Deficiency in the repair of UV-induced DNA damage in human skin fibroblasts compromised for the ATM gene
Mohammed A. Hannan1,*,
Ali Hellani2,
Fahad M. Al-Khodairy1,
Mohammed Kunhi1,
Yunis Siddiqui1,
Noujud Al-Yussef1,
Nancy Pangue-Cruz1,
Monica Siewertsen1,
Mohammed N. Al-Ahdal1 and
Abdelilah Aboussekhra1,3
1 King Faisal Specialist Hospital and Research Center Biological and Medical Research Department, MBC 03-66 and
2 Department of Pathology and Laboratory Medicine, ART Section, MBC 10, PO Box 3354, Riyadh 11211, Saudi Arabia
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Abstract
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Ataxia-telangiectasia (A-T), is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. A-T cells exhibit a greatly decreased survival and a reduction in DNA synthesis inhibition as well as p53 induction in response to ionizing radiation. Occasionally, some strains of A-T cells have been reported to manifest a slightly enhanced sensitivity with no consistent observations of a deficiency in either cell cycle control or the repair of DNA damage after treatment with ultraviolet (UV) light. In the present study it is shown that skin fibroblasts from four A-T patients, compared with the control, display enhanced sensitivity to the killing effect of UV-light, moderate radioresistant DNA synthesis, and a reduction in viral recovery in the host cell reactivation (HCR) assay. PCR based analysis indicated that three of these UV-sensitive A-T cell strains bear a large deletion in the ATM gene, and no ATM polypeptide was detected in their cell free extracts. Moreover, it is shown that, in non-replicative conditions, these A-T cells are less efficient than normal cells in repairing the T4 endonuclease V sensitive sites. These results constitute the first clear evidence showing the deficiency of A-T cells in the repair of UV-induced DNA damage, and provide further information on the relationship between cell cycle control and DNA repair in human cells.
Abbreviations: A-T, ataxia-telangiectasia; CPDs, cyclobutane pyrimidine dimers; EMEM, Eagles minimal essential medium; HCR, host cell reactivation; HSV, herpes simplex virus; IR, ionizing radiation; LZ, leucine zipper; NER, nucleotide excision repair; P1-3 K, phosphatidylinositol-3 kinase; RDS, radioresistant DNA synthesis; UDS, unscheduled DNA synthesis.
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Introduction
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Ataxia-telangiectasia (A-T) is an inherited multisystem disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, hypersensitivity to radiotherapy and predisposition to malignancies (1,2). A-T cells in vitro are known to exhibit a greatly enhanced sensitivity to the killing effects of ionizing radiation (IR), chromosomal instability, radioresistant DNA synthesis (RDS), deficiency in the activation of the tumor suppressor protein p53 after gammairradiation and possible defects in DNA repair (1,35). They are also compromised for the G1/S, S and G2/M cell cycle checkpoints in response to IR (1). ATM, the gene mutated in A-T cells, codes for a 350 kDa protein kinase, belonging to a family of high molecular weight, phosphatidylinositol-3 kinase (PI-3 K)-related proteins, involved in eukaryotic cell cycle control, DNA repair and recombination (68). In response to DNA damage, ATM protein phosphorylates several proteins including the main tumor suppressor p53 (912). In addition, ATM has DNA binding properties and might therefore function directly in DNA damage detection (13). Despite the fact that A-T cells show abnormal responses only to IR or chemicals acting like IR, certain A-T strains were shown to be quite sensitive to the lethal effects of mid UV (313 mm) and only slightly sensitive to far UV (254 mm) radiation (1416). However, the levels of both DNA synthesis inhibition and unscheduled DNA synthesis (UDS) after UV irradiation have been found to be similar in A-T and normal cells (17,18). No studies, so far, showed clear evidence of a deficiency in A-T cells to repair UV-induced DNA damage. In the course of our studies on radiosensitivity of body cells from cancer patients and cancer prone disorders, we observed that four Saudi A-Ts unexpectedly exhibited enhanced sensitivity to UV (254 mm) irradiation in a colony-forming assay. This observation stimulated further studies on UV-induced DNA synthesis inhibition and p53 accumulation in these A-T cells as well as on their capacity to repair UV-induced DNA damage. The A-T cells used herein did show less than normal levels of UV-induced DNA synthesis inhibition and a reduction in the survival of UV-damaged virus in the host cell reactivation (HCR) assay, while the level of their p53 was up-regulated upon UV-irradiation. No ATM protein was detected by immunoblotting in three cell strains derived from two different families. These strains bear a deletion spanning a large segment within the ATM gene. The most interesting observation of this study was that these UV-sensitive A-T cells were also found to be somewhat deficient in the excision of UV induced cyclobutane pyrimidine dimers (CPDs) in non-replicative conditions, an observation connecting A-T cells for the first time with UV-induced DNA damage repair.
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Materials and methods
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Cell cultures and cell survival analysis
Skin biopsies were obtained (upon consent) from (Saudi) A-T patients (clinically well-characterized for the classical hallmarks of the disease) and a healthy subject with no known history of genetic diseases. Fibroblast cell strains developed from these cases were grown in Eagles minimal essential medium (EMEM). After harvesting, cells were stored in liquid nitrogen. Two skin fibroblast strains representing the classical xeroderma pigmentosum (XP-A) syndrome were used here as positive controls for UV-sensitivity. CRL 1223 was obtained from the American Type Culture Collection, while the other one was developed from the skin biopsy of a Saudi XP patient and found to belong to the XP-A group in a complementation analysis. MRC-5 and AT5BI were used as a non-Saudi normal and A-T fibroblast strains, respectively.
For studies on cell survival after UV irradiation, fibroblast cells (passage 810) grown to confluence in Hams F12 medium with supplements, were harvested (after trypsinization), diluted and appropriate aliquots of the cell suspension were plated into 100 mm tissue culture dishes (each containing 10 ml medium). After incubation (at 37°C in a humidified incubator with 5% CO2, 95% air) for 24 h, the medium was removed and the monolayers in dishes (without lids) were exposed to a germicidal UV lamp (254 nm) at a fixed distance for different periods of time. The UV dosimetry was performed using a UV meter (Spectronics Corporation, NY). For each UV dose (012 Jm2) a set of four dishes were used, while the same number of dishes, not exposed to UV, served as controls. To each of the unirradiated and irradiated dishes, 15 ml of medium containing 60 000 feeder cells (normal human fibroblasts inactivated with 50 Gy gamma rays) were added and the dishes were incubated for 3 weeks with a weekly change of medium. Cells were then washed with a 0.9% NaCl solution, stained with crystal violet, washed with water and then colonies (>50 cells) were counted. Survival curves for each cell strain were constructed by considering plating efficiency and estimating the percent colony formation after each dose of UV irradiation compared with the respective unirradiated control.
Determination of DNA synthesis inhibition
Fibroblasts grown to confluence in F12 medium were harvested and replated at a density of 2 x 105 cells/dish. Following incubation for 24 h, the growth medium was removed and the dishes were exposed to 320 Jm2 of UV light. Following irradiation the medium (5 ml/dish) was added and the dishes were incubated for 30 min, and then [3H]-thymidine (sp. act. 5 Ci/mM) was added (5 mCi/dish) and incubation continued for another 2 h. Cells were then washed 3x with PBS, trypsinized and harvested onto glass fiber filters which were counted for radioactivity in a scintillation counter. Inhibition of DNA synthesis in irradiated cells was determined as percent of counts/min compared with those in the respective unirradiated controls analyzed in the same manner.
Host cell reactivation
HCR has been successfully used to detect cancer-prone disorders including xeroderma pigmentosum which are unable to repair UV-induced pyrimidine dimers (19). We used the assay described by Abrahams et al. (20) to examine the capability of A-T cells (compared with the cells from XP and healthy subjects) to reactivate the UV irradiated (50150 Jm2) F strain of herpes simplex virus (HSV). Briefly, monolayers of different fibroblast strains were infected with unirradiated and irradiated virus and incubated for 3 h. Monolayers were then washed with PBS, trypsinized, harvested and mixed with Vero (African green monkey kidney) cells (2.5 x 106) in dishes each containing EMEM + 10% foetal bovine serum. After incubation for 5 h, growth medium was removed and an agar (0.45%) overlay was added to each dish. Dishes with solidified agar were incubated for 34 days and then plaques were counted in dishes representing different UV doses. Percent virus survival resulting from initial infection into a host cell strain (A-T, XP or normal) was determined by comparing the numbers of plaques formed by the irradiated and unirradiated virus infected into the same strain.
Analysis of p53 and ATM proteins
Western blot technique was used to detect the p53 protein from unirradiated and UV (5 Jm2) irradiated cells of each fibroblast strain (A-T and controls), and the ATM protein from AT2HA, AT4HA and HeLa cells. Cells were lysed and treated with protease inhibitors. Cell lysates were quantitated using the Bradford color assay (Bio-Rad). 30 µg (for p53) and 100 µg (for ATM) of protein per lane were loaded on 10% SDSPAGE gels. The acrylamide:bisacrylamide ratio was 29:1 and 100:1 for p53 and ATM, respectively. After transferring the separated proteins from gel to Immobilon membranes, the non-specific binding sites were blocked by incubating for 1 h in Tris-buffered saline Tween at room temperature. The membranes were then probed with anti-p53 antibody (DO-7, PharMingen, San Diego, CA), anti-ATM antibody (H-248, Santa Cruz, CA) or anti-actin antibody (used as internal control) (Santa Cruz, CA) and subsequently with horseradish peroxidase-labelled secondary antibody. Immunoreactive bands were visualized using the super Signal system (Pierce, Rockford, IL).
Analysis of global repair by T4 endonuclease V/alkaline agarose gel electrophoresis
Fibroblasts grown to confluence were either sham irradiated (sample used as non-irradiated control) or UV-irradiated with a dose of 5 Jm2. Cells were re-incubated to allow DNA repair for different periods of time following which samples were collected for genomic DNA purification and flow cytometric analysis. Purified DNA (5 µg) was then either mock treated or digested with T4 endonuclease V, which specifically incises the unrepaired UV-induced CPDs in DNA. T4 endonuclease V cleavage products were separated on a 1.5% alkaline agarose gel for 16 h at 30 V in the cold room (4°C). The gel was then neutralized and stained with ethidium bromide (21). The gel was photographed using a Polaroid camera and the data were transferred to computer for quantification using the IP Lab Gel program.
T4 endonuclease V treatment randomly incises UV-irradiated DNA, producing a smear indicating the presence of DNA with different molecular weights. A shift of small molecular weight DNA toward larger molecular weight indicates the removal of CPDs and hence the occurrence of DNA repair.
DNA repair was quantitated as follows:
- The signal corresponding to high molecular weight DNA (intact fragment) was divided by the whole lane (+T4 endonuclease V) to yield a value normalized with respect to DNA content in that lane.
- The background signal was assessed from the same region in the corresponding (T4 endonuclease V) lane and divided by the signal of the whole lane.
- The normalized background was subtracted from the normalized signal.
- To generate repair curves, the values were normalized with respect to the initial damage (0 h, 100% PDs and 0% repair).
Flow cytometry
State and progression of cells in the cell cycle were monitored by flow cytometry. Approximately 106 cells from each sample were washed with PBS, harvested, and fixed in 70% ethanol. Cells were then treated with sodium citrate, RNase and stained with propidium iodide. Flow cytometry was carried out on a Becton Dickinson FACScan to determine the cell cycle distribution in the fibroblast strains under study.
PCR procedure for mutation detection
Genomic DNA from different A-T cell lines was used to detect deletions in the ATM gene. Primers and PCR conditions were used as previously described (22), and BC1 gene was utilized as internal control. PCR products were analyzed on a 2% ethidium bromide stained agarose gel and visualized under UV light.
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Results
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UV-sensitivity of skin fibroblasts from A-T patients
Skin fibroblast cell strains developed from four A-T patients, belonging to three different families, exhibiting classical features of the disease were analyzed for their survival to the killing effect of UV light. These cells exhibit all classical features of classical A-T cells including high sensitivity to IR (23). Figure 1
illustrates the cell survival data for fibroblasts representing one healthy subject (three more controls showed similar results), four A-T homozygotes and a highly UV-sensitive xeroderma pigmentosum (XP). The A-T cells exhibited a remarkably enhanced sensitivity to germicidal UV (254 nm) compared with the control (D10 values = doses resulting in 10% survival being 3.86.5 Jm2 for A-Ts, 1.04 Jm2 for XP and 12 Jm2 for control). Obviously, the A-Ts showed an intermediate response relative to the control and the XP fibroblasts. However, under the same experimental conditions, a reference A-T cell strain (AT5BI) exhibited a normal UV response (Figure 1
).

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Fig. 1. Effect of UV-light on colony-forming ability of A-T, XP and normal cells. Cell survival curves represent data from experiments carried out in triplicate with the bars showing upper and lower values.
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Reduced UV-induced DNA synthesis inhibition in the A-T cells
To find whether or not these A-T cells would display a radioresistant DNA synthesis in response to UV light, the incorporation of [3H]-thymidine was assessed in UV-irradiated A-T and normal cells as described in Materials and methods. Figure 2A
shows that in response to 9 Jm2,
20% of [3H]-thymidine was incorporated in normal cells while 5070% were incorporated in the different A-T cells in relation to their respective unirradiated controls. These data clearly showed that the levels of UV-induced DNA synthesis inhibition were less in A-T cells than in normal cells, indicating the occurrence of RDS in A-T cells after UV-irradiation. However, it is noteworthy that this resistance to UV-induced inhibition of DNA synthesis was less severe than what was observed in the same A-T cells after gamma-irradiation (Figure 2B
).


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Fig. 2. UV-induced DNA synthesis inhibition in A-T and normal cells. [3H]thymidine was added to the cells 30 min after irradiation and were reincubated for 2 h. Percentage of [3H]thymidine incorporated in irradiated cells was determined as percent of counts/min compared with those in the respective unirradiated controls. Bars represent the variation in data obtained from experiments in triplicates, with UV (A). The data from a single experiment with two normals and an A-T homozygote after gamma-irradiation (B).
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Deficiency in the repair of UV-induced DNA damage in A-T cells
The UV sensitivity of these A-T cells may be due to a defect in the post-irradiation processing of DNA and/or to a defect in the repair of UV-induced DNA damage.
To test the ability of these A-T cells to repair UV-induced DNA damage, we first made use of the HCR assay. This technique represents an indirect way of evaluating cellular capacity in removing photolesions from viral DNA hosted in non-irradiated cells. Figure 3
shows that all A-Ts were less capable of viral reactivation than the control, while the XP cells were the most deficient in this respect. These data suggested that the A-T cells were moderately deficient in the repair of UV-induced DNA damage, corroborating the cell survival data.

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Fig. 3. Host cell reactivation of UV-irradiated HSV in A-T, XP and normal cells. Monolayers of different fibroblast strains were infected with unirradiated and UV-irradiated F strain of HSV and incubated for 3 h. Plaque-forming ability of the virus was analyzed in Vero cells. Bars represent variation in survival data obtained in duplicate experiments.
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Analysis of the status of the ATM gene in these UV-sensitive A-T strains
To make sure that the A-T cells analyzed here bear a mutation in the ATM gene, we first made use of the amplification by PCR using primers specific for different ATM exons. In this regard, genomic DNA was extracted from normal cells, AT2HA and AT4HA strains, as well as from cells derived from the father and the mother of the AT2HA patient, utilized as a control. Figure 4A
shows the result of the amplification indicating the presence of the exons 17 and 18, in all the cells tested. However, exon 19 is absent in AT2HA and AT4HA, while present in the control cell lines derived from the parents and the normal strain. A similar result was obtained for exons 20, 39, 52 and 65 (data not shown). This clearly indicates the presence of a large deletion in the ATM gene, extending from exon 19 to exon 65.

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Fig. 4. Status of ATM gene and protein in A-T patients. The status of the ATM gene and protein in the A-T cell strains was analyzed using both, amplification by PCR from genomic DNA, and immunoblotting techniques. (A) Genomic DNA from the indicated cell lines was purified and the shown ATM exons as well as the exon 18 of the BC1 gene (internal control) were amplified by PCR. The resulting fragments were separated on a 2% agarose gel. (B) Whole cell extracts were prepared from the indicated cell strains. After SDSPAGE, ATM protein was detected by immunoblotting using anti-ATM antibody. As a control for loading of proteins the blot was reprobed with an antibody raised against ß-actin.
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As expected, the immunoblotting analysis using an anti-ATM-antibody, did not show the corresponding polypeptide in cell free extracts derived from the AT2HA and AT4HA strains, while the ATM gene product was detected in the HeLa cells (Figure 4B
). This result corroborates the previous information obtained by PCR and demonstrates the presence of a gross deletion in the ATM gene in the AT2HA and AT4HA cell strains. AT3HA cell line bears the same deletion, these cells derive from a patient that belongs to the AT2HA family. However, such deletion was not detected in the AT1HA cell strain (data not shown).
Cells derived from A-T patients are deficient in the removal of UV-induced CPDs
To further characterize the impairment of these cells in repairing UV-induced DNA damage, use was made of T4 endonuclease V which cuts DNA at the CPD sites. Since A-T cells are known to be defective in DNA damage-dependent cellular growth arrest, which is supposed to provide time for efficient DNA repair, the removal of CPDs was analyzed in non-replicating cells, in order to avoid the effect of cell cycle checkpoint defect on DNA repair efficiency. Table I
shows a flow cytometric analysis performed simultaneously with DNA repair experiments indicating that the cell cycle distribution in normal as well as A-T cells did not significantly change during the 24 h of repair study. After UV-treatment, genomic DNA was purified and analyzed at different time-points for T4-endonuclease V activity, which indicates the presence of non-repaired CPDs. Figure 5A
shows the repair ability of A-T, XP-A and normal cell lines analyzed by this method. A visual inspection of the alkaline gels indicated that the A-T cells could remove the T4 endonuclease V sensitive sites transforming the low molecular weight to high molecular weight DNA faster than the XP-A cells, however, they were less efficient in this respect than the normal cells. A similar intermediate response was obtained with two other A-T cell strains as shown in Figure 5B
that presents the quantitative data derived from the alkaline gels. We observed that all the UV-sensitive A-T cells had an altered kinetics of pyrimidine dimer removal, occupying an intermediate position between the control and the XP-A cells in general. The repair kinetics shown in Figure 5B
indicated that at 7 h post-irradiation, only
30% of CPDs were removed from the A-T cells when
80% were excised from the normal cells. Interestingly, the A-T cells, after prolonged incubation (24 h), reached the same level of repair as the control, which was not the case with the XP-A cells. An analysis of the removal of T4 endo V sensitive sites from the reference AT5BI cell strain, however, indicated a normal rate of repair as in the control cells (data not shown), consistent with its survival response to UV.


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Fig. 5. Global DNA repair in normal, A-T and XP-A fibroblasts. Confluent cells were irradiated with a UV dose of 5 Jm2 and allowed to undergo DNA repair. DNA was then isolated at different time periods, mock treated or digested with T4 endonuclease V, and separated on 1.5% alkaline agarose gel (A). CPD repair kinetics quantified from the alkaline gel experiments were conducted in triplicate. , Saudi normal; , MRC-5; , , , 3 A-Ts; , CRL 1223; , Saudi XPA (B).
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Altogether, these results represent the first clear and direct evidence that these A-T cells are not as efficient as normal cells in removing UV-induced CPDs, which would account for the enhanced lethal effects of UV light observed in the cell survival assay with the cells used in this study.
p53 induction in UV-sensitive A-T cells
A-T cells exhibit a lack of or prolonged delay in induction of p53 in response to IR. It was therefore important to test whether the UV-sensitive A-T cells exhibited an abnormal p53 induction in response to UV light. Figure 6
shows that the p53 levels increased remarkably in both AT2HA and AT4HA cell lines and normal cell, indicating that these A-T cells are not compromised for p53 induction in response to UV light, although they showed sensitivity to this genotoxic agent. Likewise, UV-dependent p21 induction is not affected in these cells (data not shown).

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Fig. 6. Effect of UV-light on the induction of p53 in A-T and normal fibroblasts. Cells were either sham treated () or UV-irradiated with a dose of 5 Jm2 (+) and harvested after 4 h of incubation. Cell extracts were prepared from two A-T cell strains and one normal cell line (C), and analyzed by western blotting using anti-p53 monoclonal antibody. 30 mg of total proteins was used in each lane.
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Discussion
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Cells respond to DNA damage by coordinating the activation of different processes which have evolved to prevent genomic instability. Transient arrests of the cell cycle and the activation of different DNA repair processes constitute the main defense mechanisms that all living cells use to maintain the integrity of their genetic information (24). The tumor suppressor gene p53 is involved in both cell cycle arrest and DNA repair, indicating that these two responses may be controlled by a common regulatory gene/pathway (2530). Likewise, the S. cerevisiae RAD9 gene, known to play a major role in cell cycle checkpoints, is also involved in excision repair of UV-induced DNA damage (31), which confirm this notion of co-regulation of the cellular responses to DNA damage in different organisms. The A-T mutated gene, ATM, connected with cancer susceptibility is also required for cellular growth arrest in response to DNA damage (1). Here we present evidence that the ATM cell cycle checkpoint gene may also be involved in DNA repair of UV-induced DNA damage.
During the characterization of four fibroblasts derived from Saudi A-T patients presented in this report, we surprisingly found all of them to be remarkably sensitive to UV-light, though not to the same extent as UV-sensitive XP cells. The fibroblasts used in this study were derived from patients with classical features of the A-T disease including cerebellar ataxia, ocular telangiectasia, chromosomal instability, immunodeficiency and cellular hypersensitivity to ionizing radiation. Moreover, these Saudi A-T cells show all the features of the classical A-T cells, including high sensitivity and the non-inducibility of p53 in response to gamma-rays. It has been therefore clear that these cells are deficient in the ATM function. This has indeed been demonstrated by showing that the ATM protein is absent from the whole cell extracts prepared from three cell lines (AT2HA, AT3HA and AT4HA) derived from two different and unrelated families (Figure 4B
). The analysis of the status of the ATM gene by PCR amplification of several exons showed that the gene bears a gross deletion that extends from exon 19 to exon 65. It is known that most of the ATM mutations truncate or destabilize the ATM protein. Several small and large deletions were previously reported and represent a large proportion of mutations found in the ATM gene (2). However, the present deletion that encompasses 48 exons of the ATM gene is, to our knowledge, a new ATM mutation, representing the largest ATM-deletion ever reported. Another gross deletion spanning several kilobases has been previously identified in a Palestinian-Arab A-T family (32), but it is different from the deletion described in the present A-T patients. Cells from the third family analyzed here do not bear the same deletion, indicating the presence of different ATM mutation in this population.
In addition to UV sensitivity, a moderate decrease in viral recovery was observed in the HCR assay, suggesting a deficiency in the repair of UV-induced lethal lesions in these A-T cells. Moreover, an altered kinetics for the removal of UV-induced T4 endonuclease V sensitive sites from the DNA of non-replicating A-T cells did indeed confirm a partial defect in the excision repair of CPDs in the Saudi A-Ts. These results constitute the only evidence of a deficiency in the repair of UV-induced DNA damage in A-T cells. Since most of these cells (>70%) were in G0/G1 phase of the cell cycle during the whole repair period, the observed reduction in the removal of CPDs is likely to be due to a defect in DNA repair per se and not to an impairment of cell cycle checkpoint. Taken together, these data suggest that these A-T cells may be deficient in NER, and prompt us to ask the following important question: Is this defect ATM-related or due to the occurrence of another gene co-segregating with ATM and accounting for UV sensitivity? The latter possibility is less probable for the following reasons:
- The four A-T cells used here were derived from three different and unrelated families, so the probability of having two mutations in two different genes co-segregating in three independent families is very low.
- In addition to their partial defect in DNA repair of UV damage, the present A-T strains exhibit a less than normal level of UV-induced DNA synthesis inhibition, corresponding to a defect in the arrest in S phase following UV irradiation. As for UV sensitivity, no UV-dependent RDS has been previously reported for the A-T mutants. This resistance is not as severe as in response to IR (Figure 2
), but it is significant and consistent with the moderate defect in the removal of UV-induced DNA damage. This also parallels the high level of DNA strand breaks and the subsequent generation of chromosome aberrations observed in UV-treated A-T cells (3336). The fact that RDS is one important feature of A-T cells, indicates that UV sensitivity as well as UV-dependent RDS observed in the Saudi A-T cells are ATM-related.
- The patients do not present any additional symptoms other than those that characterize the A-T patients.
- Other A-T strains were found to be slightly sensitive to the lethal effects of UV light (1416), and cisplatin (37).
- In these Saudi A-T families two different AT mutations were found.
Together these observations indicate that the observed defect in the processing of UV damage is related to the ATM gene. Therefore, what could be the role of ATM in excision repair of UV-induced DNA damage? The ATM gene product may not be essential for the core nucleotide excision repair (NER) reaction since it was included neither in the excision reaction (38) nor in the whole process (39) when reconstituted in vitro using pure components. In addition, the repair kinetics in the present A-T cells was slower compared with normal cells (after 7 h of repair
80% of CPDs were removed from the genome of normal cells but in the A-T cells only 30% were repaired). However, after 24 h of repair the A-T cells reached the same level as normal cells. Therefore, these mutants display a slower repair of UV-induced DNA damage. This may be due to a delay in the damage recognition and/or activation of signaling pathways rather than a defect in the repair (enzymes) process per se. Purified ATM protein has a DNA binding activity (13), and since ATM is also a phosphoprotein, it is conceivable that ATM gene product may activate some proteins, such as RPA, implicated in the repair of UV-induced DNA lesions. RPA is indeed hyperphosphorylated in normal cells in response to both IR and UV light (1). Both ATM and ATM-related protein Mec1 are required for UV-induced hyperphosphorylation of the NER protein RPA in human and S.cerevisiae, respectively (40,41). Thereby, RPA could make the liaison between ATM and the NER process.
At this stage we cannot rule out the possibility that ATM may modulate somehow the transcription coupled repair pathway of the NER mechanism. This pathway allows a faster repair of transcribed genes and strands (42,43). When defective, the repair kinetics becomes slower (this hypothesis is under investigation).
Alternatively, it is possible that the present A-T mutants possess a stable truncated ATM protein formed from the 18 exons that were not deleted. This N-terminal part of the ATM protein could have a dominant negative effect that sensitizes cells to the killing effect of UV damage. Indeed, the ATM N-terminal part that contains the leucine zipper (LZ) domain has a dominant negative effect, that sensitizes normal cells to IR (44). However, it is not known whether or not the expression of this fragment has an effect on cellular resistance to UV light. Although the LZ domain that is thought to be responsible for the observed dominant effect is absent in the present UV-sensitive A-T cells, it is still possible that the remaining ATM polypeptide could affect the cellular response to UV damage. Interestingly, despite the fact that the anti-ATM antibody that has been utilized in this study has been directed towards the N-terminal part of the protein, we did not detect any polypeptide of
100 kDa in the extracts prepared from these A-T cells (Figure 4B
).
IR-induced activation and stabilization of p53 require a normal ATM protein (2), which appears to have only a minor role in UV response, since p53 induction and phosphorylation were found to be normal in ATM-deficient cells (45). When treated with UV-light, the A-T cells analyzed here also exhibited a normal induction of p53 protein. Likewise, A-T sensitive lymphoblastoid cells to cisplatin showed normal induction of p53 by this genotoxic agent (37). Thereby, the sensitivity to UV light or cisplatin, and the repair defect observed in the present A-T cells seem to be independent of p53 up-regulation.
In summary, this study shows that A-T cells from Saudi patients bear a unique gross deletion in the ATM gene, and are defective in the removal of UV-induced DNA damage. This provides the first clear evidence that the ATM gene may be implicated in the cellular response to this carcinogenic agent. Although the present observations are of great importance for unraveling the complex phenotypegenotype connection in the A-T cells, more studies are needed to further characterize the present A-T mutants and investigate the relationship between DNA repair and cell cycle checkpoints in response to DNA photolesions.
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Notes
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* Present address: Environmental Health Science Bureau, Environmental Health Centre, HECS, Tunneys Pasture, Ottawa, ON K2M OL2, Canada. 
3 To whom correspondence should be addressed Email: aboussekhra{at}kfshrc.edu.sa 
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Acknowledgments
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The studies were supported by the King Faisal Specialist Hospital and Research Center, under the RAC project no. 93-0009. The authors thank Bayani L.Caracas for his help with the figures.
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Received February 28, 2001;
accepted July 1, 2002.