Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5–40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5β, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.
DNA helicases have important roles in cellular processes, including DNA replication, recombination, repair, and transcription, by unwinding the duplex genome strands (Lohman, 1993). Among many kinds of DNA helicases in living cells, the RecQ helicase family has unique properties apparently involved in maintaining genomic stability, although the exact biological pathways remain unknown. E. coli DNA helicase RecQ is the progenitor of this family and presumably participates in double-strand break repair and acts as a suppresser of illegitimate recombination as well as in replication restart (Courcelle and Hanawalt, 1999; Nakayama et al., 1984; Umezu and Nakayama, 1993; Umezu et al., 1990). Yeast S. cerevisiae and S. pombe have SGS1 and rqh1+helicases, respectively, that also belong to this family, and the lack of these helicases causes various disease phenotypes, such as slow growth, premature aging and aberrant assembly of chromosomes resulting primarily from the hyper- and illegitimate recombination events (Sinclair et al., 1997; Sinclair and Guarente, 1997; Stewart et al., 1997; Yamagata et al., 1998). In contrast to single-cell organisms that have only one species of RecQ helicase, higher eukaryotes contain multiple RecQ helicases. In humans the five members described to date are RecQL1 (Seki et al., 1994; Puranam and Blackshear, 1994), BLM (Ellis et al., 1995), WRN (Yu et al., 1996), RecQL4 and RecQL5 helicases (Kitao et al., 1998, 1999b). Recent studies showed that mutated BLM, WRN and RecQL4 helicases cause Bloom syndrome, Werner syndrome and a subset of Rothmund-Thomson syndrome, respectively, all of which have been known as recessive genetic disorders that have in common genomic instability, and they present complex clinical phenotypes but all with a high risk of cancer. In this paper, we use RTS (Rothmund-Thomson syndrome) in referring to the RecQL4 helicase even though it remains to be clarified whether the mutation in RecQL4 helicase is responsible for the etiology of all patients with Rothmund-Thomson syndrome. Association of mutations in the RecQL1 and RecQL5 helicases with human disorders has so far not been shown. We recently identified a new RecQL5 species formed by alternative splicing of RecQL5 gene, called RecQL5β. Because RecQL5β, but not RecQL5, localized to the nucleus (Shimamoto et al., 2000), we have focused on RecQL5β in this study.
Cumulated biochemical studies demonstrate that all RecQ helicases contain a helicase domain highly homologous to that of E. coli RecQ helicase, but that the N- and C- terminal regions differ from each other (Kitao et al., 1998). Cytochemical analyses indicate that the human RecQ helicases localize in the nucleus (Kitao et al., 1999a; Marciniak et al., 1998; Neff et al., 1999; Shiratori et al., 1999). In growing human fibroblast and K562 tumor cell lines, mRNAs coding for the five RecQ helicases are expressed throughout the cell cycle (Kitao et al., 1998), consistent with the presence of the Sp1-mediated house-keeping promoters characterized for WRN and RTS (Kitao et al., 1999a; Yamabe et al., 1998). Extensive studies on WRN helicase showed that the N- terminal domain has 5′−>3′ or 3′−>5′ exonuclease activity (Huang et al., 1997; Suzuki et al., 1999), and also DNA and RNA/DNA unwinding activity (Suzuki et al., 1997), suggesting its involvement not only in replication (Hanaoka et al., 1985) but also in transcription. WRN binds to the nuclear proteins RPA, p53, PCNA, topoisomerase I and Ku proteins, and the lack of WRN in patients' cells induces an attenuation of apoptosis (Brosh et al., 1999; Lebel et al., 1999; Spillare et al., 1999; Cooper et al., 2000). Functional interaction between the WRN helicase and DNA polymerase delta is also reported (Kamath-Loeb et al., 2000). BLM helicase is known to interact with RPA and topoisomerase IIIalpha (Brosh et al., 2000; Wu et al., 2000). Despite this biochemical knowledge, the biological role of each human helicase is still unclear, but the clinical and genetic data suggest that each helicase is involved in different tasks.
Cytogenetic studies indicate that the cells of patients with Werner, Bloom and Rothmund-Thomson syndromes undergo different types of genomic instability: pseudodiploidy involving variable structural rearrangements of chromosomes and abnormal dynamics of telomeres in Werner syndrome (Salk et al., 1985; Schulz et al., 1996; Tahara et al., 1997), an increase in sister chromatid exchange in Bloom syndrome (German, 1993) and trisomy 8 mosaicism in Rothmund-Thomson syndrome accompanied by mutations at RTS gene (Lindor et al., 1996). Thus, the RecQ helicases relevant to these disorders are predicted to maintain genomic integrity by their divergent functions that operate well in cells from healthy individuals but not in cells from patients lacking these RecQ helicases. Indeed, in a surrogate experiment using yeast cells lacking SGS1 helicase, Yamagata et al. (1998) showed that BLM and WRN genes can suppress hyper-recombination phenotypes of mutant yeast, implying that their role in human cells also is to suppress hyper-recombination. Also, Ellis et al. (1999, Neff et al., (1999) showed that the intact BLM gene transfected into the cells of a Bloom syndrome cell line reduced the increased sister chromatid exchange rate toward normal.
However, knowledge of the mechanisms regulating the expression of RecQ helicases is very limited (Yamabe et al., 1998). Related to this in an earlier study, we showed that the expression of WRN in human B cells of peripheral blood leukocytes (PBLs) and fibroblasts is increased by transformation with Epstein-Barr virus (EBV) and simian virus 40 (SV40), respectively (Shiratori et al., 1999). In the present study, we have compared the expression kinetics of the RecQ helicases WRN, BLM, RTS, RecQL1 and RecQL5β associated with the virus- or viral protein-mediated cell transformations as well as with stimulation by the tumor-promoting compound phorbol-12-myristate-13 acetate (PMA), using immunoblot analysis to measure the protein levels of individual RecQ helicases. The expression of BLM and RTS was highly dependent on cell cycle. On the other hand, RecQL5β was expressed at a relatively high level in resting B cells, and its expression was not changed during the cell cycle in normal diploid cell lines or by cell transformation. These results suggest that WRN, BLM, RTS and RecQL1 are up-regulated differentially by virus-mediated cell transformations and at certain stages of the cell cycle whereas RecQL5β plays a different role such as transcription in resting cells.
Helicase up-regulation during cell transformation by EBV and stimulation by PMA in B cells
EBV infects resting B cells in G0 and transforms them into B-lymphoblastoid cell lines (LCLs) (Miller, 1990). Figure 1a shows that the expression of the four RecQ helicases WRN, BLM, RTS and RecQL1 was up-regulated in LCLs after transformation by EBV: resting B cells (N0008R) expressed no WRN, BLM and RTS and a barely detectable level of RecQL1, but the EBV-transformed LCL N0008T expressed significant levels of these four RecQ helicases. On the other hand, resting B cells expressed a significant level of RecQL5β, which was not increased after transformation by EBV. Two EBV-transformed LCLs from WS patients WS11001 and WS11301 expressed no WRN, but expressed normal levels of other RecQ helicases BLM, RTS, RecQL1 and RecQL5β, indicating that the lack of normal WRN does not affect the expression of other RecQ helicases. The effect of PMA, a B cell mitogen (Bertoglio, 1983; Clevers et al., 1985), on the expression of the five RecQ helicases was also studied in resting B cells N0008R (Figure 1b). PMA was chosen because it stimulates the growth of human B cells. Adding PMA at a concentration of 30 ng/ml to the medium caused a marked increase in expression levels of WRN, BLM, RTS and RecQL1 although the level of RecQL5β is marginally decreased by the treatment. The levels of both RecQL1 and WRN markedly increased and reached near plateau levels within 5 h after PMA treatment, while the BLM and RTS proteins remained at the same level until 16 h and increased thereafter up to 40 h after treatment (Figure 1b). By PMA treatment, morphological changes of the B cells with notable clump formation were observed first at 5 h, and the size of the clumps increased thereafter (Figure 1c). Resting B cells purified from PBLs are in G0 of the cell cycle, and they are reported by Spender et al. (1999) to begin to express an early marker of cell cycle entry, cyclin D2, within 24 h after treatment with PMA or EBV. Given these facts, our results indicate (1) a sharp increase in WRN and RecQL1 occurs by PMA stimulation accompanied by clump formation of the B cells, which precedes PMA-induced cell proliferation (Abb et al., 1979; Clevers et al., 1985) and (2) up-regulation of BLM and RTS occurs when B cells initiate entry into the cell division cycle. This apparent correlation of the increase in the levels of BLM and RTS with entry into S phase of B cells agrees with the fact that TIG-3 and tumor cells in log phase expressed higher levels of BLM and RTS than cells in a stationary phase as will be shown later.
Table 1 shows the number of copies of the five RecQ helicase proteins per cell of resting B cells (N0008R), EBV-transformed LCL cells (N0008T) and immortalized EBV-transformed LCL cells (N6803IM). In both N0008T and N6803IM, the copy number was largest in WRN, followed by BLM, RecQ1, RTS and RecQL5β. The number of WRN copies of a LCL (N0008T) determined in this study (94 500 per cell) is near the number of an LCL (about 60 000 per cell) reported by Moser et al. (2000). Resting B cells (N0008R) are characterized by virtual absence of WRN, BLM and RTS with a level of RecQL5β comparable to that of N0008T and N6803IM. The immortalized LCL N6803IM have an abnormal karyotype with trisomy of chromosome 8 where WRN gene locates (Kataoka et al., 1997; Yu et al., 1996). Interestingly, N6803IM cells have WRN copies about twice those of pre-immortal LCL N0008T. RecQL5β is considered to play a role quite different from other RecQ helicases because the copy number of RecQL5β is much lower than that of other RecQ helicases and did not increase at all after transformation by EBV.
Up-regulation during cell transformation by SVtsT in fibroblasts and endothelial cells
We investigated the effect of cell transformation by SV40tsT on the expression of WRN, BLM, RTS, RecQL1 and RecQL5β in human diploid fibroblast TIG-3 cells (Figure 2a). Proliferating normal diploid TIG-3 cells expressed a high level of RecQL1 but lower levels of WRN, BLM, RTS and RecQL5β. By contrast, the two transformed cell lines SVts9-3 and SVts9-4, as well as the immortalized cell line SVts8, expressed markedly higher levels of WRN, BLM, RTS and RecQL5β than the untransformed TIG-3 cells, the immortalized SVts8 cell line being the highest expresser of RTS. No marked difference, however, was observed in the levels of RecQL1 helicase between pre- and post-transformed TIG-3 cells. Figure 2b shows the expression of WRN, BLM, RTS, RecQL1 and RecQL5β in various endothelial cell lines originated from HUE101-1: original HUE101-1; hT-1 and hT-2 transformed with hTERT gene, KA62, 31 and 31-2 transformed with SV40tsT; 62-5 transformed with hTERT gene and SV40tsT. KA62, 62-5 and 31-2 are immortalized cell lines. Transformation with hTERT or SV40tsT moderately increased the expression of WRN, BLM and RTS but not of RecQL1. The expression of RTS was highly augmented in all the cell lines transformed with SV40tsT. The expression of RecQL5β was not much affected by transformation with hTERT gene nor SV40tsT.
The SV40tsT gene used here originated from a mutant of SV40, tsA900, whose T-antigen was sensitive to temperature for many functions, such as virus replication, cell transformation, transcriptional repression of the SV40 early gene, and p53 binding (Kuchino and Yamaguchi, 1975; Tahara et al., 1995; Yamaguchi and Kuchino, 1975). We therefore investigated if the modulation of the expression of the five RecQ helicases by SV40tsT is sensitive to temperature. Figure 3a shows that expression of WRN at the non-permissive temperature 38.5°C was markedly lower than at the permissive temperature 34°C for the SVts9-3 cell line 51 and 84 PDLs, as well as for the immortalized cell line SVts8; for the non-ts SVOD1-2 cell line, little difference was observed between permissive and non-permissive temperatures. In addition, in three immortalized endothelial cell lines originated from HUE101-1 that were transformed with SV40tsT and hTERT, the expression of WRN alone was again down-regulated in a temperature-sensitive manner (Figure 3b). These results together indicate that the effect of SV40tsT in increasing the expression of WRN is apparently nullified at a non-permissive temperature in both TIG-3 fibroblast and endothelial cell lines transformed by SV40tsT. These data strongly suggest that SV40tsT is intimately involved in up-regulating the expression of WRN helicase. In this regard, we earlier reported that p53 suppressed the promoter activity of the WRN gene (Yamabe et al., 1998). From this observation, together with the fact that SV40tsT binds to p53 at 34°C but not at 38.5°C resulting in release of free p53 protein (Tahara et al., 1995), suppression of WRN expression at a non-permissive temperature may be mediated by p53 binding to the WRN promoter. The fact that WRN helicase binds to p53 (Spillare et al., 1999) raises an alternative possibility that p53 changes the levels of WRN by direct interaction. The possibility also remains that SV40tsT directly binds temperature-sensitively to WRN, stabilizing WRN helicase. No consistent effect of temperature, however, was observed for the expression of BLM, RTS, RecQL1 and RecQL5β. Although the expression of RTS in SVts9-3 at 84 PDLs was apparently lower in permissive than in non-permissive temperature, this phenomenon was not observed in SVts9-3 at 51 PDLs nor SVts8, and is considered to be not a consistent phenomenon (Figure 3a). The fact that the expression levels of RecQL1, BLM, RTS and RecQL5β were largely not sensitive to temperature suggests that their mechanisms of regulation are different from WRN helicase.
Changes during the cell cycle
We compared the expression levels of the five RecQ helicases in TIG-3 cell lines between log and stationary phases. Figure 4a shows that in the TIG-3 cell line the levels of WRN, BLM, RTS and RecQL1 decreased as the cells shifted from log (days 1 and 2) to stationary phases (days 3 and 4) whereas the level of RecQL5β was unaffected; the decrease was much more pronounced for BLM and RTS than for WRN and RecQL1. The levels of WRN, BLM, RTS, RecQL1 and RecQL5β were also compared in the four tumor cell lines U251MG, T98G, A2182 and KMST-6 (Figure 4c). In all the cell lines, the levels of BLM, RTS and RecQL5β apparently decreased from day 2 to day 4, whereas the change in the levels of WRN and RecQL1 was less pronounced. Analyses by flow cytometry of TIG-3 and U251MG cell lines indicated that the proportions of S phase cells were far greater when the cells were in log phase on day 2 than the cells were in the stationary phase on day 4; the proportions of cells at S plus G2/M on day 2 were 33.1% for TIG-3 and 47.6% for U251MG, whereas on day 4 they were 6.6% for TIG-3 and 11.8% for U251MG (Figure 4b, d). In these experiments the medium was not changed during the four-day culture, but similar experiments were done by changing the medium every day for four days (data not shown). These two series of experiments gave essentially the same results for immunoblot and flow cytometry analyses. Thus, the decline in the levels of RecQ helicases at late phases of culture was unlikely due to waste of nutrient or accumulation of metabolic products. The higher levels of BLM and RTS at the S, G2 and M phases in actively dividing cells imply that BLM and RTS are perhaps sepecifically synthesized or maintained in the late G1 and/or S phases.
The results obtained in this protein-kinetic study contrasted the following differential modes of expression of the five human RecQ helicases. (1) RecQL1 was expressed at a maximal level in all the cells examined, except for resting B cells in G0, which agrees with our previous observation that RecQL1 mRNA was expressed at similar levels in almost all tissues examined (Kitao et al., 1998). These facts also suggest that RecQL1 is present at all stages of the cell cycle except G0 and that RecQL1 is involved in a housekeeping function. (2) The expression of WRN increased together with cell transformation by EBV and SV40tsT, suggesting that WRN is involved in some special function required to transform cells, such as to stabilize the genome in rapidly proliferating cells as discussed later. The increase in WRN at early stages after PMA stimulation agrees with the idea that WRN may be involved in a function in G1 of dividing cells, such as in transcription mediated by RNA polymerase I or II (Balajee et al., 1999). We and others reported that WRN exists in the nucleoplasm, as well as in the nucleolus of rapidly proliferating cells (Marciniak et al., 1998; Shiratori et al., 1999; Kiao et al., 1999a). Also, WRN can unwind RNA/DNA heteroduplexes that would be formed during the transcription (Suzuki et al., 1997). (3) Increased expression of BLM and RTS at later stages of PMA stimulation, as well as at the log phase of cell culture, is likely to be intimately associated with DNA replication. This hypothesis agrees with the observation in this study that the expression of BLM and RTS was not sensitive to the temperature shift in TIG-3 cells transformed by SV40tsT, because induction of host DNA synthesis by SV40tsT was similarly insensitive to temperature: the SV40tsT used in this study can induce and maintain host DNA synthesis at both permissive and non-permissive temperatures in resting cells (Ide et al., 1983, 1984). Recently, Ellis et al. (1999); Neff et al. (1999) showed that increased sister chromatid exchange in cells from Bloom syndrome patients is suppressed by complementation with normal BLM. Our observation that BLM are up-regulated during DNA replication implies that BLM is perhaps involved in suppressing the sister chromatid exchange that is considered to occur during DNA replication. (4) The mode of the expression RecQL5β was quite different between normal cells and tumor cells. RecQL5β was unique in that this helicase was expressed at a relatively high level even in resting B cells, and its expression was not much affected by transformation with EBV or by stimulation with PMA. Furthermore, the expression of this helicase was not cell cycle dependent in the diploid human fibroblast cell line (TIG-3). It is also noted that the mRNA of RecQL5 was expressed in almost all the tissues examined including the brain, liver, skin and muscle that are considered to contain a relatively small number of replicating cells (Kitao et al., 1998). These facts suggest that in normal tissues RecQL5β plays an important role in resting cells in G0 and G1, perhaps in transcription. In the four tumor cell lines, however, the expression of RecQL5β was cell cycle-dependent, and was upregulated in log phase (Figure 4c). These results suggest that RecQL5β plays different roles in normal diploid cells and tumor cells.
Lastly, we speculate on the biological meaning of the up-regulation of RecQ helicases in general during transformation with DNA tumor viruses or stimulation with PMA. Werner, Bloom and Rothmund-Thomson syndromes are characterized by genomic instability in patient cells, accompanied by a high incidence of cancer (Ellis et al., 1995; German, 1993; Goto et al., 1996; Kitao et al., 1999b; Lindor et al., 1996; Yu et al., 1996). For instance, EBV-transformed LCLs from Werner syndrome patients show various increased chromosomal aberrations (Salk et al., 1985; Tahara et al., 1997), suggesting that WRN helicase is required for genomic stability, notably detected in actively dividing transformed cells. The fact that B cells from the patients lacking WRN can be transformed by EBV into LCLs as efficiently as those from normal individuals (Tahara et al., 1997) indicates that these RecQ helicases are not essential in the process of cell transformation. On the other hand, cells from Bloom syndrome patients often fail to give a lymphoblastoid cell line when exposed to EBV (JL German, personal communication), suggesting that BLM plays an important role in transformation of B cells. Natural infection with DNA tumor viruses is considered to induce transformation of various cells in vivo, such as acute mononucleosis caused by EBV infection. In this regard, Burkitt lymphoma accompanied by characteristic tumor-specific chromosomal translations is considered to arise in boys with X-linked lymphoproliferative disease after infection by EBV (Egeler et al., 1992).
Resting B cells become actively proliferating lymphoblasts by antigen stimulation in immune responses. RecQ helicases may be up-regulated in EBV-transformed cells and antigen-stimulated B-lymphoblasts in vivo, as observed in vitro in this study. We speculate that the increased levels of RecQ helicases in proliferating cells may be required to guarantee genomic stability of these cells. Previously, we showed that WRN helicase, when introduced into the yeast sgs1 mutant, could suppress the hyper-recombination of the mutant yeast cells which lack SGS1 protein belonging to the RecQ helicase family (Yamagata et al., 1998). In addition, we showed that LCLs from Werner syndrome patients lacking WRN helicase show augmented genomic instability accompanied by abnormal telomere dynamics (Tahara et al., 1997). These findings further support the notion that WRN helicase is involved in genome stabilization.
As the effects of PMA on normal cellular functions are mostly mediated by activating the enzyme protein kinase C (Castagna et al., 1982), the early increase in WRN and RecQL1 after PMA treatment could also be mediated by this pathway. Alternatively, it is possible that DNA tumor viruses use the same pathway or an unidentified alternative strategy to augment host RecQ DNA helicases, eventually to maintain their own genomic stability since SV40tsT seems to be directly involved in up-regulating WRN helicase.
The results of this study strongly support the idea that up-regulation of WRN, BLM, RTS and RecQL1 is firmly associated with actively dividing cells, and perhaps contributes in various ways to the genomic stability of cells that are expected to be faced with more errors in DNA replication, or entangled DNA or RNA/DNA molecules occurring during active gene expression. RecQL5β, however, may play other important function(s) in normal diploid cells in G0.
Materials and methods
Cells and cell culture
The purification of PBLs, transformation of PBLs by EBV, and the culture of established B-lymphoblastoid cell lines (LCLs) from a normal individual (N0008T) and Werner syndrome patients WS11001 and WS11301 with WRN gene mutations 6/6 and 1/4, respectively (Matsumoto et al., 1997), have been described (Tahara et al., 1997). An immortalized EBV-transformed LCL with strong telomerase activity (N6803IM) was also used. CD19-positive resting B cells were purified from PBLs from normal individuals using the pan-B Dyanabeads M450 kit (Dyanal, Oslo), and were resuspended at 106/ml in the culture medium: RPMI1640 (pH 6.5) supplemented with penicillin, streptomycin and 10% heat-inactivated fetal calf serum. In the experiments to examine the effect of PMA, B cells were incubated with 30 ng/ml PMA for 0, 5, 16 and 40 h. In the cell transformation experiment with SV40 temperature sensitive (ts) T-antigen, human fetal lung fibroblasts TIG-3 (Matsuo et al., 1982) were transformed with pMT-1ODtsA that codes for an origin-defective SV40tsT gene from which were established cell lines SVts9-3, SVts9-4 and SVts8 (Tahara et al., 1995). SVts9-3 and SVts9-4 cells had a prolonged lifespan, but senesced after serial passage around 100 population doubling levels (PDLs): SVts8 was immortalized (Tsuyama et al., 1991). SVOD1-2 fibroblasts were transformed by normal SV40 T-antigen. All these cells were seeded at 5×105 cells/100 mm dish at 34°C, and the next day the temperature was changed to 38.5°C for the cells of non-permissive temperature groups. Cells were recovered by trypsinization 24 h after the temperature change. Control cells were cultured continuously at permissive temperature 34°C. HUE101-1 (Amanuma and Mitsui, 1991), a human endothelial cell line isolated from human umbilical cord veins, was cultured at 37°C in MCDB151 medium (Sigma) supplemented with 10% fetal bovine serum (BioWhittaker), 5 ng/ml of acidic fibroblast growth factor (Wako Pure Chemicals, Osaka, Japan), 5 μg/ml of heparin sodium salt (GIBCO BRL). HUE101-1 cells ceased proliferation at approximately 65 PDLs. KA62, 7, 18, 27 and 31 cell lines were established by transforming HUE101-1 cells with pMT1-ODtsA. Transformed cells showed an extended proliferative life span, and some of them were immortalized. hT-1 and hT-2 cells were established by transformation of HUE101-1 with pIRES-hTERT-hygr which carried telomerase reverse transcriptase gene (hTERT) in pIRES-hygr vector with hygromycin resistance gene. 7–3, 18–11, 27–13, 31–2 and 62–5 cell lines were established by transforming 7, 18, 27, 31 and KA62 cells with pIRES-hTERT-hygr, respectively, and all were immortalized. The characteristics of these transformed cell lines will be published elsewhere. pIRES-hTERT-hygr was kindly donated by Professor Fuyuki Ishikawa, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology. The glioblastoma cell lines U251MG and T98G, as well as fibroblast tumor cell line A2182 and transformed fibroblast cell line KMST-6, were kindly donated by Professor M Oshimura of the Faculty of Medicine of Tottori University.
Antibodies and immunoblotting
Rabbit polyclonal antibodies against human RecQL1 helicase (Tada et al., 1996) and BLM (Neff et al., 1999) were reported previously as was mouse monoclonal antibody 4H12 against the C-terminal end of WRN helicase (Shiratori et al., 1999). Mouse monoclonal antibodies against human RTS and RecQL5β helicases were prepared by immunizing mice with a purified recombinant C-terminal fragment of RTS (amino acid residues 907–1208) or of RecQL5β (amino acid residues 848–991), respectively (the details of these antibodies will be published elsewhere). Antibodies against BLM (C-18) (Santa Cruz Biotechnology Inc., California USA) was also used. The antibodies against WRN, BLM, RTS, RecQL1 and RecQL5β stained the bands of 180, 180, 160/140, 73 and 135 kDa in immunoblot, respectively. The antibodies against WRN, BLM and RTS were shown in other studies to stain the corresponding bands or cells for samples from normal individuals, but they failed to stain the bands or cells of samples from Werner syndrome (Goto et al., 1999; Shiratori et al., 1999), Bloom syndrome (unpublished observation) and Rothmund-Thomson syndrome patients (unpublished observation), respectively. Because the antibody against RTS did not stain the major (160 kDa) and minor bands (140 kDa) of the samples from Rothmund-Thomson patients, we judged that both the major and minor bands represent specific RTS (unpublished observation). The antibody against RecQL5β mainly stained nuclei (Shimamoto et al., 2000). Mouse monoclonal IgG1 against α-actin (ICN Costa Mesa, CA, USA) was used to confirm that the same amounts of protein were applied to the sodium dodecylsulfate-polyacrylamide gels. The detailed procedure of the immunoblot was reported (Shiratori et al., 1999). Briefly, the cell pellets were lysed by RIPA buffer containing 10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 0.15 M NaCl, 1 mM ethylenediaminetetraacetate and protease inhibitors (CompleteTM Protease Inhibitor Cocktail Set, Roche Diagnostics K.K., Kamakura, Japan), and the proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred to the polyvinylidene difluoride membrane Immobilon (Millipore) and the membrane was blocked by incubating for 1 h in a buffer containing 5% skim milk and 0.2% Tween 20. RecQ helicases were detected using a specific monoclonal or polyclonal antibody followed by rabbit anti-mouse immunoglobulin, rabbit anti-goat immunoglobulin or goat anti-rabbit immunoglobulin that were conjugated with horseradish peroxidase (Dako, Denmark). The membrane was developed using enhanced chemiluminescence ECLTM (Amersham Life Science, UK).
Determination of the number of RECQ helicase copies per cell
The number of molecules per cell were roughly determined for the five RecQ helicases essentially according to the method by Moser et al. (2000). Briefly, we used quantitative immunoblotting versus purified recombinant RecQ helicase standard proteins to determine the number of copies per cell for resting B cells, EBV-transformed LCL (N0008) cells and immortalized LCL (N6803) cells. The recombinant standard proteins with 6×HIS at the N teminus were as follows: rWRN (877–1432 a.a.), rBML (1248–1415 a.a.), rRTS (907–1208 a.a.), rRecQL1 (339–649 a.a.) and rRecQL5β (848–991 a.a.).
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Kawabe, T., Tsuyama, N., Kitao, S. et al. Differential regulation of human RecQ family helicases in cell transformation and cell cycle. Oncogene 19, 4764–4772 (2000). https://doi.org/10.1038/sj.onc.1203841
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