HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/15/1970 04-1895fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by VON KOBBE, C. Articles by BOHR, V. A. Search for Related Content PubMed PubMed Citation Articles by VON KOBBE, C. Articles by BOHR, V. A.

Werner syndrome cells escape hydrogen peroxide-induced cell proliferation arrest

CAYETANO VON KOBBE^*,1, ALFRED MAY^*, CARLA GRANDORI and VILHELM A. BOHR^*,2

^* Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland, USA; and
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

²Correspondence: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA. E-mail: vbohr{at}nih.gov

SPECIFIC AIMS

Werner syndrome (WS) is a rare disease caused by lack of a functional nuclear WS protein (WRN). WS is characterized by early onset of premature aging signs and a high incidence of sarcomas. WS diploid fibroblasts display a short life span and extensive genomic instability. Mammalian cells are continuously exposed to reactive oxygen species (ROS), which represent human mutagens, and are thought to be major contributors to the aging process. Hydrogen peroxide (H₂O₂) is a common ROS intermediate generated by various forms of oxidative stress. Treatment of cells with oxidative stress by H₂O₂ produces specific DNA lesions including oxidized bases, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). This DNA damage triggers activation of intracellular pathways, including signal transduction processes and DNA repair. When the level of H₂O₂-induced DNA damage is too high, normal human diploid fibroblasts follow a pathway leading to irreversible proliferation arrest and premature senescence. This cellular response and activation of DNA damage checkpoints, ensure that those surviving, growing cells do not accumulate deleterious mutations. Deficiency in these DNA damage detection pathways leads to chromosomal instability, premature aging, and tumor formation due to incomplete assembly of DNA repair complexes on damaged DNA.

We recently showed that WS cells are deficient in the poly (ADP-ribose) polymerase-1- (PARP-1) -mediated poly (ADP-ribosyl)ation pathway after oxidative stress. Activation of PARP-1 represents an early DNA damage response that triggers cellular functions involved in sensing and processing of DNA lesions. PARP-1 has been found to be an active player in the DNA repair pathway of base excision repair (BER), and the lack of poly (ADP-ribosyl)ation in WS cells suggests a defect in oxidative DNA damage processing and/or detection pathways. Here we have investigated H₂O₂-induced DNA damage response pathway in WS cells by analyzing steps of the process: 1) how much DNA damage is induced in WS diploid fibroblasts vs. normal cells; 2) activation of other early DNA damage sensing processes such as p53 and H2AX phosphorylations; 3) final cellular response (i.e., cell death vs. cell survival and irreversible vs. reversible proliferation arrest); and 4) the presence of oxidative DNA base lesions in those surviving, H₂O₂-treated WS cells.

PRINCIPAL FINDINGS

1. Acute H₂O₂ treatment induces similar levels of DNA damage in normal and WS diploid fibroblasts
We treated cells for 15 min on ice in order to inhibit DNA repair during exposure to H₂O₂. DNA breaks were detected by comet assay in the absence of Fpg. Almost 100% of normal and WS diploid fibroblasts were damaged. Further analysis of DNA damage revealed no significant differences between normal and WS cells. Thus, acute H₂O₂ treatment damaged almost 100% of the cells and induced a similar level of DNA damage in normal and WS diploid fibroblasts.

2. DNA damage is efficiently sensed in both cell types
We analyzed two early steps in DNA damage sensing pathways, Ser20 phosphorylation of p53 and formation of DSB marker H2AX foci. Ten minutes after H₂O₂ treatment, p53 Ser20 was efficiently phosphorylated in normal and WS cells. WS cells showed higher phosphorylation levels of p53 Ser20 than normal cells. To detect H₂O₂-induced DSB formation, we used specific antibodies recognizing the phosphorylated form of histone H2AX (H2AX) in immunofluorescence assays. Normal undamaged cells showed almost no anti-H2AX reactivity whereas there was a slight background signal in WS fibroblasts. After 45 min treatment with H₂O₂, we detected numerous H2AX foci; the number and intensity of these foci were similar in normal and WS cells. After H₂O₂ treatment, normal and WS cells showed similar levels of DSB.

3. WS diploid fibroblasts are more resistant to H₂O₂-induced cell death
We analyzed H₂O₂-induced cell death 40 h after DNA damage by counting the number of viable cells. Normal cells showed a low percentage of cell survival (22%), but WS cells were surprisingly more resistant, showing 78% cell survival. Thus, WS diploid fibroblasts are more resistant to H₂O₂-induced cell death.

4. WS diploid fibroblasts escape H₂O₂-induced cell proliferation arrest
To investigate whether cells surviving H₂O₂ exposure became senescent, we performed a cell proliferation assay (Fig. 1 ) and a cell cycle analysis. Forty hours after damage, WS and normal cells were plated at the same density; the proliferation rate was measured at different days using MTT assay. The proliferation curve showed that WS cells treated with 500 µM H₂O₂ continued to grow for at least 9 days after damage at 80% of the proliferation rate of undamaged WS cells (Fig. 1 , right panel). In contrast and as expected, normal fibroblasts were completely arrested (Fig. 1 , left panel). Thus, WS diploid fibroblasts escaped H₂O₂-induced irreversible cell proliferation arrest.

View larger version (36K): [in this window] [in a new window]   Figure 1. Cells were damaged with 500 µM H2O2 for 45 min at 37°C and processed. After recovery (40 h), cells were plated at a density of 4 x 104 cells/mL. Cellular proliferation was measured on days 1, 3, 5, and 7 after plating, using MTT assay. Representative pictures of cells are shown at the top.

5. WS diploid fibroblasts do not activate the premature senescence mechanism
To further confirm the lack of premature senescent phenotype in H₂O₂-damaged WS cells, we measured specific markers for cellular senescence such as p53 and p21^WAF1 protein accumulation, stress-associated heterochromatin foci formation, SAß-gal activity, and number of nucleoli. Results (see Fig. 1 ) demonstrate that WS cells did not activate the premature senescence program after exposure to H₂O₂.

6. H₂O₂-treated WS cells proliferate despite presence of persistent, H₂O₂-induced, oxidized DNA bases
To determine whether growing, damaged WS cells contained accumulated H₂O₂-induced DNA lesions, we performed comet assays. Presence of DNA lesions was analyzed 4–5 days after damage, when surviving H₂O₂-treated WS cells continued to proliferate but damage-induced senescent normal fibroblasts did not (Fig. 1) . We noted that almost half of damaged WS cells (48.5%) displayed DNA lesions. When Fpg was not added to samples, only 9% of damaged WS cells displayed clear comet tails, indicating that >80% DNA damage in WS cells (4–5 days after H₂O₂) was due to the accumulation of Fpg sensitive DNA lesions. Thus, H₂O₂-treated WS diploid fibroblasts continue to grow in the presence of extensive DNA damage accumulation.

7. Depletion of endogenous WRN in normal diploid fibroblasts leads to the escape of H₂O₂-induced cell proliferation arrest
We used a retroviral vector expressing a short hairpin specific for WRN (siWRN) to decrease WRN protein levels in normal diploid fibroblasts by RNA interference. WRN expression levels remained almost undetectable day 2 and day 9 post-transfection (Fig. 2 A). siWRN transfected diploid fibroblasts become as big as WS cells and grew at the same proliferation rate as WS cells (Fig. 2B ). To analyze the response to H₂O₂, we damaged the cells day 2 post-transfection; 24 h later we plated them at the same density. Cellular proliferation was analyzed at different times (Fig. 2C ). Normal fibroblasts transfected with the vector alone stopped growing after damage (Fig. 2C , left panel). In contrast and as described for WS cells, siWRN transfected fibroblasts continued to proliferate after damage (Fig. 2C , right panel). Thus, depletion of WRN in normal fibroblasts bypasses H₂O₂-induced cell proliferation arrest in a uniform genetic background. We analyzed p53/p21^WAF1 induction levels 2 days after H₂O₂ treatment. Vector alone transfected cells efficiently accumulated both proteins. In contrast, siWRN-transfected cells showed no accumulation of p53 and a modest p21 accumulation resembling the damage response of primary WS fibroblasts. We compared p53/p21^WAF1 induction levels with those reported in nontransfected primary fibroblasts by quantifying intensities of blots after normalizing with loading control lamin B. p53/p21^WAF1 induction levels in damaged vector transfected cells were similar to those observed in normal fibroblasts. Thus, siWRN transfected cells behaved almost identically to WS diploid fibroblasts by displaying no significant accumulation of either protein. These data are in accord with the bypass of H₂O₂-induced cell proliferation arrest observed above for siWRN transfected cells.

View larger version (28K): [in this window] [in a new window]   Figure 2. WRN expression levels are critical in normal cellular growth rate and escape of H2O2-induced irreversible growth arrest. A) Normal diploid fibroblasts (MRC-5 cells) were transfected with retroviral pBabe-siWRN vector (lanes 1, 2) or retroviral pBabe vector alone (lane 3, vector), and collected for immunoblotting analysis 2 and 9 days after puromycin selection. Expression levels of WRN, p53, and lamin B were analyzed with corresponding antibodies. B) Nontransfected diploid fibroblasts (MRC-5 cells) or fibroblasts transfected with pBabe alone (MRC-5 vector) or pBabe-siWRN (MRC-5 siWRN) and WS cells (WS) were plated (second day post-transfection) at a density of 4 x 104 cells/mL. MRC-5 transfected cells were grown in the presence of puromycin (2 µg/mL). Proliferation rate was measured using the MTT assay after plating. Note that WS cells and pBabe-siWRN transfected MRC-5 cells are bigger than normal fibroblasts and reached confluence 5–7 days after plating, showing no further proliferation. Similar results were obtained using AG00780H or AG03141C (WS diploid fibroblasts). C) After 2 days of puromycin selection, MRC-5 cells transfected with pBabe alone (vector) or pBabe-siWRN (siWRN), were treated with different concentrations of H2O2 or medium alone (control) for 45 min at 37°C. After washing, cells were incubated in complete medium + puromycin for 24 h (recovery time). Cells were plated at a density of 4 x 104 cells/mL in the presence of puromycin. Proliferation rate was measured using MTT assay after plating. , 50 µM H2O2, , 250 µM H2O2, x, 500 µM H2O2.

These results suggest a central role of WRN in the H₂O₂-induced cellular damage response pathway and in processing oxidative DNA lesions.

CONCLUSIONS AND SIGNIFICANCE

Our results suggest that WRN is an important mediator in the cellular response after DNA damage (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Exposure of cells to H₂O₂ generates SSB, DSB, and oxidized DNA bases. WRN is thought to be involved in different DNA metabolic pathways, including recombination and DNA repair. Our data show that undamaged WS primary fibroblasts contain more Fpg sensitive sites than undamaged normal primary fibroblasts, consistent with a role for WRN as sensor of oxidative DNA lesions.

Oxidized DNA bases and SSB are mainly repaired by the BER pathway. WRN interacts physical and functionally with several proteins involved in this DNA repair pathway. Present and earlier data strongly suggest an active role of the WRN/PARP-1 complex in detection, processing, and/or repair of H₂O₂-induced DNA lesions, such as SSB and oxidized bases.

DSB can be repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR);d WRN has physical and functional interactions with components of these two pathways. WS cells display defects in DSB repair, suggesting an active role of WRN in handling this type of lesion.

Fibroblasts are prototypical mesenchymal cells and our results demonstrate that, in contrast to normal diploid fibroblasts, WS diploid fibroblasts are more resistant and continue growing after extensive DNA damage caused by H₂O₂ (Fig. 3) .

FOOTNOTES

¹ Present address: Centro Nacional de Investigaciones Oncologicas (CNIO), Telomeres and Telomerase Group, Madrid 28029, Spain.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1895fje;

This article has been cited by other articles:

				R. M. Brosh Jr and V. A. Bohr Human premature aging, DNA repair and RecQ helicases Nucleic Acids Res., December 3, 2007; 35(22): 7527 - 7544. [Abstract] [Full Text] [PDF]

				A. Das, I. Boldogh, J. W. Lee, J. A. Harrigan, M. L. Hegde, J. Piotrowski, N. de Souza Pinto, W. Ramos, M. M. Greenberg, T. K. Hazra, et al. The Human Werner Syndrome Protein Stimulates Repair of Oxidative DNA Base Damage by the DNA Glycosylase NEIL1 J. Biol. Chem., September 7, 2007; 282(36): 26591 - 26602. [Abstract] [Full Text] [PDF]

				M. S. Eller, X. Liao, S. Liu, K. Hanna, H. Backvall, P. L. Opresko, V. A. Bohr, and B. A. Gilchrest A role for WRN in telomere-based DNA damage responses PNAS, October 10, 2006; 103(41): 15073 - 15078. [Abstract] [Full Text] [PDF]

				F. M. Hisama, V. A. Bohr, and J. Oshima WRN's Tenth Anniversary Sci. Aging Knowl. Environ., June 28, 2006; 2006(10): pe18 - pe18. [Abstract] [Full Text]

				A. M. Szekely, F. Bleichert, A. Numann, S. Van Komen, E. Manasanch, A. Ben Nasr, A. Canaan, and S. M. Weissman Werner Protein Protects Nonproliferating Cells from Oxidative DNA Damage Mol. Cell. Biol., December 1, 2005; 25(23): 10492 - 10506. [Abstract] [Full Text] [PDF]

				J. Dairou, F. Malecaze, J.-M. Dupret, and F. Rodrigues-Lima The Xenobiotic-Metabolizing Enzymes Arylamine N-Acetyltransferases in Human Lens Epithelial Cells: Inactivation by Cellular Oxidants and UVB-Induced Oxidative Stress Mol. Pharmacol., April 1, 2005; 67(4): 1299 - 1306. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/15/1970 04-1895fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by VON KOBBE, C. Articles by BOHR, V. A. Search for Related Content PubMed PubMed Citation Articles by VON KOBBE, C. Articles by BOHR, V. A.