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1 Department of Immunology and Molecular Genetics, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan
2 Department of Biochemistry, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-1692, Japan
3 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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Saccharomyces cerevisiae has four additional Sir2-like genes termed ‘HST’ (homologue of Sir two), whereas humans have seven obvious homologues termed ‘SIRT.’ Given the potential link with longevity (Picard et al. 2004; Wood et al. 2004), physiological substrates of vertebrate SIRT genes are under extensive investigation. Although several of them deacetylate histones in vitro (Imai et al. 2000), Sir2 homologues have non-histone targets. For example, SIRT2 and SIRT3 were shown to have a role in tublin deacetylation (North et al. 2003) and a role in mitochondria (Onyango et al. 2002), respectively. Furthermore, several studies now identified important substrates of the SIRT1 in the regulation of apoptosis, including tumor suppressor p53 (Langley et al. 2002; Luo et al. 2001; Vaziri et al. 2001), FOXO transcription factors (Brunet et al. 2004; Daitoku et al. 2004; van der Horst et al. 2004; Motta et al. 2004), ku70 (Cohen et al. 2004), and NF-
B (Yeung et al. 2004).
To gain more insight into the function of SIRT1 and SIRT2, we established cells deficient in either SIRT1 or SIRT2 as well as SIRT1/2 double deficient cells using gene targeting technology in the chicken B cell line DT40 (Buerstedde & Takeda 1991). In particular, we wished to elucidate a role of SIRT1 and SIRT2 in the DNA damage response, given the relocalization of the Sir2 complex to sites of DSBs upon DNA damage in S. cerevisiae (Mills et al. 1999). We found that both SIRT1- and SIRT2-deficient cells are sensitive to the DNA damaging agent cisplatin, while only SIRT1–/– cells are hypersensitive to ionizing radiation (IR). Curiously, SIRT1–/–/SIRT2–/– double deficient cells did not show any additive sensitivity to cisplatin compared to either single mutant. DNA DSB repair and the G2/M checkpoint appeared to be intact in SIRT1–/– cells, but these cells are more susceptible to IR-induced cell death, explaining the hypersensitivity of SIRT1–/– cells to IR. Both SIRT1–/– and SIRT2–/– cells are more susceptible than controls to cell death induction by cisplatin and staurosporine but not by UV treatment. These results suggest that SIRT1 and SIRT2 participate in stress responses by regulating cell death through the deacetylation of target proteins.
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Results |
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We isolated full-length chicken SIRT1 or SIRT2 cDNAs (DDBJ accession number AB160952 or AB160951) by combination of EST searching, reverse transcriptase-polymerase chain reaction (RT-PCR), and library screening as described in the Experimental procedures.
The open reading frames of chicken SIRT1 and SIRT2 cDNA encode predicted proteins of 757 or 389 amino acids, respectively. The overall identity between the human and chicken Sirt1 proteins is 
69%, whereas the catalytic domains of the two species share 95% identity. Chicken Sirt2 is overall 67% identical to its human counterpart with 79% identity in the catalytic domains. Among human SIRT family proteins, chicken Sirt1 or Sirt2 amino acid sequence has highest degree of identity to the corresponding human Sirt1 or Sirt2 (Fig. 1B).
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To characterize the SIRT mutants, we looked at proliferative kinetics and cell cycle distribution by flow cytometeric analysis. Both SIRT1–/– and SIRT2–/– cells grew more slowly than wild-type cells. Furthermore, SIRT1/SIRT2 double mutant grew more slowly than either single mutant (Fig. 4A). The distribution of these mutants in each cell cycle stage and the percentage of subdiploid cells did not appear to be significantly different from those of wild type (data not shown). These observations suggest that the slower proliferation rate of the mutant cells might be explained by a prolonged cell cycle time. Indeed, pulse-chase experiment of cells labeled with bromodeoxyuridine (BrdU) revealed that the duration of a single cell cycle of SIRT1–/– cells was slightly lengthened (11 h in SIRT1–/– vs. 10.5 h in wild type).
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To measure the ability of mutant cells to cope with stress by DNA damage, colony formation capacity was assayed following exposure to DNA damaging agents. SIRT1–/– cells were moderately sensitive to cisplatin compared with wild type and SIRT2–/– cells exhibited mild sensitivity to the same drug (Fig. 4B). In contrast, SIRT1–/– but not SIRT2–/– cells were sensitive to ionizing radiation. Such a role for SIRT1 in IR sensitivity was previously reported using SIRT1-deficient mouse splenocytes (Cheng et al. 2003) and cells transfected with dominant negative constructs (Vaziri et al. 2001). In contrast to the additive effects in growth profile, SIRT1/2 double deficient cells were no more sensitive to either cisplatin or ionizing radiation than the SIRT1 single mutant (Fig. 4B). In addition, neither SIRT1–/– nor SIRT2–/– cells were sensitive to UV compared to wild type (Fig. 4B). To prove that the observed defects were indeed caused by specific gene disruption of SIRT1, we expressed chicken SIRT1 cDNA in SIRT1–/– cells. SIRT1 expression was verified by EGFP fluorescence that was expressed from the same construct (bicistronic expression). Full-length chicken SIRT1 cDNA was able to normalize cisplatin sensitivity (Fig. 4B). SIRT2 complementation has been unsuccessful because no colony was obtained after repeated transfections with the expression vector, suggesting that over-expression of SIRT2 has inhibitory effects on cell growth.
DSB repair pathways are intact in SIRT1–/– cells
The main mechanism of DSB repair in yeast or vertebrates consists of two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). The elevated IR sensitivity of SIRT1–/– cells (Fig. 4B) might be compatible with the idea that, similar to S. cerevisiae Sir2, vertebrate SIRT1 may regulate the expression of a key factor in NHEJ or DSB repair. To examine the effect of SIRT1 deficiency in NHEJ, we generated cells deficient in both SIRT1 and KU70. Ku70 is a key regulator in NHEJ by forming stable heterodimer with Ku80 protein. Ku70/80 complex is thought to bind to DSB ends and initiate NHEJ pathway with other proteins including Dna-Pk, Artemis/SNM1C, Xrcc4, and Ligase IV (van Gent et al. 2001; Lieber et al. 2003). In previous studies, it was demonstrated that cells rendered doubly deficient in KU70 and DNA-PKcs or KU70 and LIGASE IV exhibited exactly the same IR sensitivity curve as KU70 single mutants, indicating that Dna-Pk and Ligase IV are involved in NHEJ and that their activities are Ku70-dependent (epistasis) (Adachi et al. 2001; Fukushima et al. 2001). Furthermore, deletion of the HR factor Rad54 in KU70 mutant leads to significantly additive levels of IR sensitivity (Takata et al. 1998), confirming the validity of this approach. The increased resistance to higher dose IR in KU70–/– cells has been interpreted as being the result of increased availability of DNA ends to HR machinery in the absence of the DNA end binding factor Ku70 (Takata et al. 1998; Adachi et al. 2001; Fukushima et al. 2001). As shown in Fig. 5A, two clones of SIRT1–/–/KU70–/– double deficient cells clearly exhibited higher IR sensitivity compared with KU70–/– cells, indicating that SIRT1 or its target gene do not function in the NHEJ pathway.
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The previous observations in turn raised a possibility that another DSB pathway, HR, might be affected in the absence of SIRT1. To test directly whether SIRT1–/– cells are defective for HR-mediated repair of DSBs, we used SIRT1–/– cells that carry the artificial recombination substrate, SCneo (Johnson et al. 1999), at the OVALBUMIN locus (Fukushima et al. 2001). By using transient transfection of the plasmid, encoding the rare restriction enzyme I-SceI, DSBs can be produced in one of two tandem non-functional neo genes. If the DSB is repaired by HR with another tandem neo as a template, a functional neo gene can be reconstituted. Thus, the frequency of G418-resistant colonies represents the HR-directed DSB repair capacity. No difference between SIRT1–/– and wild-type cells was seen in this assay (Fig. 5C), indicating that HR repair appears to work normally. Furthermore, we noticed that the efficiency of gene targeting in SIRT1–/– background was not compromised during SIRT2 disruption (data not shown). Finally, we looked at the number of chromosomal breaks after IR, as DSB repair mutants generally have increased number of breaks following IR. X-ray-irradiated SIRT1–/– and wild-type cells were examined in four consecutive time slots by sampling at 3-h intervals. SIRT1–/– cells did not exhibit more chromosomal aberrations than wild-type cells (Fig. 5D). Taken together, these data indicate that the radiosensitivity of SIRT1–/– cells was not the result of defective DSB repair.
Absence of G2/M cell cycle checkpoint defects in SIRT1–/– and SIRT2–/– cells
Next we looked at whether SIRT mutants have any defects in G2/M checkpoint. To this end, cells were either irradiated (2 Gy) or left unirradiated, and then mitotic index was determined every hour by Hoechst staining in the continuous presence of colcemide. Overall, IR-induced delay in the increase in mitotic index was similar in both wild-type and mutant cells, indicating that deficiency in both SIRT1 and SIRT2 does not affect G2/M DNA damage checkpoint (Fig. 6A). Interestingly, in SIRT1–/–, SIRT2–/– and SIRT1–/–/SIRT2–/– cells, the mitotic index at time 0 appeared elevated compared with wild type (Fig. 6B). Except for SIRT2–/– cells, this difference was statistically significant.
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SIRT1 has been previously shown to negatively regulate the pro-apoptotic factors such as p53 or FOXO3a (Luo et al. 2001; Vaziri et al. 2001; Langley et al. 2002; Motta et al. 2004). The IR sensitivity of SIRT1–/– and the cisplatin sensitivity of SIRT1–/– or SIRT2–/– cells might be because of increased cell death following DNA damage, although DT40 cells lack p53. Therefore, we examined the effect of SIRT1 or SIRT2 deficiency on cell viability after DNA damage or other stress stimuli. At appropriate hours after various treatments, cells were stained with PI and immediately examined using FACSCalibur. PI-positive cells were scored as dead cells.
As shown in Fig. 7A, the percentage of surviving cells in single and double mutants decreased significantly after exposure to cisplatin compared to wild type, whereas IR induced more cell death only in SIRT1–/– and SIRT1–/–/SIRT2–/– cells. Furthermore, all mutant cells showed a reduced fraction of surviving cells after staurosporine or oxidative stress treatment (H2O2) but not after UV irradiation (Fig. 7A). These results are consistent with the cell survival data described previously. Taken together, we conclude that SIRT1 and SIRT2 likely regulate cell death in response to various stress stimuli including DNA damage.
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Transcriptional activity of human p53-related genes in SIRT1 or SIRT2-deficient cells
The p53-family members, p63, p53 or p73, are important regulators for apoptotic cell death (Ishida et al. 2000). Although it is well established that p53 is regulated by deacetylation by SIRT1 (Luo et al. 2001; Vaziri et al. 2001; Langley et al. 2002), a role of SIRT1 or SIRT2 in p63 or p73 regulation is less clear. We introduced the human p63
, p53, or p73ß expression vectors into wild-type and mutant DT40 cells, and measured transcriptional activation of co-transfected luciferase reporter. We observed that, compared to wild-type cells, p53 expression activated particularly robust luciferase transcription in SIRT1–/–, SIRT2–/– and HDAC1–/– cells (Fig. 8). By p73 expression, SIRT1–/– and HDAC1–/– cells induced much stronger transcription of the reporter than wild-type cells. Interestingly, in any of the tested mutants, reporter activation by p63 was not significantly elevated compared to wild-type cells (Fig. 8). These results confirm the previous reports (Luo et al. 2000, 2001; Vaziri et al. 2001; Langley et al. 2002), and may suggest that p73 is strongly down-regulated by Sirt1 and HDAC1 in vertebrate cells.
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Discussion |
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We have also found that SIRT1- or SIRT2-deficient cells are moderately or mildly sensitive to cisplatin, respectively. Only SIRT1-deficient cells are sensitive to IR. The levels of IR sensitivity of SIRT1/2 double deficient cells were the same as that of SIRT1-deficient cells, indicating that SIRT2 plays no significant role in IR sensitivity. Interestingly, cisplatin sensitivity in SIRT1/2 double mutant cells was the same as that of SIRT1-deficient cells (see succeeding discussion).
We tested the DSB repair capability of SIRT1–/– cells, because they are sensitive to IR and because the yeast SIRT1 ortholog, Sir2, affects DSB repair by regulating an essential component of the pathway. We conclude that the IR sensitivity of SIRT1–/– cells is not the result of defects in DSB repair from several observations. First, KU70/SIRT1 double deficient cells showed clearly additive sensitivity compared to KU70–/– cells (non-epistatic relationship). This is in sharp contrast to the NHEJ factors such as DNA-PK or LIGASE IV that were previously examined in this manner. Second, direct measurements of HR capability utilizing integrated HR substrate SCneo in SIRT1–/– cells were entirely normal. Third, unlike any DSB repair mutants, SIRT1–/– cells did not show increased levels of IR-induced chromosome aberrations. Consistently, a previous study found no obvious defects in two classical NHEJ events, V(D) J and class switch recombination, in SIRT1-deficient mice (Cheng et al. 2003). The fourth, and perhaps most importantly, we found that neither SIRT1–/– nor SIRT2–/– cells displayed significant defects in intracellular re-ligation of introduced plasmid using our novel assay. We also showed that SIRT1–/– cells did not have defects in G2/M checkpoints after IR exposure. We included SIRT2- and SIRT1/SIRT2 double deficient cells in this analysis and the results were similar to SIRT1–/– cells.
We confirmed that both SIRT1- and SIRT2-deficient cells displayed increased susceptibility to apoptotic cell death induced by the drug. These cells were more sensitive to cell death induction by number of stimuli including cisplatin, staurosporine and oxidative stress, but not by UV exposure. In addition, SIRT1-deficiency, but not SIRT2-deficiency, imposed more susceptibility to IR. The mechanism of cell death involves a number of factors, and SIRT1 and SIRT2 could participate in this pathway by deacetylating some components in a context-specific manner. Alternatively, they might affect target protein levels by transcriptional silencing (through histone modification). In any case, as DT40 lacks expression of p53 (Takao et al. 1999), regulation of cell death by SIRT1 and SIRT2 should be through p53-independent mechanism.
Interestingly, the SIRT1/2 double mutant cells did not show any additive effects on cell death induction, relative to either single mutant under all stimuli tested, while they did show additive effects in cell growth. SIRT1 and SIRT2 may have distinct targets that work in the same cell death pathway, thus leading to epistasis. These targets might vary depending on the stimulus. For example, SIRT2 seems to have no role in IR-induced cell death. Alternatively, they may have a common deacetylation target and the absence of either one of them results in full down-regulation of the target's activity.
It was reported that Sirt1 resides in the nucleus whereas Sirt2's localization is limited to cytoplasm (Afshar & Murnane 1999). We confirmed this distinct localization by transient transfection into HeLa cells of human SIRT1 or SIRT2 green fluorescent protein (GFP)-fusion constructs (data not shown). These localizations did not undergo any changes after treatment with cisplatin (data not shown). Hence, if SIRT1 and SIRT2 may have a shared target/substrate, this target should be freely distributed in both compartments, although the exact localization of chicken Sirt proteins has not been explored. In keeping with this idea, yeast HST2 (SIRT2 homologue) and yeast Sir2 (SIRT1 homologue) are proposed to share possible ligands for telomere position effects, which shuttles between the nucleus and the cytoplasm (Perrod et al. 2001).
It is likely that both SIRT1 and SIRT2 have a relatively limited number of substrates. There were numerous bands in immunoblotting of whole cell lysates with an anti-acetylated lysine antibody in DT40 cells. We could not detect any notable change in acetylated protein levels in SIRT1–/– or SIRT2–/– cells compared to wild-type cells (our unpublished observation). It has also been reported that there is no global defect in gene silencing in cells from SIRT1-deficient mice (McBurney et al. 2003). In addition to p53, recent studies expand our knowledge about proteins modulated by acetylation/deacetylation, which include p73 (Costanzo et al. 2002), E2F (Martinez-Balbas et al. 2000; Pediconi et al. 2003), Forkhead transcription factors (Brunet et al. 2004; Daitoku et al. 2004; van der Horst et al. 2004; Motta et al. 2004), and Ku70 (Cohen et al. 2004). In the absence of SIRT1, Ku70 might be hyper-acetylated, leading to disruption of Ku70-BAX interaction. This in turn releases more Bcl-2 associated x protein (BAX), which promotes apoptosis (Cohen et al. 2004). However, hyper-acetylated Ku70 is an unsatisfactory explanation for IR sensitivity of sirt1-deficient cells, because ku70/sirt1 double deficient cells showed higher IR sensitivity than sirt1-deficient cells (additive phenotype). Alternatively, p73 might be a good candidate for a target of SIRT1 and SIRT2, because it is induced only by a subset of DNA-damaging agents, including IR and cisplatin (Agami et al. 1999; Gong et al. 1999; Yuan et al. 1999), and not by UV (Kaghad et al. 1997). Indeed, transiently expressed human p73 in our SIRT1–/– and SIRT2–/– cells exhibited increased transcriptional activity compared to wild-type cells. In any case, DNA damage sensitivity of the cells deficient in SIRT1 or SIRT2 could be caused by sum of effects on modulators of apoptosis induction.
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Experimental procedures |
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DT40 cells were cultured at 39.5 °C with 5% CO2 in RPMI1640 media supplemented with 10% foetal calf serum, 1% chicken serum, and 10–5ß-mercaptoethanol (Buerstedde & Takeda 1991). Stable or transient transfections were done as previously described (Buerstedde & Takeda 1991; Fukushima et al. 2001). Generation of KU70–/–, HDAC1–/– or HDAC2–/– cells were previously described (Takata et al. 1998; Takami et al. 1999).
Cloning of chicken SIRT1 and SIRT2
We amplified part of the chicken SIRT1 cDNA from DT40 using degenerate primers based on human and mouse SIRT1 amino acid sequences, and used this PCR product as a probe in screening a DT40 
ZAP II cDNA library as described (Takami et al. 1999). The extreme N-terminal part of chicken SIRT1 cDNA was identified by sequencing a SIRT1 genomic clone (see succeeding discussion), and was fused with the rest of the cDNA by PCR.
A partial chicken cDNA sequence for SIRT2 was identified in the chicken Web Bursal EST database <http://www.chick.umist.ac.uk/index.html>, and was subsequently used as a probe in screening a chicken intestinal mucosa cDNA library (Stratagene, La Jolla, CA). To isolate full-length SIRT2 cDNA, the N-terminal portion of chicken SIRT2 was PCR-amplified from DT40 cDNA on the basis of the sequence of another chicken expressed sequence tag (EST) clone. Identity of the cDNA clones was confirmed by sequencing using the dye terminator method (Applied Biosystems Division, Perkin-Elmer, Wellesley, MA). Genomic DNA clones of SIRT1 and SIRT2 were isolated by screening a chicken genomic library (Stratagene) with chicken cDNAs as probe. Genomic DNA fragments of the SIRT1 or SIRT2 loci were amplified either from these genomic clones or DT40 genomic DNA using a pair of specific primers. Amplified fragments were subsequently subcloned into the TOPO-XL cloning vector (Invitrogen, Carlsbad, CA).
The SIRT1 or SIRT2 expression vectors were constructed by inserting the full-length chicken cDNAs into pCR3-loxP-IRES/EGFP-loxP vector (Fujimori et al. 2001). Expressing clones were identified by measuring enhanced green fluorescent protein (EGFP) fluorescence by flow cytometry.
Generation of SIRT1 and SIRT2 deficient cells
To construct SIRT1 targeting vectors, upstream (1.5 kb) and downstream (2.5 kb) fragments of the SIRT1 locus were excised from genomic clones and transferred into pBS vector (Stratagene). The bsr or hisD drug resistance cassette flanked by loxP sequences on both sides was created and inserted into BamHI site in the vector. Gene targeting by these constructs was expected to delete exon 5 and part of exon 4 together with the intervening intron, resulting in deletion of the coding sequence corresponding to chicken SIRT1 amino acids 318–379.
SIRT2 gene targeting vector was also constructed in pBS vector by subcloning upsteam (4 kb) and downstream (2.5 kb) genomic fragments. Then the hisD or neo drug resistance cassette (Sugawara et al. 1997) was inserted into BamHI site. Gene targeting with this construct was expected to replace the exons that encode chicken SIRT2 amino acids 84–389 with the selection markers.
Generation of SIRT1–/–/SIRT2–/– double deficient cells
The loxP-flanked bsr and hisD resistance markers used in generation of SIRT1–/– cells were removed by Cre recombinase-mediated excision. SIRT1–/– cells were transiently transfected with Cre expression plasmid pBS185 (Invitrogen), and then subcloned by limiting dilution. Loss of the drug resistance was screened by exposing a fraction of expanded subclones to both blasticidin and histidinol for 72 h. Among 192 clones tested, 10% regained sensitivity to both drugs. Resistance cassette removal was confirmed by Southern blot analysis using a SIRT1 probe. The SIRT2 gene was disrupted in one such clone using the SIRT2-hisD and then SIRT2-neo targeting vectors described above.
Flow cytometric analysis of cell number, cell cycle, and EGFP expression
All flow cytometric analyses were carried out using a FACSCalibur (Beckton-Dickinson, Mountain View, CA) as described (Takata et al. 1998). Briefly, cell numbers were counted by flow cytometer by mixing fixed numbers of plastic beads (Polysciences, Inc.,Warrington, PA) with cells. For cell cycle analysis, cells were cultured in the presence of BrdU for 10 min, then fixed and stained with anti-BrdU antibody (Pharmingen, San Diego, CA) and propidium iodide. EGFP expression was monitored by FACSCalibur as green fluorescence-positive cells.
Measurement of sensitivity of cells to X-rays, UV, and cisplatin
Clonogenic survival was monitored by colony formation assay as previously described (Takata et al. 1998). Serially diluted cells were plated in medium containing methylcellulose and then irradiated with 4 MV of X-rays (linear accelerator; Mitsubishi Electric Inc., Tokyo, Japan). For exposure of cells to UV, 3 x 105 cells were suspended in 0.5 mL of phosphate-buffered saline containing 1% foetal calf serum, spread on to six-well cluster plates and irradiated with UVC (wavelength, 254 nm), followed by immediate addition of 1 mL of complete medium. Sensitivity to cisplatin (Nihon-Kayaku, Tokyo, Japan) was measured by plating cells on to methylcellulose plates containing cisplatin.
Measurement of homologous recombination repair of I-SceI-induced DSBs
The I-SceI expression vector pcBASce was kindly provided by Dr Maria Jasin (Sloan-Kettering Institute, New York, NY). The recombination substrate, modified SCneo (Johnson et al. 1999; Fukushima et al. 2001), was targeted into the OVALBUMIN locus in wild-type and SIRT1–/– DT40 cells using puromycin selection. Analysis of recombination frequencies was carried out as described (Fukushima et al. 2001). Briefly, cells (5 x 106) carrying SCneo recombination substrate were transfected with 30 µg of either the I-SceI expression vector (Johnson et al. 1999) or the control plasmid, pBluescript SK. Cells were subsequently selected with G418. The number of G418-resistant colonies was counted after 10–14 days in each cell line.
Plasmid re-ligation assay
Expression vectors of the firefly (pGL3) or Renilla luciferase (phRL-CMV) were purchased from Promega (Madison, WI). The coding sequence of the firefly luciferase was blunt-ligated to the multicloning site of pcDNA3.1 (Invitrogen) so that EcoRI digestion was able to linearize the plasmid between the CMV promoter and the open reading frame of the luciferase. The EcoRI-digested (20 µg) or non-digested (10 µg) plasmid was electroporated into DT40 cells in parallel. In all cases, the expression vector of the Renilla luciferase (2 µg) was co-transfected as an internal control. These two luciferase activities were measured by a single tube assay (Dual Luciferase Reporter Assay System, Promega) 24 h after the transfection. The firefly luciferase activity was normalized by the Renilla luciferase activity. The re-ligation efficiency was calculated as percentage of the firefly luciferase activity obtained with the linearized vector relative to the activity with the non-linearized vector.
Analysis of irradiation-induced chromosome aberrations or G2/M checkpoint
Chromosome analysis was done with or without X-ray (2 Gy) treatment as previously described (Takata et al. 1998). To monitor the G2/M checkpoint, cells were either untreated or irradiated with 2 Gy X-rays, then incubated in the presence of colcemide (0.1 µg/mL). Aliquots of the culture were taken at 1 h intervals. Cells in mitosis were identified by Hoechst staining and fluorescence microscopy. Statistical analysis was performed using Student's t-test.
Cell death assay
Wild-type and mutant cells were continuously exposed to different concentrations of either cisplatin (24 h), H2O2 (12 h), or staurosporine (8 h). In the case of UV or X-ray, wild-type and mutant cells were irradiated with different doses and analyzed 18 h later. Cells were stained with 5 µg/mL propidium iodide (PI) or Annexin V-FITC (Clontech) then analyzed by flow cytometry.
p53 luciferase reporter assay
Plasmids that contain three copies of the consensus p53-binding sequence or its non-binding mutant upstream of luciferase gene (pGL3-p53CBSwt or pGL3-p53CBSmt, respectively) and the expression plasmids for human p63, p53 and p73ß were kindly provided by Prof Takashi Tokino (Sapporo Medical College, Hokkaido, Japan) (Ishida et al. 2000; Sasaki et al. 2002). Cells were transiently transfected with the expression vector (10 µg) together with pGL3-p53CBSwt or pGL3-p53CBSmt (10 µg). The expression vector for the Renilla luciferase (2 µg) was co-transfected as an internal control. Lysates were prepared 24 h later and analyzed using the dual luciferase reporter assay system.
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Acknowledgements |
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Footnotes |
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*Correspondence: E-mail: mtakata{at}med.kawasaki-m.ac.jp
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References |
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Received: 20 August 2004
Accepted: 11 December 2004
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