) also revealed contacts between DNA damage sites and PML bodies (green) in the dermis of human skin. (E) Confocal image of a human skin section immunostained with antibodies against ANX5 (green) and pATM (red). DNA was stained with DAPI. (F) Quantitation of the cells positive for the indicated events from experiments shown in panel E. Mean values and SEM are depicted (n = 10 individual healthy skin sections from 3 different middle-aged donors).

FIG 6

MDC1, 53BP1, pNBS1, p16, p21, and p53 do not accumulate at persistent DNA damage-associated PML NBs. WI-38 fibroblasts were gamma irradiated and fixed at the indicated time points after DNA damage induction (A to H) or passaged until senescence (I). The fixed cells were subjected to immunofluorescence staining. An antibody against Sp100 was used as a marker for PML NBs, combined with a γH2AX antibody to stain DNA damage foci. Additionally, the DNA damage response proteins Mdc1, 53BP1, and phosphorylated Nbs1 were stained. (J to L) WI-38 fibroblasts were gamma irradiated with 15 Gy, fixed after 6 days, stained with antibodies against p16, p21, or p53, and costained with an antibody against PML. Insets show enlargements of the boxed area in each image. The line scans (right) show the pixel intensity distribution along the lines indicated in the merged image. Scale bars = 5 μm.

FIG 7

Superresolution imaging of PML nuclear bodies and DNA damage sites. Primary human fibroblasts (at low PD) were irradiated with 10 Gy, fixed after 90 min, and subsequently immunostained to detect the indicated proteins. 3D SIM superresolution images of single nuclei were collected using an Elyra structured-illumination microscope (Zeiss). The white boxes within the overview images are shown enlarged on the right. Each image on the right also shows the individual channels in monochrome.

FIG 8

DNA damage signaling is not altered in PML knockout MEFs. (A) PML wild-type, heterozygous, and knockout MEFs were gamma irradiated and lysed at the indicated time points, and Western blot analyses were carried out using the indicated antibodies. (B and C) Relative protein levels as measured by densitometry of Western blot bands were quantified using ImageJ software. All experiments were done in triplicate. Mean values ± SEM are depicted. Note that the blots shown in panel A have been spliced from two Western blots each. The splice sites are indicated by a thin white line between 3 and 8 h or 0.5 and 3 h within the left and the right blots, respectively. All Western blots shown in panel A originate from the same gel run and were developed simultaneously under the same conditions, which also allows comparison of the relative band intensities even between the 2-Gy- and 15-Gy-irradiated cells.

FIG 9

IRIF formation in PML-depleted cells. (A) Freshly isolated MEFs from PML wild-type, heterozygous, and knockout embryos were irradiated with 2 Gy and immunolabeled 30 min later using antibodies against γH2AX (green) and 53BP1 (green). The images show representative midnucleus confocal sections. Bars = 5 μm. (B and C) MEFs as in panel A were irradiated with 2 Gy or 15 Gy, and samples on coverslips were taken at different time points after irradiation. The cells were immunolabeled as in panel A to quantify the DNA damage foci. The data points represent mean values ± SEM. (D and E) Same as in panels B and C, using primary human fibroblasts stably expressing a vector control, a vector expressing control siRNA, and a vector expressing siRNA against PML. The data points represent mean values ± SEM. ***, P ≤ 0.001 (t test statistical analysis comparing data points for siControl and siPML).

FIG 10

PML depletion does not alter fibroblast senescence. (A and B) Primary human fibroblasts with a stable PML knockdown (siPML) or control-infected cells (vector and siControl) were gamma irradiated with 15 Gy and fixed after 6 days (A) or treated with 50 μM BrdU and 10 μM DMA and fixed at the indicated time points (B). To monitor senescence induction, cells were stained for SA–β-Gal. (C) pMEFs isolated from PML wild-type, heterozygous, and knockout embryos were treated with 50 μM BrdU and 10 μM DMA or 20-Gy irradiation, fixed after 6 days, and stained for SA–β-Gal activity. In all experiments, at least 50 cells were monitored, and all experiments were done in triplicate. Mean values ± SEM are depicted. (D and E) HFFs with a stable PML knockdown (siPML) or control-infected cells (vector and siControl) (D), as well as wild-type (+/+), heterozygous PML knockout (+/−), and PML knockout (−/−) pMEFs (E), were cultured until they reached senescence, and population doublings over time were monitored. For pMEFs, 2 or 3 cell lines per genotype were used, and mean values ± SEM are depicted.