Functional interaction of p53 and BLM DNA helicase in apoptosis.

The Bloom syndrome (BS) protein, BLM, is a member of the RecQ DNA helicase family that also includes the Werner syndrome protein, WRN. Inherited mutations in these proteins are associated with cancer predisposition of these patients. We recently discovered that cells from Werner syndrome patients displayed a deficiency in p53-mediated apoptosis and WRN binds to p53. Here, we report that analogous to WRN, BLM also binds to p53 in vivo and in vitro, and the C-terminal domain of p53 is responsible for the interaction. p53-mediated apoptosis is defective in BS fibroblasts and can be rescued by expression of the normal BLM gene. Moreover, lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to both gamma-radiation and doxorubicin-induced cell killing, and sensitivity can be restored by the stable expression of normal BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and in response to DNA damage normal accumulation of p53, normal induction of p53 responsive genes, and normal G(1)-S and G(2)-M cell cycle arrest. BLM localizes to nuclear foci referred to as PML nuclear bodies (NBs). Cells from Li-Fraumeni syndrome patients carrying p53 germline mutations and LCLs lacking a functional p53 have a decreased accumulation of BLM in NBs, whereas isogenic lines with functional p53 exhibit normal accumulation. Certain BLM mutants (C1055S or Delta133-237) that have a reduced ability to localize to the NBs when expressed in normal cells can impair the localization of wild type BLM to NBs and block p53-mediated apoptosis, suggesting a dominant-negative effect. Taken together, our results indicate both a novel mechanism of p53 function by which p53 mediates nuclear trafficking of BLM to NBs and the cooperation of p53 and BLM to induce apoptosis.

Bloom syndrome (BS) 1 is an autosomal recessive genomic instability syndrome characterized by growth retardation, immune deficiency, and cancer predisposition (1,2). The gene responsible for the BS has been identified as BLM, which encodes a 1417-residue protein with ATP-dependent 3Ј-5Ј DNA helicase activity, and it belongs to the RecQ DNA helicase family (3)(4)(5). In addition to BLM, the RecQ family in mammalian cells contains at least four more proteins, i.e. RecQL, WRN, RTS, and RecQL5 (4,6). Germline mutations in BLM, WRN, and RTS are known to be associated with cancer predisposition syndromes, BS, Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), respectively (4). Furthermore, both BLM and WRN also contain signature motifs that belong to the DExH-containing DNA helicase superfamily, which includes XPB and XPD. Germline mutations in these genes have been linked to cancer predisposition syndromes, WS, xeroderma pigmentosum syndrome B (XP-B), and D (XP-D), respectively (5)(6)(7)(8). Similarly, germline mutations in p53 are responsible for the cancer predisposing LFS (9). Therefore, both p53 and members of the RecQ family play an essential role in maintaining genomic stability and preventing the risk of cancer.
Apoptosis is an important process in normal development and in stress-mediated signaling pathways. Defective regulation of apoptosis often contributes to the etiology of cancer. Consequently, cells with mutations in tumor suppressor genes such as p53 and the promyelocytic leukemia protein (PML) display a deficiency in stress-induced apoptosis (10 -15). BLM protein is a nuclear protein and is present in both foci and a diffuse, micro-speckled distribution (16). The nuclear foci have been identified as PML nuclear bodies (NBs) (17)(18)(19), structures that also contain PML, hypophosphorylated Rb, SUMO-1, p53, and other proteins (20 -22). Cells from PMLϪ/Ϫ mice are resistant to apoptosis induced by many stimuli, including Fas, tumor necrosis factor-␣, ceramide, and ␥-radiation (14,15). These data indicate that NBs may play a role in multiple apoptotic pathways.
We recently reported that cells from XP-B, XP-D, or WS individuals have an attenuated p53-dependent apoptotic pathway and p53 binds to XPB, XPD, and WRN (23)(24)(25). In addition, similar to p53-deficient cells, the XP-D LCLs have a deficiency in DNA damage agent-induced apoptosis (23,25). In this study, we investigated the physical and functional interactions of p53 and BLM in an apoptotic pathway. We hypothesized that similar to WRN, BLM is also a target for p53 binding and p53-mediated apoptosis. We found that BLM-deficient cells have an attenuated DNA damage-activated and p53-mediated apoptosis. However, induction of p53 and p53mediated transcriptional activation of downstream targets including Gadd45, Bax, and p21 waf1 was normal. p53 bound to BLM both in vivo and in vitro. Cells that lack p53 had normal numbers of NBs and normal levels of BLM, however, BLM transit to the NBs was reduced and ectopic expression of p53 in these cells restored BLM transit to the NBs to normal. More-over, overexpression of BLM mutants that have a deficiency in localization to NBs in normal cells can block wild type BLM localization to NBs and inhibit p53-mediated apoptosis. Our results indicate a novel mechanism of p53-mediated apoptosis involving a p53-BLM-PML signaling pathway.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-GM01310, GM02184, and HG1943 are normal human LCLs and GM3403, GM09960, GM04408, and HG1525 are BS patients-derived LCLs. All the BS LCLs, with an exception of GM04408 (heterozygous with a low sister chromatid exchange (SCE) rate), are homozygous in BLM with a high SCE rate. All of the GM lines were obtained from Coriell Cell repositories (Camden, NJ) while the HG lines were obtained from New York Blood Bank Cell repositories. These cells were maintained in a density greater than 3 ϫ 10 5 cells/ml in RPMI medium supplemented with 15% fetal bovine serum, penicillin, and streptomycin (BIOFLUIDS, Rockville, MD). Primary normal human fibroblasts (GM07532, GM08402, and GM00038) and primary BS fibroblasts (GM03498 and GM01492) were obtained from Coriell Cell Repositories. GM01492 is homozygous for a protein chain-terminating mutation BLMc.2207-2212delATCTGAinsTAGATTC, referred to as blm Ash (26). Cell culture conditions were described previously (23).
Indirect Immunofluorescence and Western Blot Analyses-Cells were seeded onto coverslips, fixed with 4% paraformaldehyde in phosphatebuffered saline for 10 min, permeabilized with methanol for 20 min, blocked with phosphate-buffered saline-plus solution (phosphate-buffered saline, 0.15% glycine, 0.5% bovine serum albumin), and incubated with primary antibody for 1 h at 37°C. The slides were then incubated with the corresponding secondary antibodies conjugated with either Texas red or fluorescein isothiocyanate (Vector Laboratories) at a 1:300 dilution for 1 h at room temperature. After being washed with phosphate-buffered saline, coverslips were mounted in VectaShield solution containing 0.5 g/ml 4Ј,6-diamidino-2-phenylindole (Sigma). Cells were examined with a Zeiss Axioskop fluorescence microscope equipped with a high-performance CCD imaging system (IP Lab Spectrum). Alternatively, confocal fluorescent images were collected with a Bio-Rad MRC 1024 confocal scan head mounted on a Nikon Optiphot microscope with a ϫ60 planapochromat lens. Sequential excitation at 488 and 568 nm was provided by a krypton-argon gas laser. Emission filters of 598/40 and 522/32 were used for sequentially collecting red and green fluorescence, respectively, in channels 1 and 2. Z-sections were collected at 0.5-m intervals for each cell using LaserSharp software. Images were analyzed by Confocal Assistant software. To quantify nuclear BLM foci between NHF and LFS fibroblasts, images that only showed cells with BLM foci were randomly collected. Western blot analysis was performed similarly as described previously (23).
Microinjection, Apoptosis, and Cell Viability Analysis-The microinjection protocol and determination of apoptosis were essentially as described (23). For analyzing the viability of LCLs, exponentially growing cells were plated at a density of 6.25 ϫ 10 5 cells/ml, treated with various doses of either doxorubicin (Sigma) or ␥-radiation, and incubated for a designated time. Cell viability was assessed by trypan blue exclusion as described previously (23). The ability of normal LCLs undergoing apoptosis as measured by caspase-mediated cleavage of poly(ADP)-ribose polymerase and by annexin V staining as well as by morphologic examination of the nuclei was confirmed previously (23,25). In the experiments with Fas-mediated apoptosis, a Fas-specific antibody (C3H) at a concentration of 1.4 g/ml was added to the culture media that contain LCLs. Cell viability was monitored at 4, 8, and 24 h.
For flow cytometric analysis, cells were exposed to 5 Gy of ␥-radiation and incubated for 4, 8, or 24 h. Prior to harvest, cells were incubated in the presence of bromodeoxyuridine at a final concentration of 10 M for 30 min. After harvest by trypsinization, cells were fixed in 70% ethanol until the day of analysis. Cells were treated with 0.1 M HCl containing 0.5% Triton X-100 to extract histones, followed by boiling and rapid cooling to denature the DNA. Cells were then incubated with antibromodeoxyuridine-fluorescein isothiocyanate (PharMingen, San Diego, CA) and counterstained with propidium iodide containing RNase. Samples were run on a Becton-Dickinson FACSCalibur. Data analysis was performed with CellQuest software for the Macintosh (Becton-Dickinson). Cellular debris and fixation artifacts were gated out, and the G 0 /G 1 , S, and G 2 /M fractions were quantified.

p53-mediated Apoptosis Is Attenuated in BS Cells-To inves-
tigate the response of BS fibroblasts to p53-dependent apoptosis, we microinjected p53 and BLM cDNA expression constructs separately or together into normal human fibroblasts (NHF) and BS fibroblasts, and then monitored their ability to undergo apoptosis. The BS fibroblasts that were used in these experiments, GM03498 and GM01492, are null for BLM (see "Experimental Procedures") and exhibit a high SCE phenotype characteristic of BS cells. GM01492 fortuitously is negative for p53 expression (27). p53-mediated apoptosis was attenuated in two BS fibroblasts (GM03498 and GM01492) when compared with three normal NHF examined (GM07532, GM00038, and GM08402) ( Table I). Coexpression by microinjection of a normal BLM expression vector caused a significant increase in p53-mediated apoptosis of GM01492 cells (Fig. 1A), which is evidence of complementation of the attenuated apoptotic phenotype. Expression of a mutant BLM protein that contains a cysteine to serine amino acid substitution at residue Normal a Cells that stained positively for p53 and exhibited typical features of apoptosis, including cytoplasmic blebbing, chromatin condensation, and nuclear fragmentation, were counted. Both the in situ terminal deoxynucleotidyltransferase end-labeling assay (TUNEL) and the annexin V staining assay confirmed that the morphological changes in these cells, following microinjection of the wild type p53 expression vector, were markers of apoptosis (data not shown). b n, total number of p53 immunopositive cells that were counted at 24 h following microinjection of a wild type p53 expression vector and were obtained from at least three independent experiments. c 2 test was used for analyzing the statistical significance between one normal strain (GM07532) and cells from other normal strains or BS donors. pendent ATPase activities (28), did not cause an increase in p53-mediated apoptosis. These data indicate that an enzymatically active BLM product is required for the p53-dependent apoptotic effect. In contrast, the mutant BLM (C1055S) acts as a dominant-negative mutant to block p53-mediated apoptosis in NHF (Fig. 1A). Similarly, an N-terminal deletion mutant of BLM (⌬133-237) also blocks p53-mediated apoptosis in NHF (Fig. 1A).
To determine the role of BLM in apoptosis at the physiological level, sensitivity to cell death induced by ␥-irradiation or doxorubicin was investigated in four BS LCLs. Three BS LCLs (GM03403, GM09960, and HG1525) feature a high-SCE phenotype and are homozygous for null mutations in BLM. The fourth (GM04408) is a low-SCE cell line in which the BLM gene was spontaneously corrected in vivo by a somatic intragenic recombination event, thereby becoming heterozygous for one of its BLM mutations (3). We have previously shown that cell viability, monitored by trypan blue exclusion, in LCLs treated with ␥-irradiation or doxorubicin is tightly correlated with apoptosis in a p53-dependent manner, as evidenced by annexin V positivity, nuclear condensation, and DNA fragmentation (23,25). Using trypan blue exclusion assay, we found that the three high-SCE BS LCLs examined were less sensitive to both of the genotoxic agents when compared with normal human LCLs (Fig. 1, B and C, and data not shown). In contrast, the low-SCE BS LCL (GM04408) exhibited the same sensitivity to both agents as normal LCLs (Fig. 1, B and C). Expression of a normal BLM cDNA expression vector construct into the high-SCE BS LCL HG1525, which reduces the SCE rate toward normal (16), sensitized the cells to ␥-radiation-induced cell killing (Fig. 1D). BS LCLs exhibit a normal sensitivity to Fas antibody-induced apoptosis (data not shown), indicating that the resistance obtained in BS cells to DNA damage-induced cell killing is not due to a general defect or inability to undergo apoptosis.
Normal p53-mediated Activation of Its Downstream Genes in BS LCLs-Because DNA damage-induced cell killing of normal cells is predominantly dependent on the induction of p53 (10 -13, 25), we compared p53 levels in normal and BS LCLs following treatment with 5 Gy ␥-radiation or 1 g/ml doxorubicin. Both normal and BS LCLs had similar expression profiles after the induction of p53 in response to DNA damage ( Fig. 2A). Similar results were obtained with another BS LCL GM09960 (data not shown). To determine whether a defect in p53-mediated transcriptional transactivation of downstream genes associated with a loss of BLM, we examined the mRNA levels of p21, Gadd45, Bax, and p53 by RNase protection assay in BS LCLs following treatment with 5 Gy ␥-radiation. Both normal and BS LCLs displayed a similar induction of p21, Gadd45, and Bax (Fig. 2B), indicating that p53-dependent transcription is normal in BS cells. Consistent with this finding, BS LCLs underwent normal cell cycle arrest at G 1 and G 2 (Fig. 2, C and D, data not shown) and accumulation of cyclin B1 and hypophosphorylated Rb upon treatment with ␥-radiation (data not shown). Collectively, these results indicated that p53-dependent transactivation in response to DNA-damaging agents is normal in BS cells.
BLM Localization in the PML Nuclear Bodies Is Modulated by p53-By indirect immunofluorescence microscopy, focal concentrations of BLM can be detected in the NBs in all cells in which BLM protein can be detected (the co-localization is Ͼ90%). Because the NBs are known to play an important role in apoptosis and p53-mediated apoptosis was defective in BS cells, we investigated whether BLM localization to the NBs could be modulated by p53 function. To test this possibility, we analyzed three LFS fibroblast cell strains, two heterozygous for missense mutations of p53 at codons 248 and 175, respectively, and one homozygous for a frameshift mutation in p53 that results in a p53-null phenotype (see "Experimental Procedures").
The average numbers of BLM foci per nucleus were significantly lower in the p53-deficient fibroblasts compared with the numbers in normal fibroblasts (Fig. 3), whereas the average numbers of NBs per nucleus as determined by staining with anti-PML was the same in all fibroblast cell lines examined ( Fig. 3F and data not shown). We also examined BLM localization in two LCLs (TK6 and WTK1) that differ in the status of p53. TK6 cells contain a normal p53 gene and are sensitive to radiation-induced cell killing, whereas its isogenic derivative (WTK1) contains a mutant p53 and is radiation-resistant (29).
Consistent with the results from fibroblasts with a mutant p53, the average numbers of BLM foci per nucleus in WTK1 cells was lower than in TK6 cells (Fig. 3D). The numbers of BLM foci per nucleus in normal fibroblasts was unchanged by ␥-irradiation (Fig. 3, B and C), suggesting that post-translational modification or stabilization of a normal p53 does not affect BLM localization to the NBs. Similar results were obtained with ␥-irradiated TK6 and WTK1 cells (Fig. 3D). Because the steady-state levels of BLM were the same regardless of p53 status or radiation treatment (Fig. 3H), the reduction in BLM foci in the LFS fibroblasts was not explained by decreased BLM protein.
To test whether expression of a normal p53 protein in the mutant fibroblasts could overcome the defect in BLM localization, we examined a 041-derived clone that contains a tetracycline-inducible p53 expression construct (30). Upon induction of p53, the average numbers of BLM foci per nucleus was increased to levels similar to those in normal fibroblasts (Fig.  3E), whereas the number of NBs per nucleus remained the same (Fig. 3F). BLM levels were not changed by induction of p53 (Fig. 3G). Because the steady-state levels of BLM are the same under all conditions tested, the data indicate that a greater proportion of BLM in the LFS cells is in a diffuse rather than a focal distribution compared with normal cells. Taken together, our data show that p53 functions in the localization of BLM to the NBs.
Expression of a normal GFP-BLM in normal fibroblasts resulted in efficient localization of the protein to the NBs (Fig. 4)

FIG. 3. Subcellular localization of BLM. A, fibroblasts (left 4 panels) from a normal donor (GM08402) (NHF), a BS donor (GM03498) (BS)
, and a LFS donor (041) (LFS) were treated without (top panels) or with 5 Gy of ␥-radiation for 2 h (bottom 3 left panels). TK6 (wild type p53) and WTK1 (mutant p53, M237I) cells were prepared by cytospin (right panels). Cells were stained with anti-BLM polyclonal antibody followed by a fluorescein isothiocyanate-conjugated anti-rabbit antibody. Representative images were obtained by confocal laser microscopy, with magnification at ϫ630 for fibroblasts and magnification at ϫ200 for TK6 cells. B, BLM and PML colocalize similarly in ␥-irradiated (bottom panels) and untreated (top panels) NHF. GM08402 cells were treated with 5 Gy of ␥-radiation, incubated for 2 h, and co-stained with anti-BLM polyclonal antibody revealed with an fluorescein isothiocyanate-conjugated anti-rabbit antibody (green, left panels) and anti-PML monoclonal antibody revealed with a Texas red-conjugated anti-mouse antibody (red, middle panels). Superimposition of BLM and PML staining (yellow) is shown in the right panels. Representative images are shown. Numbers of BLM foci in each nucleus were counted in one NHF (GM08402) and three LFS fibroblast lines (041, homozygous for p53, Ϫ/Ϫ; 087, heterozygous for p53, wild type/248trp; and 172, heterozygous for p53, wild type/175his) (C), or in TK6 and WTK1 cells (D), either without or treated with 5 Gy of ␥-radiation followed by 2 h incubation (C). E, numbers of BLM foci were quantified in a tet-inducible p53 cell line derived from LFS041 (041-TR), incubated either with (ϩTet) or without (ϪTet) tetracycline for 24 and 48 h. F, numbers of PML foci were quantified in the cells (E). G, Western blot of 041-TR cells. H, fibroblast cells from normal (GM08402) and three LFS donors were irradiated at 5 Gy and incubated for 2 h. Cellular proteins were Western blotted with anti-BLM antibody and anti-p53 antibody. (31); however, expression of the GFP-BLM C1055S or ⌬133-237 mutant resulted in either a lower efficiency of localization or almost no localization to the NBs, respectively. Interestingly, the ⌬133-237 mutant was mainly localized in the nucleolus as large aggregates as evidenced by their morphologically distinct features and by their co-localization of nucleoli-associated protein nucleolin with a monoclonal nucleolin-specific antibody (data not shown). (A full report of the molecular analysis of the segments of BLM necessary for localization to the NBs will be published elsewhere.) As mentioned above, overexpression of the GFP-BLM mutants C1055S or ⌬133-237 inhibited p53-mediated apoptosis in normal fibroblasts (Fig.  1A). To test whether the dominant effect of these mutants could be explained by a disruption of BLM localization, we co-expressed by microinjection normal and mutant GFP-BLM proteins in normal fibroblasts and counted fluorescent green BLM foci. We detected a significant reduction of BLM foci per nucleus in the cells that expressed normal and mutant BLM proteins compared with cells that expressed normal GFP-BLM and GFP as a control. These data suggest that the inhibition of p53-mediated apoptosis in normal cells by expression of either of these mutant proteins may be explained by a reduction in the localization of normal BLM to the NBs.
Physical Interaction between p53 and BLM-We have shown recently that p53 binds to WRN, a DNA helicase that shares homology with BLM, and p53 cooperates with WRN to induce apoptosis (24). To explore mechanism(s) through which BLM and p53 might cooperate to induce apoptosis, we examined a potential protein-protein interaction between p53 and BLM. Cell lysates prepared from GM01310 (normal LCL) and GM03403 (BS LCL) with or without treatment with 5 Gy ␥-radiation were subjected to immunoprecipitation with a polyclonal anti-BLM antibody, and BLM-bound proteins were analyzed by Western blotting with an anti-p53 monoclonal antibody (DO-1). Anti-BLM antibody immunoprecipitates p53 only from extracts of ␥-irradiated normal LCL but not from any other extracts tested (Fig. 5A). It is possible that DNA damage leads to an increase in p53 levels that provides sufficient sensitivity for the detection, rather than the requirement for the radiation. Consistently, a recombinant GST-p53 fusion protein (1-393), as well as N-terminal-truncated proteins (94 -393 and 155-393) but not C-terminal-truncated proteins (94 -293 and 1-293) or the GST control, bind to an in-vitro-translated fulllength BLM (Fig. 5B). These results indicated that p53 can physically interact with BLM. DISCUSSION We have shown here that BLM-null BS LCLs are resistant to DNA damage agent-induced cell killing. The genotoxic resistance associated with BS LCLs can be reversed by expression of a normal BLM. We have shown recently that p53-mediated apoptosis is attenuated in cells with mutations in other DExH family DNA helicases, including XPB, XPD, and WRN (23)(24)(25). DNA damage-induced cell killing in normal LCLs is largely dependent on p53 (10 -13, 25). It is possible that the resistance of BS LCLs to cell killing is the result of a defect in a p53modulating pathway, similar to XP-D and WS cells (23)(24)(25). One of the potential mechanisms is that p53 binds to XPB, XPD, and WRN and cooperates with these DNA helicases to induce apoptosis. Consistent with this hypothesis, we found that BS fibroblasts were resistant to apoptosis induced by ectopic expression of p53, whereas the introduction of normal BLM restored them to normal sensitivity. Further genetic evidence of the cooperation of BLM and p53 is the dominant negative activity of two BLM mutants in the attenuation of p53-mediated apoptosis in NHF. Interaction between p53 and BLM can be detected in ␥-irradiated cells, suggesting that p53 may cooperate with BLM to induce apoptosis.
A potential mechanism by which p53 and these DNA helicases cooperate to induce apoptosis is through a direct proteinprotein interaction. Consistent with this hypothesis, p53 has been shown to bind to XPB, XPD, WRN, and now BLM. Remarkably, the C terminus of p53 is the common domain required for binding to these DNA helicases. These results imply that p53 may be a common upstream molecular trigger of apoptosis. However, the different DNA helicases probably act differently in response to various types of DNA damage in their cooperation with p53, and most likely other proteins, in modulating apoptosis. An alternative and not mutually exclusive hypothesis is that mutations in these DNA helicases lead to an imbalance in the transcriptional expression of pro-and antiapoptotic genes and diminishes p53-mediated apoptosis (25,32). Certain DNA helicases, e.g. WRN, may also act as cotranscription factors with p53 and enhance transcription-dependent p53-mediated apoptosis (33). We consider it likely that multiple mechanisms are responsible for the attenuation of p53-mediated apoptosis in cells with germline mutations in XPB, XPD, WRN, or BLM. For example, the data presented here indicate that a p53-mediated recruitment of BLM to the NBs is a possible mechanism.
The mechanism for the functional interaction between p53 and BLM may involve PML NBs. The NB coordinates multiple apoptotic pathways that are either dependent or independent of caspase activation (14,15). A chromosomal translocation leading to the formation of a fusion protein between PML and retinoic acid receptor ␣ is responsible for disruption of the NBs and the development of acute promyelocytic leukemia, possibly due to the disruption of an apoptotic pathway(s) (34). Cell death susceptibility is associated with the recruitment of certain proteins to the NBs (15). NBs are nuclear matrix-associated structures in which PML appears to be the structural entity that recruits other cellular proteins, including Rb, SUMO-1, and UBC9 (20). In addition, both PML and p53 can be modified by SUMO-1, and p53 has been shown to form nuclear foci juxtaposed to NBs (35). Recent studies indicate that NBs contain p53 (21,22). Furthermore, BLM localizes to NBs (17)(18)(19). Because p53-deficient cells have a reduced localization of BLM to the NBs and the reintroduction of wild type p53 increases BLM localization to NBs, p53 may control intranuclear trafficking of BLM to the NBs. A potential role of BLM nuclear trafficking to NBs on p53-mediated apoptosis was further supported by our findings that two BLM mutants (C1055S and ⌬133-237) were able to block p53-mediated apoptosis. These dominant negative mutants have a deficiency in the localization in NBs and are able to inhibit wild type BLM trafficking to NBs. Collectively, our results indicate that BLM may be one of the mediators in NBs that senses p53-dependent apoptotic stimuli.
The level of BLM protein is cell cycle dependent, which peaks in S and G 2 phases (37)(38)(39)(40)(41)(42). However, we showed here that BS cells appear to have intact G 1 /S and G 2 /M cell cycle checkpoints in response to ␥-radiation. We also showed that there is a decrease in BLM foci formation in p53-deficient cells. In addition, we did not observe any change in BLM levels in normal LCLs and in LFS fibroblasts at 2 h post-␥-radiation. Therefore, it is unlikely that defects of the BLM foci formation in p53deficient cells are due to cell-cycle-dependent BLM expression.
Interestingly, earlier studies demonstrated that 2 of 11 primary BS fibroblast strains tested show delayed p53 induction following both UV and ␥-radiation exposures (43), and that one strain (GM01492) lacks the p53-dependent G 1 cell cycle checkpoint (44). Collister et al. (45) compared the kinetics of DNA damage-induced p53 accumulation in a NHF strain (GM0038) and a fibroblast strain (GM02932) from a BS patient. Exposure of the cells to 2.5 Gy ␥-radiation rapidly increased p53 levels as well as p21 and hdm2 to peak values at 2 h in both normal and BS cells, but a slower decline in p53 as well as p21 and hdm2 was shown in the BS cell strain when compared with the fairly rapid declines observed in the NHF. Our results are similar to these published data in that p53 is induced efficiently and peaks at 2 h post-␥-radiation in both normal and BS LCLs. We also observed a relatively high basal level of p53 at 0 or 24 h after DNA damage. However, these differences may not be the same in all the BS cells. Importantly, we showed that the BS cells studied here have both normal p53-dependent induction of p21, Gadd45, and Bax, and normal G 1 and G 2 cell cycle checkpoint controls in response to ␥-radiation.
BLM is a 3Ј to 5Ј DNA helicase (28, 46 -49). It contains a helicase domain that is 40 -50% identical to members of the RecQ family, a subfamily of the DExH-containing DNA helicases, which includes WRN, RecQL, RTS, and RecQL5 (3,6) in mammalian cells, Sgs1 and Rqh1 in yeasts (50), and recQ in bacteria. Similar to its mammalian counterparts, Sgs1 mutant cells show mitotic hyper-recombination (50), which can be partially suppressed by expression of normal BLM and WRN (51). Loss of the helicase activity may be responsible for hyperrecombination in BS cells (28). These results indicate that BLM and WRN may share a similar pathway in the modulation of homologous recombination repair and in controlling genomic stability. Data supporting this hypothesis include a defect in p53-mediated apoptosis in BS and WS cells and a cancer predisposition in both BS and WS patients. Although WRN is predominantly localized in the nucleolus (52), BLM is localized in NBs and in the nucleolus during S phase of the cell cycle (19). In addition, WRN contains an exonuclease motif at its N terminus and shows an exonuclease activity in vitro, a property unique to WRN (36). These data imply that BLM and WRN may utilize different mechanisms and intranuclear locations to regulate genomic stability. Although the relationship between the localization of BLM to the NBs and its potential functional role as a helicase and modulator of homologous recombination remain unclear, our data are consistent with the hypothesis that the cancer-proneness of BS patients may be in part due to the attenuation of an apoptotic pathway involving a p53-BLM-PML signaling cascade.