Abstract

J Cell Mol Med

J. Cell. Mol. Med

jcmm

Journal of Cellular and Molecular Medicine

1582-1838 1582-4934

Blackwell Publishing Ltd

Oxford, UK

PMC3837611

3837611

19840190

10.1111/j.1582-4934.2009.00945.x

Articles

Telomere attrition and genomic instability in xeroderma pigmentosum type-b deficient fibroblasts under oxidative stress

Ting

Aloysius Poh Leong

* Low

Grace Kah Mun

Gopalakrishnan

Kalpana

Hande

M Prakash

* Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore

Singapore

*Correspondence to: M. Prakash HANDE, Ph.D., Genome Stability Laboratory, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD9, 2 Medical Drive, Singapore 117597, Singapore. Tel.: +65-6516-3664 Fax: +65-6778-8161 E-mail: phsmph@nus.edu.sg

Jan-Feb 2010

16 10 2009

14 1-2 403 416 02 10 2008 06 3 2009

2009

Abstract

Xeroderma pigmentosum B (XPB/ERCC3/p89) is an ATP-dependent 3′→5′ directed DNA helicase involved in basal RNA transcription and the nucleotide excision repair (NER) pathway. While the role of NER in alleviating oxidative DNA damage has been acknowledged it remains poorly understood. To study the involvement of XPB in repair of oxidative DNA damage, we utilized primary fibroblasts from a patient suffering from XP with Cockayne syndrome and hydrogen peroxide (H₂O₂) to induce oxidative stress. Mutant cells retained higher viability and cell cycle dysfunction after H₂O₂ exposure. Cytokinesis blocked micronucleus assay revealed increased genome instability induced by H₂O₂. Single cell gel electrophoresis (comet) assay showed that the missense mutation caused a reduced repair capacity for oxidative DNA damage. Mutant fibroblasts also displayed decreased population doubling rate, increased telomere attrition rate and early emergence of senescent characteristics under chronic low dose exposure to H₂O₂. Fibroblasts from a heterozygous individual displayed intermediate traits in some assays and normal traits in others, indicating possible copy number dependence. The results show that a deficiency in functional XPB paradoxically renders cells more sensitive to the genotoxic effects of oxidative stress while reducing the cytotoxic effects. These findings have implications in the mechanisms of DNA repair, mutagenesis and carcinogenesis and ageing in normal physiological systems.

xeroderma pigmentosum B nucleotide excision repair oxidative DNA damage genome instability telomere dysfunction ageing

Introduction

Damage resulting from exposure to both endogenous and exogenous genotoxic agents can alter DNA and interfere with DNA replication and protein transcription, which ultimately predisposes cancer, and developmental defects [1]. DNA damage may also occur at telomeres, a process which can compromise their integrity. As telomeres are important factors in genome stability and replication competency, damaged telomeres can lead to outcomes such as genomic instability and premature senescence [2–6]. Mammals have evolved to be equipped with DNA repair mechanisms that specifically operate for individual base damages as well as genome repair through recognizing specific DNA helix distortions, one of which is the most versatile nucleotide excision repair (NER) pathway [7].

Reactive oxygen species (ROS) are known to cause oxidative damage to cellular components, as well as a multitude of DNA lesions [8]. Hydrogen peroxide (H₂O₂) is an endogenously produced ROS, generated during oxidative energy metabolism and exposure to exogenous factors such as lead compounds. Similar to UV-induced DNA damage, oxidative DNA damage gives rise to helix distortions that hinder base pairing, transcription and replication. Accumulation of unrepaired oxidative lesions predisposes cancer [1] and shortens telomeres [9], leading to premature senescence and possibly ageing.

Xeroderma pigmentosum (XP) is a rare autosomal recessive congenital DNA repair disorder stemming from defects in the NER. This syndrome manifests as segmental progeria, sunlight hypersensitivity, a 1000-fold increase in risk of cutaneous cancers and a host of other developmental and neurological abnormalities. Defects in the NER can also cause Cockayne’s syndrome and trichothiodystrophy [1, 10, 11].

The NER pathway engages a multiplex of proteins in a spatially and temporally specific manner to excise primarily bulky lesions including UV-induced pyrimidine dimers [11]. Though oxidative lesions are mainly resolved by the base excision repair (BER) pathway, the NER pathway has also been implicated in the repair of some of these lesions [1, 12, 13]. This process is however still poorly characterized.

XPB is a 3′→5′ helicase in the NER pathway and a subunit of the RNA polymerase II holoenzyme TFIIH. XPB plays a key role in the NER by unwinding the DNA helix surrounding the lesion and thus allowing access by subsequent factors. Studies have also shown roles for XPB in the incision [14] and excision [15] of lesions and in the recruitment of other NER factors to lesion sites [16]. As a subunit of TFIIH, XPB also plays a role in basal transcription [17]. As such, XPB mutation can give rise to all three NER dysfunction syndromes [18]. XPB is a member of the helicase domain containing family of proteins. Other members of this family including WRN and BLM have been shown to have a role in telomere maintenance [19–22]. Additionally, it has been reported that the NER endonuclease XPF is involved in telomere dynamics in mice overexpressing telomere restriction fragment (TRF)2 [23].

The hitherto poorly defined role of the NER and its helicase XPB in oxidative DNA lesion repair and telomere dynamics in contrast to the known and reported functions of the BER and its helicase WRN in these processes led us to investigate the possible role of the XPB in genome stability and telomere dynamics under oxidative stress.

Materials and methods Cell culture and H2O2 treatment

Our study utilizes primary fibroblasts from individuals heterozygous for dysfunctional XPB as a result of a splice donor mutation in intron 3 (c.471+1G>A) with the second allele being either dysfunctional as well due to a missense mutation (p.F99S) or normal (henceforth designated XPB^−/− and XPB^+/−, respectively) and normal primary fibroblasts [24, 25]. There are clinical differences among XPB patients with the same p.F99S mutation. Primary human diploid fibroblasts from a normal individual (normal GM03651E; female, 25 years; passage 10), an individual suffering from XP complementation group B (XPB^−/− GM13026; male, 25 years; passage 10) and the unaffected mother of the XPB patient (XPB^+/− GM13027; female, 63 years; and passage 10) were purchased from Coriell Cell Repositories (Camden, NJ, USA), and cultured in minimal essential medium (Gibco, Grand Island, NY, USA) supplemented with 15% foetal bovine serum (FBS; Hyclone, Logan Town, UT, USA), 100 U/ml penicillin/streptomycin, 1% vitamins, and 2% essential and 1% non-essential amino acid. All cells were grown in a humidified 5% CO₂ incubator at 37°C and maintained in a log phase. All other supplements unless otherwise stated were from Gibco. Cells at passages between 13 and 15 were used in the experiments. Exponentially growing cells were exposed to 20 μM, 40 μM, 60 μM, 80 μM and 100 μM of H₂O₂ (Kanto Chemical Co. Inc., Tokyo, Japan) for 2 hrs, following which the medium was replaced with fresh medium for a 22-hr recovery period.

Crystal violet assay

Crystal Violet dye binds electrostatically to nuclear proteins and thus stains DNA. Upon elimination of excess dye and solubilization of the dye, the relative density of adhered cells can be measured, which correlates to live cell number. Following treatment with different doses of H₂O₂, cells were washed gently in PBS (NUMI supplies, Singapore). Crystal Violet solution (0.75% crystal violet in 50% ethanol: distilled water with 1.75% formaldehyde and 0.25% NaCl) was gently added to the wells and incubated at room temperature, then washed in PBS. Thereafter, the wells were air-dried. A total of 1% SDS (NUMI supplies) in PBS was added to lyse the cells and solubilize the dye. Solution absorbance at 595 nm was measured in an ELISA plate reader.

Cell cycle analysis by fluorescence activated cell sorting

Control and treated cells were fixed in 3:1 70% ethanol: PBS, and subsequently stained with propidium iodide (PI, Sigma, St Louis, MO, USA): RNase A (Roche, Indianapolis, IN, USA) solution (2 mg PI and 2 mg RNaseA/100 ml 0.1% BSA in 1×PBS). Samples were analysed by flow cytometry at 488 nm excitation λ and 610 nm emission λ. Ten thousand events were collected and the data obtained was analysed using WINMDI software.

Cytokinesis blocked micronucleus analysis

After 2 hrs exposure to H₂O₂, cells were incubated in a fresh medium with 4.0 μg/ml cytochalasin B (Sigma) for 22 hrs. The protocol used is adapted from Hande et al. [26, 27]. Cells were subjected to hypotonic swelling in cold 0.075 M potassium chloride (KCl) and processed as previously described [28]. One thousand binucleated cells with/without micronuclei were scored under the Axioplan 2 imaging fluorescent microscope (Carl Zeiss, Oberkochen, Germany) with the appropriate filters.

Chromosome aberration analysis by peptide nucleic acid- fluorescence <italic>in situ</italic> hybridization (PNA-FISH)

Treated cells were allowed to grow in fresh medium for 24 hrs before being arrested at metaphase with 0.1 μg/ml colcemid for chromosome preparation. FISH and analysis were performed as described before [28] using Cy3-labelled PNA-telomeric and FITC-labelled PNA-centromeric probes (Applied Biosystems, Foster City, CA, USA) [29].

Alkaline single cell gel electrophoresis (Comet) assay

Cells were harvested for Comet assay after 2-hr exposure to H₂O₂ and after 22 hr recovery. Cells were resuspended in Hank’s balanced salt solution (HBSS; Sigma), mixed with 0.7% low melting point agarose (Conda, Madrid, Spain) and applied on Comet slides (Trevigen, Gaithersburg, MD, USA). Embedded cells were subjected to in lysis (2.5 M NaCl, 0.1 M pH 8 ethylenediaminetetraacetic acid [EDTA], 10 mM Tris base, 1% Triton X) at 4°C for 1 hr. After lysis, the slides were loaded onto a gel electrophoresis tank and immersed alkaline electrophoresis buffer (0.3 M NaOH buffered at pH 13–13.8 with 0.5 M EDTA pH 8.0) for 40 min. to allow DNA denaturation before being run at a constant 25 V/300 mA for 20 min. Following run, samples were neutralized with 0.5 M Tris-HCl pH 7.5 (NUMI supplies) for 15 min., dehydrated in 70% ethanol for 5 min., and then dried at 37°C. DNA was stained using SYBR Green (Trevigen). Analysis of comets was performed with Comet Imager Software (Metasystems, Altlussheim, Germany). Extent of DNA damage was expressed as a measure of the percentage of DNA in the comet tails. One hundred randomly selected cells were examined per sample.

Cellular kinetic studies Treatment

Cells were seeded in T75 flasks at a density of 2 × 10⁵ cells per flask and subjected to a 30-day long-term chronic treatment. One set of cells was treated with 20 μM H₂O₂ every 48 hrs, with media changed prior to addition of drug; another set of cells was maintained in a 40% O₂ incubator, with media changed every 48 hrs; media for control cells was also changed every 48 hrs.

Morphological analysis

Cells were observed under a light microscope at 40×, 100× and 200× magnifications before media changes. Morphology of the cells was photographed using an Olympus C-7070 WZ (Tokyo, Japan) digital camera.

Population doubling (PD) study

Cells were harvested whenever 90% confluency was reached for untreated cells of each cell type, and on the final day of the experiment. This occurred on days 6, 12, 18, and 30. Harvested cells were counted using a haemocytometer. A fresh tissue culture flask was then reseeded with 2 × 10⁵ cells or all cells if cell numbers were less than 2 × 10⁵.

The population doubling number (PDN) was calculated as follows:

where N₀= number of cells at harvest and N_x= number of cells seeded [30].

A portion of the remaining cells was seeded in 6-well culture plates (NUNC) for senescence-associated β-galactosidase (SA-β-gal) assay and the rest kept for DNA extraction for TRF analysis.

<italic>Senescence-associated</italic>β<italic>-galactosidase (SA-</italic>β<italic>-Gal) assay</italic>

Expression of SA-β-gal was performed with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Denvers, MA, USA) following manufacturer’s instructions. Cells were observed under a light microscope at 40×, 100× and 200× magnifications, and cell photographs taken with an Olympus C-7070 WZ digital camera.

Telomere restriction fragment length analysis

DNA was extracted from the cells using DNeasy Tissue Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. The telomere lengths of these cells were measured using TeloTAGGG Telomere Length Assay Kit (Roche Applied Science, USA). Two μg of purified DNA was digested with HinfI and RsaI for 2 hrs at 37°C. DNA fragments were separated by gel electrophoresis in 0.8% agarose gel at 60 V for 3 hrs, transferred via over night Southern blot onto a nylon membrane and cross-linked onto the membrane using a UV cross-linker (Stratagene, La Jolla, CA, USA). TRFs were hybridized with telomere-specific digoxigenin (DIG)-labelled probe and incubated with Anti-DIG alkaline phosphatase and tetramethylbenzidine according to manufacturer’s protocol. Chemiluminescence signal was detected using X-ray film and telomere length was analysed using GeneTools software (SYNGENE, Cambridge, UK). Decrease in telomere length was then expressed as a function of PDN to assess the rate of telomere shortening, indicating the effect of drug treatment on telomeres.

Western analysis for XPB and p53 proteins

Cells were seeded, as described above and harvested at 2, 24 and 48 hrs after exposure to H₂O₂. Total cellular protein was extracted by lysing cells in 100–200 μl lysis buffer (10 mM Tris-HCl [pH 7.4], 1% SDS, 1 mM sodium ortho-vanadate in ddH₂O). Released DNA was sheared by passing the lysate through a 0.4 × 12 mm syringe (100 Sterican, B. BRAUN, Melsungen, Germany). Lysed cells were centrifuged at 13.2 rpm, for 6 min. at 4°C, and the supernatants collected. Protein concentrations were determined using the micro BCA Protein Assay kit (Pierce, Rockford, IL, USA). One μl of cell lysate was diluted in 249 μl of dH₂O and 250 μL micro BCA working reagent (25 parts solution A: 24 parts solution B: 1 part solution C) and quantified against known concentrations of BSA (1 μg/ml −40 μg/ml). Forty micrograms protein from each sample was loaded into and run on 7.5% and 10% SDS-Polyacrylamide gel and then electro-blotted onto a nitrocellulose membrane (BioRad Co., Hercules, CA, USA). Transfer of proteins was checked by Ponceau Reagent (0.5% Ponceau S (Sigma), 1% glacial acetic acid (Sigma) in dH₂O). Membranes were blocked for 60 min. with 5% non-fat milk in TBS-T (0.1% Tween-20 (Sigma) in 1× TBS (0.1 M NaCl, 0.1 M Tris pH 7.4 in ddH₂O) (All purchased from Numi, National University of Singapore) or 5% Bovine Serum Albumin (BSA, Amresco, Salem, OH, USA) in 1× TBS-T, depending on the protein probed for. Subsequently, membranes were probed with primary antibodies against target proteins overnight at 4°C. Mouse monoclonal antibodies used were phosphorylated p53 (Ser 15) (p-p53) (Cell Signaling; 1:1000) and p53 DO-1 (Santa Cruz, Santa Cruz, CA, USA; 1:500) and actin (Chemicon, USA; 1:5000). Rabbit polyclonal antibody used was XPB (Santa Cruz; 1:100). The membrane was then incubated in goat anti-mouse IgG (H+L)-HRP (Pierce; 1:5000) or sheep anti-rabbit IgG (H+L)-HRP (Pierce; 1:5000) secondary antibody for 1 hr at room temperature. The protein bands were visualized after incubation of membranes in Chemiluminescence Reagent Plus (Perkin Elmer Life Science Inc., Waltham, MA, USA) followed by exposure to X-ray film (Pierce).

Statistical analysis

Statistical significance between and among data sets was assessed using two-way ANOVA, using Graphpad Prism (Graphpad, La Jolla, CA, USA). The difference was considered to be statistically significant when P < 0.05.

Results XPB−/− cells are less sensitive to the cytotoxic effects of oxidative stress

All three cell types exhibited a common trend of dose dependant decrease in cell viability (Fig. 1). The normal cells appeared to be more sensitive to H₂O₂ induced stress with significantly lower viability compared to the other two cells types from concentrations of 40 μM and above (P < 0.05). XPB^−/− cells appeared to be the least sensitive cell type with XPB^+/− exhibiting intermediate sensitivity. Interestingly, there was a levelling off of viability decline in normal and XPB^+/− cells but not in XPB^−/− cells. The differences in the viability trends prompted our investigation of cell cycle changes in the cells following exposure to H₂O₂.

Fig 1

Dose-dependent decrease in cell viability in H₂O₂ treated cells. XPB^−/− and XPB^+/− cells are significantly less sensitive to cell death compared to normal cells at concentrations above 20 μM. *P < 0.05; **P < 0.01 (two-way ANOVA). Data are represented as mean ± S.E. XPB^−/− cells are also significantly less sensitive to cell death compared to XPB^+/− cells at 40 μM and 60 μM (†P < 0.05).

Dysfunctional XPB causes deficiency in cell cycle checkpoint function

Figure 2 A illustrates the cell cycle profiles of normal, XPB^+/− and XPB^−/− cells after 24 hrs of exposure to H₂O₂. Normal cells displayed a dose dependent shift in cell cycle profiles (Fig. 2B). There was an increase in G2/M phase of cells after 20 μM and 40 μM H₂O₂ in normal cells with a corresponding decrease in G₁ and S phases. Above 40 μM, the G1 and Sub-G₁ populations were greatly increased. XPB^+/− cells displayed a similar increase in G2/M followed by G1 populations in XPB^+/− cells at the same doses as in normal cells, however there no change in the Sub-G1 population (Fig. 2C). XPB^−/− cells presented a little change in profiles up to 40 μM and above this concentration, there was a slight decrease in the G₂/M population with concomitant increases in the G₁ and Sub-G₁ populations (Fig. 2D). To determine if cell cycle profile changes in the cells or lack thereof were associated with DNA damage, we employed the alkaline single cell electrophoresis or comet Assay.

Fig 2

Cell cycle analysis by FACS. (A) Cell cycle histograms 24 hrs after exposure to H₂O₂. Clear profile changes and phase shifts observed in normal and XPB^+/− cells. Changes in XPB^−/− cells not easily discriminated. (B)–(C). Percentages of cells in each phase of the cell cycle. (B) H₂O₂ affects a G2/M increase at 20 to 40 μM with increases in G1 and sub-G1 populations at subsequent concentrations. (C) Similar increase in G2/M followed by G₁ populations in XPB^+/− cells at the same doses as in normal cells. Minimal sub-G₁ population observed. (D) XPB^−/− cells no not display the G₂/M increase observed for the latter two cells at 20 to 40 μM. G₁ and sub-G₁ populations increase slightly at subsequent concentrations.

XPB deficient cells show compromised repair capacity for oxidative DNA lesions

A pair of single cell gel electrophoresis (comet) assays was done to determine initial DNA damage at 2 hrs following H₂O₂ exposure and persisting DNA damage after a 22-hr recovery period. Nuclei with undamaged DNA appear round; nuclei with damaged DNA in the form of strand breaks result in DNA fragments which migrate faster during gel electrophoresis and give rise to a ‘tail’. The percentage of DNA in these ‘tails’ (% tail DNA) was used as a measure of DNA damage.

At 2 hrs, normal cells displayed significant dose dependent increases (P < 0.001) in % tail DNA at concentrations from 40 μM. XPB^−/− and XPB^+/− cells displayed a similar trend from 20 μM (P < 0.05). Normal cells displayed significantly lower (P < 0.001)% tail DNA compared to the other two cell types (P < 0.01) (Fig. 3).

Fig 3

Alkaline single cell gel electrophoresis. Tail DNA percentages immediately following 2 hrs H₂O₂ treatment (treated) and 22 hrs recovery in fresh medium (recovery). Treatment of 40 μM H₂O₂ and above resulted in significantly increased percentages (P < 0.01) which decreased significantly following recovery (P < 0.001) in all cell types. Only XPB^−/− cells displayed significantly increased percentages compared for normal cells at 20 μM H₂O₂ (P > 0.05). Both XPB^−/− and XPB^−/− cells exhibited higher percentages compared to the normal cells at all subsequent concentrations. Following recovery, there was no significant difference between normal and XPB^+/− cells at all concentrations (P > 0.05) except 80 μM. XPB^−/− cells showed significantly higher percentages than normal cells at 40–100 μM. *P < 0.05 and **P < 0.001 indicate significantly greater tail DNA percentage when comparing XPB^−/− and XPB^+/− cells to their normal counterparts (two-way ANOVA). Data are represented as mean ± S.E.

Following the recovery period, there were significant reductions (P < 0.001) in % tail DNA for all conditions under which there was damage at 2 hrs (Fig. 3). This returned back toward the baseline is indicative of damage repair. In normal cells there was no significant difference between treated at all concentrations and untreated cells (P > 0.05). Treated XPB^+/− cells displayed significant differences from untreated cells only from 80 μM (P < 0.001). XPB^−/− cells exposed to H₂O₂ retained significantly higher (P < 0.001)% tail DNA than untreated cells at all doses. Between cell types, XPB^−/− cells displayed significantly higher% tail DNA recovery as compared to normal (P < 0.05; 20 to 100 μM) and XPB^+/− cells (P < 0.01; 40 to 100 μM). There was no such significant difference (P > 0.05) between recovered normal and XPB^+/− cells apart from at 80 μM (P < 0.01).

Lack of functional XPB increases incidence of oxidative-stress induced genomic instability

Genomic instability markers were used to assess if loss of XPB function renders cells more susceptible to H₂O₂-induced genomic instability. Micronuclei which are the result of lagging chromosomes and acentric chromosomes being excluded from daughter nuclei following cytokinesis are markers of genome instability. Utilising cytochalasin B, an actin polymerization inhibitor, we arrested cells that completed a single nuclear division at cytokinesis. The numbers and distribution of micronuclei in the resulting binucleates were scored to determine genome stability following exposure to H₂O₂ (Fig. 4). We reduced the H₂O₂ dose range to a maximum of 40 μM as higher concentrations yielded very few mitotic cells and binucleated cells. In congruence to the ability of H₂O₂ to induce DNA strand breaks, all three cell types displayed significant increases in micronuclei frequency following exposure. XPB^−/− cells showed significantly higher micronuclei frequency than normal cells when exposed to H₂O₂ (P < 0.05). There was no significant difference between XPB^+/− and normal cells or between XPB^+/− and XPB^−/− cells (P > 0.05).

Fig 4

(A) Cytokinesis blocked micronucleus assay. Percent MN per 1000 BN scored following H₂O₂ treatment. There was no significant difference between XPB^+/− and normal cells (P > 0.05). XPB^−/− cells exhibit significantly more MN presence in BN cells compared to control cells. *P < 0.05 (two-way ANOVA). Data are represented as mean ± S.E. (B) Chromosome analysis by PNA-FISH: Chromosome analysis was done in metaphase spreads following H₂O₂ treatment. All three cell types displayed an increase in chromosomal aberrations in the form of breaks following H₂O₂-induced oxidative stress (see results for explanation). Chromosome aberrations per cell are displayed for each sample. Chromosome aberrations detected include fragments and breaks. *P < 0.05 compared to untreated sample of respective cell type. **P < 0.05 – Compared to normal and XPB^+/− cells after 40 μM H₂O₂ treatment.

Similar to the micronucleus results, all three cell types displayed an increase in chromosomal aberrations in the form of breaks following H₂O₂-induced oxidative stress (Fig. 4B). Importantly, although there were not any significant difference between XPB^+/− and normal cells with or without treatment, H₂O₂ proved to be more clastogenic on XPB^−/− cells. Following H₂O₂ treatment, XPB^−/− cells exhibited significantly (P < 0.05) higher chromosome aberrations than normal cells.

XPB deficient cells display early appearance of senescent characteristics

Based on the results of the crystal violet assays and cell cycle profiles, we selected a dose of 20 μM to utilize for low dose chronic oxidative stress exposure. This dose caused only slight non-significant decrease in cell viability in all three cell types and did not greatly perturb cell cycle profiles. We also included a set of cells cultured under hyperoxia (40% O₂) for a broader perspective of chronic oxidative stress.

Senescent characteristics included enlarged and flattened cell morphologies, increased cell volume, expression of SA-β-gal and reduced population doubling rate. In the absence of oxidative stress, the enlarged morphologies in XPB^−/− cells appeared at day 23 of treatment compared to normal fibroblast morphologies throughout the entire period in normal cells (Fig. 5A, B). SA-β-gal expression in XPB^−/− was detected at day 24 compared to no expression in normal cells (Fig. 5A, B). XPB^+/− cells also displayed typical fibroblastic morphologies and no SA-β-gal expression when not exposed to H₂O₂ or 40% O₂ (Fig. 5A, B).

Fig 5

Cellular kinetics study. (A) Morphologies of fibroblasts under chronic oxidative stress at 40× magnification. Typical elongated fibroblast morphology is replaced with enlarged and flattened senescent morphology (arrows) over time and with oxidative stress. Pictures show cells at the start of treatment, on the first day of morphology change. Where no change occurs before the final day, pictures from day 30 are shown. Morphology change takes place earlier in XPB^−/− than in the other two cell types in all conditions and hastened by oxidative stress. (B) SA-β-gal expression at 100× magnification. Pictures show cells at the end of treatment. Higher incidence of positive staining (bluish; arrows) is present XPB^−/− cells than for the other two cell types in all conditions and hastened with oxidative stress. XPB^−/− cells also developed blue staining earlier in the treatment period (data not shown) (C) Total population doublings undergone by cultures on indicated days throughout the 30-day treatment period. Treated cells displayed reduced total population doublings compared to untreated cells. Treated XPB^−/− cells possessed the lowest total population doublings out of all three cell types throughout the investigation.

Exposure to oxidative stress conditions resulted in the appearance of senescent characteristics in normal and XPB^+/− cells and hastened their appearance in XPB^−/− cells. The latter displayed cell body enlargement as early as day 6 for H₂O₂ and day 11 for 40% oxygen compared to days 9 (H₂O₂) and 17 (40% O₂) for normal cells and days 11 (H₂O₂) and 21 (40% O₂) for XPB^+/− cells. SA-β-gal expression in stressed XPB^−/− cells was observed by day 12. Minimal staining was only observed in H₂O₂ treated normal and XPB^+/− cells by day 18 and absence of staining with 40% O₂. Interestingly, treatment with H₂O₂ results in earlier appearance of senescent morphologies and positive SA-β-gal staining than 40% O₂. With the latter treatment regime, minimal staining was observed even by the end of the 30-day period. We also observed that XPB^+/− cells first manifested senescent morphological changes later than both the other two cell types.

Total population doublings in treated XPB^−/− cells were also lower than those in treated normal cells. XPB^+/− cells were intermediate in the appearance of these features (Fig. 5C).

Cells lacking XPB exhibit higher telomere attrition

All cells under untreated conditions and exposure to H₂O₂ and 40% O₂ displayed telomere shortening as revealed by TRF length analysis. The raw decreases in TRF length were greater for XPB^−/− and XPB^+/− cells than for normal cells (Fig. 6B). After taking into account population doubling numbers for the samples, XPB^−/− and XPB^+/− cells had substantially greater telomere attrition rates than normal cells under oxidative stress conditions (Fig. 6C).

Fig 6

Telomere restriction fragment analysis (A) Southern blot of TRFs obtained by digest of genomic DNA with Hinf1 and RsaI. (B) Total TRF length decreases after completion of 30-day period. Both XPB deficient cell types displayed greater decreases of TRF length than normal cells under all conditions. (C) Telomere attrition rate derived by dividing the TRF length decrease by the number of population doublings. Attrition rate increases in all cell types under conditions of oxidative stress similar trend to raw TRF length decrease. XPB deficient cell types exhibited a greater attrition rate than normal cells. Attrition rate for XPB^−/− was also markedly greater than that for XPB^+/− cells at 40% O₂.

XPB up-regulation occurs as part of normal cellular response to oxidative stress

In normal cells, up-regulation of XPB is evident at 24 hrs after treatment. p53 phosphorylation increased within 2 hrs and p53 up-regulation was observed at 24 hrs; both levels returned to control levels at 48 hrs (Fig. 7A and C). Phosphorylation and up-regulation of p53 phosphorylation and up-regulation was observed at 2 hrs and persisted till 48 hrs in XPB^+/− cells. Similarly, up-regulation of XPB occurred in XPB^+/− cells at 2 hrs with return towards control levels at 24 and 48 hrs (Fig. 7B and E). In XPB^−/− cells p53 up-regulation was seen at 2 hrs with a return to control levels by 48 hrs. XPB up-regulation and a profound increase in p53 phosphorylation were observed at 48 hrs (Fig. 7C and F).

Fig 7

Western blot analysis for XPB and p53 following exposure to H₂O₂: Following treatment with H₂O_2, whole cells were lysed, equal amounts of proteins were separate using 4–20% SDS-PAGE, transferred to PVDF membrane and immunoreacted with antibodies against XPB, p53 and phospho-p53. Actin was used as loading control. Histograms represent fold differences in protein levels normalized against actin in treated cells as compared to untreated cells. (A, D) Normal cells. Treatment with H2O2 results in phosphorylation of XPB (2 hrs) followed by up-regulation of XPB and p53 (24 hrs). (B, E) XPB^+/− cells. XPB and p53 up-regulation take place at an earlier time-point (2 hrs) than in normal cells. p53 up-regulation persists till 48 hrs. A lower fold increase in p-p53 than in normal cells was also observed. (C, E) XPB^−/− cells. p53 up-regulation occurs earlier than in normal cells (2 hrs). XPB up-regulation and p53 phosphorylation is delayed till 48 hrs.

Discussion

It is known that the NER pathway has a function in the repair of DNA lesions induced by oxidative stress. Our data show that XPB^−/− cells are resistant to viability decline than normal cells. The ability of XPB^−/− to survive in spite of oxidative damage hinted at possible cell cycle checkpoint dysfunction. Obligatory cell cycle arrest occurs when DNA is damaged so as to allow repair or execution of apoptosis if the damage is irreparable. Cell cycle profile shifts upon exposure to genotoxic agents are indications of DNA assault [8, 31]. Lack of cell cycle profile changes in XPB^−/− fibroblasts pointed to a cell cycle dysfunction. It was recently reported that XPB has a partially redundant role in p53-mediated apoptotic signalling [32, 33]. Our results of persistence in cell viability and checkpoint dysfunction in XPB^−/− fibroblasts are in agreement with these findings. This is significant in light of XPB dysfunction already compromising cell cycle checkpoint response to UV-induced DNA damage [34]; there is now also evidence of checkpoint dysfunction in response to oxidative lesions.

There is an implication that in XPB^−/− fibroblasts, the large proportions of cells which have sustained genomic assault and survived also fail to properly repair the damage and can pass this onto progeny. Using alkaline single cell gel electrophoresis or comet assay, we measured tail DNA percentages at two time-points; immediately after the 2-hr exposure period to determine the extent of immediate DNA damage [35] and 22 hrs after removal of H₂O₂ to determine DNA repair or damage recovery [36]. All cells displayed increased tail DNA percentages at the 2-hr mark which subsequently decreased after the recovery period. This is within expectations as all three cell types have a functional base excision repair pathway, the main pathway for alleviating oxidative DNA lesions. Notably, while normal and XPB^+/− fibroblasts were able to decrease tail DNA percentages to baseline values, XPB^−/− cells retained higher damage after recovery. This is in line with our hypothesis that XPB is involved in proper cellular response to and repair of oxidative lesions. The unrepaired damage in XPB^−/− cells could constitute two functional scenarios. The first is that this damage arises from a subset of oxidative lesions that are specifically or preferentially repaired by XPB and its partner factors [13]. However, it was recently reported that a common oxidative DNA adduct, 8-oxoguanine did not require NER factors including XPB for proper excision [37]. The second scenario involves XPB and its partner proteins acting in concert with the BER to expedite repair of oxidative DNA lesions and XPB dysfunction causes a delay in repair. The exhibition of higher tail DNA percentages by XPB^+/− and XPB^−/− fibroblasts immediately after H₂O₂ exposure is another interesting trend as it is also indicative of a role for XPB in detecting initial oxidative damage, restricting its extent or starting the repair process. Support for this phenomenon comes from studies which indicate XPB is important for proper recruitment of other NER factors to UV lesion sites [16] and that the particular mutation (F99S) in our cells reduces opening of DNA surrounding UV-induced lesions [38].

Various syndromes including XP and Ataxia Telangiectasia are caused by DNA repair deficiencies and result in increased disposition to cancers [1, 10, 39–41]. Compromised repair in such cells may result in inheritance of DNA damage by their progeny. DNA damage such as chromosomal breaks can result in potentially oncogenic changes through the formation of truncated or fusion proteins and misregulation of tumour suppressors and oncogenes [29, 42]. We utilized the micronucleus assay [26, 43] to investigate the presence of accompanying genomic instability. Exposure to oxidative stress induced by H₂O₂ resulted in increased micronuclei frequency which is an expected result as H₂O₂ induces DNA strand breaks. It is notable that this frequency is greater in XPB^−/− cells than the other two cell types.

Chromosome aberrations detected support this observation. The results from this assay indicate that lack of repair combined with cell cycle checkpoint dysfunction contributes to increased numbers of daughter cells with genomic damage. This agrees with XP-B patients having increased predisposition to developing cancers and implicates oxidative stress as a contributing factor in carcinogenesis in these patients.

It has been widely shown that oxidative stress causes decline in cell viability, cell cycle arrest and DNA damage including enhanced telomere attrition [9, 36]. Induction of oxidative stress affects the proliferative capacity of cells and predisposes premature senescence and ageing. Notably, cells defective in repair mechanisms such as Ataxia Telangiectasia have been shown to exhibit faster rates of telomere attrition and earlier senescence than normal cells [21, 44]. We performed a group of cellular kinetics studies to address the effects of chronic low level exposure to oxidative stress in relation to XPB. Untreated XPB^−/− cells displayed enlarged and flattened cell morphology, SA-β-gal expression and reduced population doubling rates towards the end of the treatment period whereas normal and XPB^+/− cells did not. This is significant as it agrees with the segmental progeria presented by XP patients and implicates factors other than UV for this symptom as cell cultures were not exposed to UV sources. Consistent with known findings that H₂O₂ and hyperoxia induces premature senescence in cells [36, 45], exposure to H₂O₂ and 40% O₂ resulted in morphological changes and SA-β-gal expression in all three cell types. Compared to untreated cells, these changes appeared earliest in XPB^−/− cells. We observed that H₂O₂ had a more pronounced effect on all three cell types than 40% O₂. H₂O₂ is only one of a plethora of ROS generated under normal metabolism. Introduction of exogenous H₂O₂ may thus constitute a greater imbalance in cellular redox potential than hyperoxia which in turn gave rise to more pronounced effects. We also found that XPB^+/− were almost as adept as normal cells in coping with chronic oxidative stress. The above mentioned partially redundant role of XPB in apoptotic signalling may offer an explanation as one functional copy of the gene is able to compensate along with the other partially redundant factors for the dysfunctional copies.

As well as these morphological changes and marker expression, we observed a heightened telomere attrition rate under oxidative stress, especially when combined with XPB dysfunction. Telomeres cap chromosomal ends and are important for chromosome integrity and segregation [29, 42]. They have been regarded as a determinant in cellular replication, aging and lifespan [2–6]. Telomeres have been shown to preferentially accumulate single stranded regions induced by oxidative stress [46]. Various factors, such as the telomere repeating binding factors TRF1 and TRF2 and telomerase, modulate telomere dynamics and integrity [5]. It has been found that various DNA repair factors function in telomere maintenance and repair of damage sustained at telomeres [19, 47]. These include the helicase domain containing family of proteins [20, 22, 48] of which XPB is a member [20]. As such, the presence of increased telomere attrition in XPB^−/− cells is not an unexpected result. Further support comes from studies that show XPF, one of the partner proteins of XPB in the NER pathway is involved in telomere dynamics in mice [23].

The various functional observations made led us to look into the molecular basis behind the previous results. In all cells, XPB was up-regulated following exposure to H₂O₂. Interestingly, up-regulation occurred at divergent time-points in all cells. Up-regulation in XPB^+/− cells was present as early as 2 hrs. This might be explained by lower levels of functional XPB in XPB^+/− cells compared to normal cells. These cells would thus require an earlier increase in XPB levels following oxidative stress whereas normal cells have a sufficient baseline reservoir allowing for later up-regulation. In XPB^−/− cells, up-regulation only occurred at 48 hrs. This is likely due to the very low levels of only dysfunctional protein in these cells. A lag time could arise as other repair factors would be initiated first as the cells attempted to build up sufficient amounts of XPB. We observed both up-regulation and phosphorylation of p53, a cell cycle checkpoint protein and a DNA damage marker, in all three cell types with exposure to H₂O₂. In addition, p53 expression and phosphorylation patterns were different between normal and XPB^−/− cells. In normal cells, increased phosphorylation was observed first at 2 hrs with up-regulation following at 24 hrs, with reversion of both to baseline levels at 48 hrs. XPB^−/− cells displayed up-regulation at 2 hrs while phosphorylation was delayed till 48 hrs. XPB^+/− cells displayed similar trends to XPB^−/− cells. XPB was reported to be able to bind to p53 [49, 50] and have a partially redundant role in p53-mediated apoptotic signalling [32, 33]. Lack of functional XPB in XPB^−/− cells might compromise the ability of these cells to properly phosphorylate p53 and trigger DNA damage and possibly apoptotic signalling cascades. This is in agreement with the results of the crystal violet assay showing higher viability in XPB^−/− cells following H₂O₂ treatment. The presence of increased p53 phosphorylation in XPB^−/− cells at 48 hrs also corroborates the results of the comet assay which indicate compromised recovery and persistence of DNA damage after resolution in normal cells. The earlier increased expression of p53 in these cells might be an attempt to overcome their inability to phosphorylate p53.

In summary, our study has shown that XPB dysfunction sensitizes cells to the genotoxic effects of oxidative stress while reducing the cytotoxic effects. Such a phenomenon can result in genome instability which can predispose cancer and accelerated telomere attrition which may influence aging. This is congruent with XP patients being extremely sensitive to UV-induced skin lesions and cancers. It is however hard for UV-induced DNA damage to explain the full range of XP symptoms. Our findings implicate oxidative stress as a possible major contributor to such manifestations, particularly at tissues away from the body surface and hence protected from UV exposure. This is not surprising given that oxidative stress via ROS generation is downstream to a various other genotoxic agents including UV irradiation [8]. It has been shown that polymorphisms in the XPB gene can rise to diseases of varying severities and phenotypes [24, 25]. Additionally, the inclusion of heterozygous cells in our study hint as well at a possible copy number dependence. Our results suggest a role for XPB in the resolution of oxidative stress induced DNA lesions and in telomere dynamics. A recent report suggests that the helicase activity of XPB in NER repair of UV-induced DNA lesions is dispensable while its ATPase activity is essential [38]. Whether this is also true of repair of oxidative lesions and telomere maintenance, and whether these activities are performed exclusively by XPB or in concert with other factors including the rest of the NER and telomere binding proteins is the subject of further research.

M.P.H. acknowledges the grant support from Academic Research Fund, National University of Singapore.

Conflict of interest statement

The authors have no conflicts of interest to declare.

References 1

Hoeijmakers

Genome maintenance mechanisms for preventing cancer

Nature 2001 411 366 74

11357144

Gilley

Tanaka

Herbert

Telomere dysfunction in aging and cancer

Int J Biochem Cell Biol 2005 37 1000 13

15743674

Klapper

Parwaresch

Krupp

Telomere biology in human aging and aging syndromes

Mech Ageing Dev 2001 122 695 712

11322993

Levy

Allsopp

Futcher

Telomere end-replication problem and cell aging

J Mol Biol 1992 225 951 60

1613801

Rodier

Kim

Nijjar

Cancer and aging: the importance of telomeres in genome maintenance

Int J Biochem Cell Biol 2005 37 977 90

15743672

Shay

Wright

Telomeres and telomerase: implications for cancer and aging

Radiat Res 2001 155 188 93

11121233

Wood

Nucleotide excision repair in mammalian cells

J Biol Chem 1997 272 23465 8

9295277

Finkel

Holbrook

Oxidants, oxidative stress and the biology of ageing

Nature 2000 408 239 47

11089981

Von Zglinicki

Oxidative stress shortens telomeres

Trends Biochem Sci 2002 27 339 44

12114022

Norgauer

Idzko

Panther

Xeroderma pigmentosum

Eur J Dermatol 2003 13 4 9

12609773

Van Steeg

Kraemer

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Mol Med Today 1999 5 86 94

10200950

Rybanska

Pirsel

Involvement of the nucleotide excision repair proteins in the removal of oxidative DNA base damage in mammalian cells

Neoplasma 2003 50 389 95

14689058

Satoh

Jones

Wood

DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions

Proc Natl Acad Sci USA 1993 90 6335 9

8327515

Coin

Auriol

Tapias

Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity

EMBO J 2004 23 4835 46

15549133

Wakasugi

Sancar

Assembly, subunit composition, and footprint of human DNA repair excision nuclease

Proc Natl Acad Sci USA 1998 95 6669 74

9618470

Imoto

Boyle

Influence of XPB helicase on recruitment and redistribution of nucleotide excision repair proteins at sites of UV-induced DNA damage

DNA Repair 2007 6 1359 70

17509950

Akoulitchev

Makela

Weinberg

Requirement for TFIIH kinase activity in transcription by RNA polymerase II

Nature 1995 377 557 60

7566158

Lehmann

Nucleotide excision repair and the link with transcription

Trends Biochem Sci 1995 20 402 5

8533152

Crabbe

Jauch

Naeger

Telomere dysfunction as a cause of genomic instability in Werner syndrome

Proc Natl Acad Sci USA 2007 104 2205 10

17284601

Shen

Kugan

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

Mol Cell Biol 2004 24 8437 46

15367665

Lee

Harrigan

Opresko

Pathways and functions of the Werner syndrome protein

Mech Ageing Dev 2005 126 79 86

15610765

Opresko

Mason

Podell

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates

J Biol Chem 2005 280 32069 80

16030011

Munoz

Blanco

Flores

XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer

Nat Genet 2005 37 1063 71

16142233

Vermeulen

Scott

Rodgers

Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

Am J Hum Genet 1994 54 191 200

8304337

Khan

Jaspers

Phenotypic heterogeneity in the XPB DNA helicase gene (ERCC3): xeroderma pigmentosum without and with Cockayne syndrome

Hum Mutat 2006 27 1092 103

16947863

Hande

Boei

Natarajan

Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridization. II. Micronuclei

Int J Radiat Biol 1996 70 375 83

8862448

Hande

Boei

Natarajan

Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridization. III. Chromosome malsegregation/aneuploidy

Mutagenesis 1997 12 125 31

9175636

Newman

Banerjee

Fang

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells

J Cell Physiol 2008 214 796 809

17849448

Hande

Samper

Lansdorp

Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice

J Cell Biol 1999 144 589 601

10037783

Greenwood

Hill

Sun

Population doubling: a simple and more accurate estimation of cell growth suppression in the in vitro assay for chromosomal aberrations that reduces irrelevant positive results

Environ Mol Mutagen 2004 43 36 44

14743344

Barzilai

Yamamoto

DNA damage responses to oxidative stress

DNA Repair 2004 3 1109 15

15279799

Spillare

Wang

Von

Redundancy of DNA helicases in p53-mediated apoptosis

Oncogene 2006 25 2119 23

16288211

Bernstein

Payne

DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Mutat Res 2002 511 145 78

12052432

Marini

Nardo

Giannattasio

DNA nucleotide excision repair-dependent signaling to checkpoint activation

Proc Natl Acad Sci USA 2006 103 17325 30

17088560

Collins

The comet assay for DNA damage and repair: principles, applications, and limitations

Mol.Biotechnol 2004 26 249 61

15004294

Duan

Zhang

Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening

Int J Biochem Cell Biol 2005 37 1407 20

15833273

Jan

Wang

8-Oxoguanine DNA glycosylase and MutY homolog are involved in the incision of arsenite-induced DNA adducts

Toxicol Sci 2007 95 376 82

17101720

Coin

Oksenych

Egly

Distinct roles for the XPB/p52 and XPD/p44 subcomplexes of TFIIH in damaged DNA opening during nucleotide excision repair

Mol Cell 2007 26 245 56

17466626

McKinnon

ATM and ataxia telangiectasia

EMBO Rep 2004 5 772 6

15289825

Orren

Werner syndrome: molecular insights into the relationships between defective DNA metabolism, genomic instability, cancer and aging

Front Biosci 2006 11 2657 71

16720342

Mor-Gueret

Bloom syndrome, genomic instability and cancer: the SOS-like hypothesis

Cancer Lett 2006 236 1 12

15950375

Slijepcevic

Hande

Bouffler

Telomere length, chromatin structure and chromosome fusigenic potential

Chromosoma 1997 106 413 21

9391214

Fenech

The in vitro micronucleus technique

Mutat Res 2000 455 81 95

11113469

Tchirkov

Lansdorp

Role of oxidative stress in telomere shortening in cultured fibroblasts from normal individuals and patients with ataxia-telangiectasia

Hum Mol Genet 2003 12 227 32

12554677

Chen

Ames

Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells

Proc Natl Acad Sci USA 1994 91 4130 4

8183882

Petersen

Saretzki

Von Zglinicki

Preferential accumulation of single-stranded regions in telomeres of human fibroblasts

Exp Cell Res 1998 239 152 60

9511733

Hande

DNA repair factors and telomere-chromosome integrity in mammalian cells

Cytogenet Genome Res 2004 104 116 122

15162024

Crabbe

Verdun

Haggblom

Defective telomere lagging strand synthesis in cells lacking WRN helicase activity

Science 2004 306 1951 3

15591207

Leveillard

Andera

Bissonnette

Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations

EMBO J 1996 15 1615 24

8612585

Wang

Yeh

Schaeffer

p53 modulation of TFIIH-associated nucleotide excision repair activity

Nat Genet 1995 10 188 95

7663514