Carcinogenesis vol.23 no.6 pp.959–965, 2002
DNA damage responses protect xeroderma pigmentosum variant from UVC-induced clastogenesis
Marila Cordeiro-Stone1,2,3,5, Alexandra Frank1, Miriam Bryant1, Ikechukwu Oguejiofor1, Stephanie B.Hatch1, Lisa D.McDaniel4 and William K.Kaufmann1,2,3 1Department of Pathology and Laboratory Medicine, 2Lineberger Comprehensive Cancer Center, 3Center for Environmental Health and Susceptibility, University of NC at Chapel Hill, Chapel Hill, NC 27599– 7525 and 4McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390–8591, USA 5To
whom correspondence should be addressed Email:
[email protected]
Lack of DNA polymerase η and the attendant defect in bypass replication of pyrimidine dimers induced in DNA by ultraviolet light (UV) underlie the enhanced mutagenesis and carcinogenesis observed in xeroderma pigmentosum variant (XP-V). We investigated whether diploid XP-V fibroblasts growing in culture are also more susceptible to UV-induced clastogenesis than normal human fibroblasts (NHF). This study utilized diploid fibroblasts immortalized by the ectopic expression of human telomerase. The cell lines displayed checkpoint responses to DNA damage comparable with those measured in the parental strains. Shortly after exposure to low doses of UVC (≤4 J/m2), XP-V cells accumulated daughter strand gaps in excess of normal controls (>25-fold). Daughter strand gaps generated in UV-irradiated S phase cells are potential precursors of chromatid-type chromosomal aberrations. Nonetheless, chromatid-type chromosomal aberrations were only 1.5 to 2 times more abundant in XP-V than in NHF exposed to the same UVC dose. XP-V cells, however, displayed S phase delays at lower doses of UVC and for longer periods of time than NHF. These results support the hypothesis that aberrant DNA structures activate S phase checkpoint responses that increase the time available for postreplication repair. Alternatively, cells that cannot be properly repaired remain permanently arrested and never reach mitosis. These responses protect human cells from chromosomal aberrations, especially when other pathways, such as accurate lesion bypass, are lost. Introduction Checkpoint responses to DNA damage and pathways of DNA repair co-operate to maintain genomic stability (1). Structural alterations in DNA, such as double strand breaks and daughter strand gaps that result from the replication of damaged DNA are expected to induce checkpoint responses in S and G2 phases of the cell cycle to minimize the risk for transmission of chromosomal aberrations in mitosis (2). Xeroderma pigmentosum variant (XP-V) cells are prone to mutations induced Abbreviations: hTERT, human telomerase; NHF, normal human fibroblasts; UV, ultraviolet light; XP-V, xeroderma pigmentosum variant. © Oxford University Press
by ultraviolet light (3–5) and their donors are susceptible to skin cancers in areas exposed to the sun (6). The molecular defect underlying this disease was first recognized as interfering with the replication of DNA containing UVC-induced lesions (7). Accumulation of daughter strand gaps was inferred from the appearance of abnormally small nascent DNA strands within the first hour after irradiation (7–9). These cellular characteristics were later correlated with the deficiency of extracts from XP-V cells to catalyze the bypass replication of UVC-induced cyclobutane thymine dimers (10–13). Now it is known that XP-V cells carry mutations in the gene encoding pol η (14–16). This special DNA polymerase catalyzes efficient and accurate translesion synthesis across the cyclobutane thymine dimer (17–20). These phenotypic and molecular characteristics of XP-V identified this cell type as an interesting model system for investigating the relationship between bypass replication activity and activation of the S phase checkpoint, and how these two processes contribute to the maintenance of genetic stability in human cells. Fibroblasts immortalized by expression of human telomerase (hTERT) have stable telomeres, normal control of growth, and retain the phenotypic characteristics of their parental cells (21–26). The availability of diploid cell lines of normal human fibroblasts (NHF) and XP-V fibroblasts with indefinite life span and lacking transformation-associated changes facilitated the studies reported here. NHF and XP-V fibroblasts expressing hTERT responded to UVC damage as predicted by the presence or absence of pol η, respectively. Their proliferative capacity and fraction of cells in S phase (higher than displayed by the parental strains) offered the opportunity to evaluate checkpoint responses without the interference of oncogenic viral proteins, which were used often to immortalize human cell lines in the past. Furthermore, the hTERT-expressing fibroblasts made possible the re-evaluation of whether the absence of pol η in XP-V cells enhances the risk for accumulating UVC-induced chromosomal aberrations (27–30), in addition to increasing sensitivity to UVC-induced gene mutations (3–5). Materials and methods Cell culture The two telomerase-expressing cell lines, hTERT-GM01604 and hTERTGM02359, were derived in the laboratory of Dr Roger A.Schultz (UT Southwestern Medical Center) (25). They were made available to the CordeiroStone laboratory through a material transfer agreement with Geron Corporation. GM01604 originated from lung fibroblasts of an apparently normal fetus. The hTERT-GM01604 cell line is referred to throughout the text as NHF, the control cell line of normal human fibroblasts. hTERT-GM02359 (designated simply as XP-V in this report) originated from dermal fibroblasts obtained from the XP-V patient XP115LO and catalogued as GM02359 by the NIGMS Coriell Mutant Cell Repository. The parental XP-V cells harbor a nonsense mutation in the gene encoding pol η that leads to premature termination of the protein (15), so they lack any activity of this DNA polymerase. These cell lines have remained stable and free of Mycoplasma contamination beyond population doubling levels 200 (XP-V) and 370 (NHF). Cultures were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Saint Louis, MO) supplemented with 2⫻ the concentration of MEM non-essential amino acids (GIBCOBRL, Life Technologies, Grand Island, NY), 2 mM
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10–15% fetal calf serum (Hyclone Laboratories, Logan, UT). Cultures were maintained in Falcon® polystyrene tissue culture dishes (Becton Dickerson Labware, Franklin Lakes, NJ) at 37°C in a humidified atmosphere of 5% CO2 in air.
Karyotype Metaphase spreads were prepared by conventional methods. Chromosomes were banded at the 500-band level by trypsinization and Giemsa staining (31). Images were captured on a Nikon Microphot FXA microscope, equipped with an Optronics DEI-750 CCD video camera system, and attached to an Apple Power Macintosh G3 computer with a Scion CG-7 card. Image processing and analysis were done in Adobe Photoshop 5.5. PCR DNA purified from XP-V cells was amplified with primer sets (Research Genetics, Huntsville, AL) for polymorphic microsatellites mapping to either the p or the q arms of chromosome 8. Five loci were analyzed: D8S639 (8p), D8S311 (8p21-p11), D8S320 (8q), D8S347 (8q22.3-q24.3), and D8S348 (8q24.13-q24.3). PCR was carried out with forward primers that were endlabeled with [γ-33P]ATP and polynucleotide kinase (New England Biolabs). PCR products were then fractionated by gel electrophoresis (7% acrylamide, 7 M urea) and analyzed on a Fuji FLA-2000 Multifunctional Imaging System. Alkaline sucrose gradient centrifugation Logarithmically growing cells were plated at about 2.5⫻105 per 60 mm plate (two plates per treatment condition) and incubated for ~32 h in medium containing 10 nCi/ml of [14C]thymidine (53–59 mCi/mmol; ICN Radiochemicals, Irvine, CA) to label DNA uniformly. The radiolabeled medium was replaced with fresh medium and the cultures incubated overnight. For treatment with UVC (0–3 J/m2), the culture medium was removed and reserved, the plates were rinsed once with warm Hanks’ balanced saline solution (HBSS), drained and placed uncovered under a short-wave UV lamp emitting mostly 254 nm radiation (UVC). The incident fluence rate was 0.2 to 0.5 J/m2/s, as measured by an UV radiometer (UV Products, San Gabriel, CA). The reserved medium was added back and the cultures incubated for 30 min at 37°C, then pulse-labeled for 15 min with 25–50 µCi/ml of [3H]thymidine (50 Ci/mmol; ICN Radiochemicals). Cells were rinsed twice with ice-cold saline and scraped from the plates with a rubber policeman into 0.5 ml/plate of 0.1 M NaCl containing 0.01 M EDTA (pH 8). Cell suspensions from two identically treated plates were combined and passed five times through a 22-gauge needle to disrupt cell clumps. An aliquot of 0.5 ml of the cell suspension was added to the lysis layer (0.5 ml of 1 M NaOH, 0.02 M EDTA) on top of a 36 ml linear sucrose gradient (5–20%) containing 0.4 M NaOH, 2 M NaCl, 0.01 M EDTA. The gradients were left under standard fluorescent laboratory illumination for 1 h to facilitate cell lysis and DNA unwinding. The gradients were centrifuged in a Beckman SW28 rotor at 25 000 rpm and 20°C for 5 h. About 28 equal-volume fractions were collected from the bottom of the gradient. DNA-associated radioactivity in each gradient fraction was determined by scintillation counting after collecting acid-precipitable macromolecules onto glass filters. The 3H counts were corrected for spillover of 14C radioactivity. Determination of breaks in nascent DNA (daughter strand gaps) The number average molecular weight (Mn) of the nascent DNA pulse-labeled with [3H]thymidine was determined by the expression Mn ⫽ Σri/Σ(ri/Mi), where ri is the amount of 3H radioactivity in fraction i and Mi is the molecular weight of the DNA banding in fraction i. The alkaline sucrose gradients were previously calibrated with supercoiled and linear SV40 DNA (32) and Mi was determined from the Studier’s expression Si ⫽ 0.0528Mi0.400 (33). The number of breaks per 108 Da was then determined from the equation {(108/MnSham)*[(MnSham/MnTreated)-1]} (34). Breaks in 3H-labeled DNA were presumed to reflect daughter strand gaps in nascent DNA, created when replication forks failed to bypass UVC-induced template lesions. Flow cytometry Cells in logarithmic growth were seeded at 1⫻106 per 100 mm plate (two plates per condition) and incubated for 2 days. Cultures were exposed to UVC, or sham-treated in parallel. Incubation with 10 µM BrdU for 1 or 2 h occurred either immediately after irradiation or following increasing times of incubation in fresh medium, as indicated in figure legends. In experiments in which cells were irradiated with UVC (0 or 2 J/m2) and pulsed immediately with BrdU, the cultures were fed with fresh medium and harvested by trypsinization after increasing periods of time. Cells were resuspended in cold saline, fixed in 67% ethanol, then stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ), as described (35). Flow cytometric analyses were done on a FACScan station with Cyclops software (Becton Dickinson).
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Results Analyses of karyotype and heterozygosity We analyzed at least 25 Giemsa-banded metaphases from the two major cell lines used in this study (NHF: hTERT-GM01604 and XP-V: hTERT-GM02359). This confirmed that the NHF line retained its diploid karyotype. Among the XP-V metaphases, however, we discovered that 76% contained an extra chromosome 8 (47, XY ⫹ 8) and 24% were 46, XY. It is unclear at this point when the first trisomic-8 cell appeared in the XP-V population. Upon replating at low density and expanding isolated colonies, we identified XP-V clone 1B in which 100% of the cells contained 46, XY chromosomes. This clone is identified in the text as XPV-1B. PCR analysis of five polymorphic microsatellites (see Materials and methods) demonstrated that hTERT-GM02359 cells were heterozygous for three of them. The diploid clone (XPV-1B) retained heterozygosity for each of these three markers. These findings established that XPV-1B contained one maternal and one paternal chromosome 8. Effect of UVC on DNA synthesis The gold standard for inclusion in the XP-V complementation group, prior to the cloning of the XP-V gene, was the finding of an abnormal size distribution of nascent DNA in cells exposed to low doses of UVC. Figure 1 illustrates this differential effect of UVC on DNA synthesized 30–45 min after irradiation of hTERT-expressing NHF and XP-V. Note that the inhibition of incorporation of [3H]thymidine in NHF was primarily in nascent DNA of low molecular weight (fractions 13–22), even when the incident dose was increased to 2 J/m2 (Figure 1A–C). This finding reflects the S checkpoint response of inhibition of initiation of new replicons after DNA damage (36). Figure 1D illustrates the same phenomenon in XP-V cells that were irradiated with only 0.3 J/m2. Together these results confirm previous observations (36) that UVC induces inhibition of replicon initiation in both NHF and XP-V before severe inhibition of DNA strand growth is detected. At doses of UVC in the range of 1–2 J/m2, only a very mild inhibition of DNA strand growth (inhibition of incorporation of [3H]thymidine in large intermediates of DNA replication; fractions 5–13) was seen in the bypass-proficient NHF (Figure 1B,C). Contrast these results with the strong inhibition of DNA synthesis in large intermediates of DNA replication and accumulation of small nascent DNA fragments, when XP-V cells were exposed to 1 and 2 J/m2 of UVC (Figure 1E,F). This observation reflects the increased amount of daughter strand gaps created in newly replicated DNA (7,8) because of the XP-V defect in bypass replication of UVC-induced cyclobutane thymine dimers (10–13,17). The UVC-induced effects on DNA synthesis observed with the XP-V cell line expressing telomerase and reported here were identical to those previously obtained with the parental GM02359 (XP115LO) fibroblasts (9,36). These results were reproduced also with the diploid XPV-1B clone (results not shown). This clone represents an immortalized diploid cell line of the XP-V mutant XP115LO. Results identical to those illustrated in Figure 1 were observed with other hTERTexpressing human fibroblasts (not shown). They were derived in our laboratory from normal neonatal foreskin fibroblasts (NHF1) and the XP-V strain CRL1162 (XP4BE). The generation and characterization of these hTERT-expressing cell lines will be described elsewhere. The accumulation of daughter strand gaps during replication
UVC-induced clastogenesis in XP-V
Fig. 2. UVC-induced breaks in nascent DNA (daughter strand gaps). The average number of breaks per 108 Da of nascent DNA was determined as described in Materials and methods. We presume that these breaks reflect the formation of daughter strand gaps opposite template photoproducts that were not bypassed during DNA replication 30–45 min after exposure of fibroblasts to the indicated fluences of UVC. Data points represent experiments conducted with XP-V (filled symbols; the filled triangle indicates XPV-1B) and NHF (open circle). Fig. 1. Alkaline sucrose gradient profiles of nascent DNA in hTERTexpressing NHF and XP-V fibroblasts after exposure to increasing fluences of UVC. Logarithmically growing cultures were incubated with [14C]thymidine for at least two cell generations to label nuclear DNA uniformly and then overnight in non-radioactive medium. Cultures were sham-treated or exposed to 0.3 (A, D), 1 (B, E) or 2 (C, F) J/m2 of UVC. Afterwards, they were incubated for 30 min at 37°C, then pulse-labeled with [3H]thymidine for 15 min. Normalized 3H CPM represents the radioactivity from 3H in each fraction, corrected for 14C spillover, and divided by the total 14C radioactivity in the gradient, which is proportional to the number of cells. Sedimentation was from right to left. Open symbols depict size distributions of labeled nascent DNA in sham-treated populations and closed symbols the equivalent distributions in populations exposed to the indicated fluences of UVC.
of UVC-damaged DNA can be evaluated from the average molecular weight of the pulse-labeled nascent DNA in irradiated cells and sham-treated controls (Materials and methods) determined in velocity sedimentation experiments, such as those illustrated in Figure 1. As UVC fluence is increased, the number of daughter strand gaps also increases and the average molecular weight of the nascent DNA is reduced. In the absence of bypass replication, the accumulation of daughter strand breaks shortly after irradiation depends on the number of photoproducts encountered by the DNA replication machinery and correlates with the frequency of photoproducts deposited on template DNA. In Figure 2 we plotted the average number of breaks per 108 Da of nascent DNA against the UVC fluence used to irradiate XP-V cells. Linear regression through these data points defined a slope of 1.7 breaks per 108 Da per J/m2 UVC. This value compares well with estimates of the frequency of cyclobutane dimers in DNA (1 to 2 per 108 Da per J/m2 UVC) published by several laboratories (9,37). We also included in Figure 2 the frequency of breaks observed after irradiating NHF with 3 J/m2 UVC (the lowest dose for which a reduction in the average molecular weight of nascent DNA was detected in NHF). The value of 0.2 breaks per 108 Da in NHF is ~25-fold lower than that observed in XP-V cells exposed to the same UVC fluence (4.9 breaks per 108 Da).
Therefore, in addition to inhibition of replicon initiation and nucleotide excision repair (NER), S phase XP-V cells must rely on post-replication repair pathways, other than pol η-dependent translesion synthesis of UVC-induced thymine dimers, to seal a large number of daughter strand gaps and survive following exposure to UVC. Effect of UVC on the transition of hTERT-expressing fibroblasts through the cell cycle Logarithmically growing cultures were irradiated with 0, 2 or 4 J/m2 of UVC and pulsed for 2 h with BrdU, beginning 6 h after irradiation. Results of flow cytometric analyses (Figure 3) confirmed that XP-V cells were distinctly more sensitive to UVC than NHF. In several experiments of this type, we noticed that the mean FITC fluorescence (BrdU incorporation) decreased in the XP-V populations of S phase cells as the UVC fluence increased. Even 4–8 J/m2 did not inhibit BrdU incorporation in NHF as much as 1–2 J/m2 did in XP-V cells. This inhibition of DNA synthesis was most noticeable in cells that were in S phase at the time of irradiation (Figure 3F inset). The early S phase compartment in the irradiated XP-V populations included cells in which the incorporation of BrdU was equivalent to that seen in early S phase cells in the shamtreated cultures (compare panels D-F in Figure 3; see blackfilled area in Figure 3F inset). These cells were in G1 at the time of irradiation and nucleotide excision repair (NER) reduced their DNA lesion burden before they entered S phase. Next, we examined in more detail the response of hTERTexpressing fibroblasts that were sham-treated or irradiated with 2 J/m2 of UVC. We either incubated the cells in BrdUcontaining medium for 1 h, starting the pulse at different times after irradiation (Figure 4), or pulsed them during the first hour after the UVC treatment and incubated the cultures in fresh medium for extended periods of time (Figure 5). The first protocol displayed the UVC- and time-dependent changes in the relative distribution of cells in G1, S and G2 in the irradiated populations (Figure 4). Except perhaps for a slightly 961
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Fig. 4. Response time of hTERT-expressing fibroblasts to UVC. Log cultures of NHF and XP-V fibroblasts were treated with 0 (sham) or 2 J/m2 of UVC and pulsed with BrdU for 1 h at the indicated time intervals following irradiation. Cells were harvested at the end of the BrdU pulse. Bivariate flow cytometric analyses were performed as described in the legend to Figure 3.
Fig. 3. Log cultures of hTERT-expressing NHF (A–C) and XP-V (D–F) fibroblasts were exposed to the indicated fluences of UVC, incubated at 37°C for 6 h, and pulsed with BrdU (10 µM) for 2 h. Cells were harvested by trypsinization, fixed in 67% ethanol, and stained with propidium iodide and anti-BrdU FITC-conjugated antibody (Becton Dickinson, Franklin Lakes, NJ). Flow cytometric analyses were done on a FACScan station with Cyclops software (Becton Dickinson). In these profiles, the ordinate shows green fluorescence (FITC) relative to BrdU incorporation. DNA content is indicated by propidium iodide (red fluorescence) in the abscissa. Filled areas shown in the panel F inset indicate cells in S phase at the time of labeling with BrdU. These cells were either in G1 (black-filled area) or in S (grayfilled area) at the time the cultures were exposed to UVC.
lower BrdU incorporation by the irradiated S phase cells, we could not discern obvious differences in cell cycle distribution between sham and irradiated cultures during the first hour after treatment, either in NHF or XP-V fibroblasts. Over the next 12 h period we noticed a gradual, but substantial, reduction of DNA synthetic activity in XP-V cells in mid- to late-S phase (Figure 4). It is also clear that the S phase compartment in the XP-V population was gradually enriched with cells moving from G1 at apparently normal rates. Although the percentage of XP-V cells in S phase in sham-treated populations remained around 23% at all times, in the irradiated population this percentage increased from 23% during the first hour after 962
Fig. 5. hTERT-expressing NHF and XP-V fibroblasts were irradiated with either 0 (sham) or 2 J/m2 of UVC and immediately pulsed with BrdU for 1 h. Cultures were washed, fed with fresh medium and incubated for increasing periods of time. Cells were stained for bivariate flow cytometric analysis of DNA content and BrdU fluorescence. For this analysis we followed the transition through the cell cycle of the BrdU-labeled cells only in the sham-treated (open circles) and irradiated (closed circles) populations. The percentage of total cycling cells displaying BrdU fluorescence and DNA content equivalent to mid S phase (A, D, propidium iodide fluorescence between the G1 and G2 compartments), late S/G2 (B, E, above the G2 compartment) or early S/G1 (C, F, above the G1 compartment), were plotted against the time elapsed from UVC irradiation.
UVC exposure to 43% 12 h later. These results suggest that many XP-V cells damaged by UVC while in S were still in this phase of the cell cycle after the 12 h incubation. These are the cells in which BrdU incorporation was reduced in a time-dependent fashion, creating a distinct population of cells that separated from those actively synthesizing DNA (cells that were in G1 at the time of irradiation and moved into the
UVC-induced clastogenesis in XP-V
S phase during the following 12 h). By comparison, the response of NHF to the same UVC treatment revealed only a mild and transient increase of cells in S phase (Figure 4). The results in Figure 5 summarize the transitions through different cell cycle compartments of cells that incorporated BrdU during the first hour following UVC irradiation (2 J/m2) or sham treatment. In this pulse-chase experiment, we followed the fate of BrdU-labeled cells that were in or about to enter S at the time the population was exposed to UVC. Note the similarity between the lines illustrating the changes with time of the percentage of BrdU-labeled cells in mid S (Figure 5A), late S/G2 (Figure 5B) or G1/early S (Figure 5C) phases in sham-treated and irradiated populations of NHF. UVC irradiation caused at best a 2 h delay in the exiting of BrdU-labeled NHF from mid-S phase and this compartment was emptied completely at the 12 h time point. Contrast the contiguous lines for NHF with the well-separated lines in sham- and UVC-treated XP-V fibroblasts (Figure 5D–F). The lines are out of phase by at least 6 h. In addition, 6–8% of the irradiated XP-V cells were still in mid S at 12 h and in late S/G2 18–24 h after UVC exposure. Taken together, the results illustrated in Figures 4 and 5 indicate that a fraction of the irradiated XP-V fibroblasts were very sensitive to UVC and were arrested in S phase (6–8%, perhaps representing the cells that were most damaged by UVC). Two thirds of the S phase XP-V cells exposed to 2 J/m2 UVC, however, were delayed in S phase, then recovered from the radiation effects and continued to cycle. UVC-induction of chromosomal aberrations in hTERTexpressing fibroblasts Diploid XP-V cells were reported to have normal sensitivity to clastogenesis (27) and normal induction of SCE after exposure to UVC (28,29). Given the hypersensitivity of S phase XP-V to UVC-induced inhibition of DNA chain elongation (Figure 1), daughter strand gap formation (Figures 1 and 2), and S phase delays (Figures 3–5), it was important to reevaluate UVC clastogenesis in the hTERT-expressing XP-V and NHF cell lines. Cultures of hTERT-expressing fibroblasts were grown to confluence to reduce the number of cells in S phase. Upon replating at lower density, these fibroblasts reentered the cell cycle and progressed toward S phase with kinetics similar to those previously published for other NHF (38,39). Because of the strong UVC-induced S phase delay in XP-V cells, it was important to chart the kinetics of cellular progression through S and G2 phases into mitosis for both sham-treated and irradiated cells. XP-V and NHF were released from confluence arrest and 12 h later sham-treated or irradiated with 2 or 4 J/m2 of UVC. The irradiation time was selected to damage cells as they entered the S phase. At different times after irradiation, the cultures were pulsed for 1 h with BrdU to determine the fractions of cells in S phase. Mitotic indices were also determined in the same cultures by enumerating cells in metaphase. As expected, we observed an extension of the S phase in irradiated cells. There was also a significant UVC-induced delay in entry into mitosis. Both responses were more pronounced in XP-V than in NHF treated with the same doses of UVC (not shown). The UVC-induced delay in entry into mitosis was likely due to the induction of the G2 checkpoint in cells with chromatid breaks, in addition to effects of UVC during the S phase. Under these same experimental conditions, metaphases were collected from sham-treated and irradiated cultures during 3 h incubations in 30 ng/ml of colcemid,
starting at 24, 27 or 30 h after replating from confluence. Mitotic indices at these time points were determined in parallel cultures stained with Giemsa and the first cells reaching mitosis were collected, as these were in S phase at the time of UVC exposure or entered S phase shortly after irradiation. Table I summarizes the data on percentages of metaphases with aberrations and frequencies of aberrations per mitotic cell. The aberrations observed most frequently were chromatid gaps and breaks (91–100%). In cultures irradiated with 2 J/m2 of UVC, very few metaphases were found 24–27 h after replating. Therefore, the chromosomal aberration analyses in both NHF and XP-V were done in metaphases trapped by colcemid in the period of 27–30 h after replating. At this time window, there were numerous metaphases in NHF cultures exposed to 4 J/m2 of UVC. This was not the case, however, for XP-V cultures irradiated with the same UVC dose. Inhibition of entry into mitosis was stronger in XP-V than NHF and the first metaphases appeared between 30 and 33 h after replating. Table I shows that chromosomal aberration frequencies in XP-V fibroblasts were 1.5 to 2 times higher than those observed in NHF. We also collected and analyzed the metaphases trapped in the 33–36 h window. XP-V cells reaching metaphase at these later times carried a reduced frequency of aberrations (0.11 UVC-induced aberrations per metaphase) compared with those from the 30–33 h sample (0.28 UVC-induced aberrations per metaphase). These cells were probably in G1 at the time of irradiation (12 h after replating) and benefited from the removal of DNA lesions by NER prior to DNA replication (38). Discussion The data presented in this report illustrate the important contribution of DNA damage checkpoints to the protection of human cells against chromosomal instability. The induction of chromosomal aberrations in UVC-irradiated cells is dependent on the replication of DNA containing unrepaired photoproducts (38). Single stranded DNA regions at replication forks or across from daughter strand gaps are thought to be the precursors to chromatid-type aberrations detected in metaphase chromosomes. Intermediates of DNA replication containing regions of single stranded DNA accumulate in XP-V cells exposed to UVC. Absence of pol η activity severely hampers the ability of XP-V cells to replicate past cyclobutane thymine dimers, which are strong blocks to DNA replication by the principal DNA polymerases (α and δ). Accordingly, the data illustrated in Figure 1 show that the size distributions of nascent DNA in UVC treated XP-V cells that were immortalized by the ectopic expression of human telomerase, shifted to lower molecular weights after doses of 1–2 J/m2. These low doses of UVC barely caused any inhibition of DNA elongation in normal fibroblasts (Figure 1), which are competent for bypass replication of cyclobutane pyrimidine dimers. The estimated frequency of UVC-induced daughter strand gaps in XP-V actually approximated the frequency of cyclobutane pyrimidine dimers deposited on parental DNA and was 25-fold higher than the frequency of gaps detected in normal fibroblasts (Figure 2). After a strong delay in progression through the S phase (Figures 3 and 4), most of the XP-V cells exposed to 2 J/m2 UVC were able to recover and transit through the cell cycle (Figure 5). Thus, XP-V must be able to tolerate about three daughter strand gaps per 108 Da (Figure 2) by using pol η-independent post-replication repair pathway(s) to seal these gaps prior to mitosis. Otherwise, the frequency of metaphases 963
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Table I. Frequency of chromosomal aberrations in hTERT-expressing fibroblasts that were sham-treated or irradiated with UVC as they entered the S phase (12 h after replating from confluence arrest) Cell type
UVC dose (J/m2)
Total mets analyzed
Number of mets with aberrations
Percent of mets with aberrations
Number of aberrations
Aberration per metaphase
UVC-induced aberrations
NHF
0 2 4
300a 200b 100c
34 38 24
11 19 24
35 39 31
0.12 0.20 0.31
– 0.08 0.19
XP-V
0 2 4
299a 318b 100c
26 70 30
9 22 30
27 79 37
0.09 0.25 0.37
– 0.16 0.28
Detected most frequently were chromatid-type aberrations (91–100%). Among these 90–100% were gaps and breaks. The other chromatid-type aberrations were tri-radials (1 in NHF, 1 in XP-V), quadri-radials (2 in XP-V) and fragments (3 in NHF, 1 in XP-V). The chromosomal-type aberrations observed were breaks (1 in NHF, 4 in XP-V), terminal deletions (1 in XP-V), acentric fragments (3 in NHF, 2 in XP-V) and minute chromosomes (2 in NHF). aResults from three experiments were grouped. About 100 metaphases (mets) were analyzed in each experiment. bResults from two independent experiments. cResults from a single experiment. XP-V metaphases were collected 3 h later than NHF metaphases to compensate for the stronger UVC-induced mitotic delay in the mutant cells.
containing chromatid breaks and gaps would be much higher in UVC treated XP-V fibroblasts than recorded in Table I. Thus, a strong S phase checkpoint response appears to cooperate with post-replication repair to avoid the accumulation of damaged chromosomes in mitosis. SV40-transformed XP-V fibroblasts were shown to display frequencies of UVC-induced SCE (29) and hMRE11/PCNA foci (30) substantially above those detected in SV40-transformed NHF. These observations suggest that XP-V cells are proficient in double strand break repair and homologous recombination between sister chromatids. Why, then, do nontransformed XP-V cells display normal frequencies of UVC-induced SCE (28,29) and clastogenesis (27)? At least two of the factors responsible for the enhanced sensitivity of transformed XP-V cells to UVC are the abrogation of the S phase checkpoint response of inhibition of replicon initiation (40) and the inactivation of p53-dependent regulatory pathways by SV40 large T antigen (29,41). Even though diploid XP-V cells accumulate abnormal numbers of daughter strand gaps during replication of UVC-damaged DNA (Figures 1 and 2), the burden of these secondary lesions would be even greater in the absence of inhibition of replicon initiation. Thus, the pool of intermediates of DNA replication representing precursors of chromatid strand breaks is presumed to be larger in SV40-transformed XP-V cells. The size of the target pool for chromatid breaks, however, might not be sufficient to explain the unexpected findings of normal or near normal frequencies of chromatid-type aberrations in non-transformed XP-V cells. Another contributing factor might be the potential role of checkpoint proteins in stabilizing blocked replication forks until bypass replication can take place. In this scenario, both breaks and homologous recombination at blocked replication forks and daughter strand gaps would be reduced. In addition, the p53-dependent death of S phase cells that fail to repair daughter strand gaps and the long S phase delays, during which post-replication repair pathways fill the daughter strand gaps prior to entry of cells in G2 and mitosis, should reduce the frequency of mitotic cells carrying chromosomal aberrations. In summary, the results of this study lend support to the hypothesis that post-replication repair defects (e.g. lack of pol η in XP-V, leading to deficient translesion synthesis of cyclobutane thymine dimers) cause the accumulation of aberrant DNA structures in S phase cells. These structures are 964
strong signals for the activation of the intra S phase checkpoint, which slows progression through S by reducing the rate of replicon initiation. Damage-induced delays in S phase (and later in G2) increase the time available for repair of blocked replication forks and daughter strand gaps. In a fraction of daughter strand gaps, scission of the template strand produces double-strand breaks, which activate the G2 checkpoint. If the burden of aberrant replicating structures and double strand breaks cannot be managed adequately by repair pathways, then cells remain permanently arrested and never reach mitosis. It is only when breaks are not repaired and checkpoint mechanisms fail to stop cells from reaching mitosis that metaphase chromosomes carrying chromatid breaks or gaps can be visualized. Acknowledgements We are grateful to Dr Roger A.Schultz (McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center) and Dr Jerry W.Shay (Department of Cell Biology, University of Texas Southwestern Medical Center) for the gift of cell lines hTERT-GM01604 and hTERT-GM02359, which were created with telomerase reagents from Geron Corporation (Menlo Park, CA). We thank Dr Rosann A.Farber for expert advice on PCR-based analyses of heterozygosity. This work was supported by PHS grants CA55065 (MCS) and CA81343 (WKK). Support from the UNC-CH Center for Environmental Health and Susceptibility (ES10126) to WKK is also recognized.
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