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Genes to Cells (2007) 12, 853-861. doi:10.1111/j.1365-2443.2007.01097.x
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Ser46 phosphorylation of p53 is not always sufficient to induce apoptosis: multiple mechanisms of regulation of p53-dependent apoptosis

 Akira Kurihara1,2,3, Hirokazu Nagoshi1, Masato Yabuki1,2,3, Ryuhei Okuyama4, Masuo Obinata2,3 and Shuntaro Ikawa1,2,3,*

1 Center for Interdisciplinary Research, Tohoku University, Aramaki, 6-3 Aoba, Aoba-ku, Sendai, Japan 980-8578
2 Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Japan 980-8575
3 Laboratory of Cell Differentiation, Graduate School of Life Sciences, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Japan 980-8575
4 Department of Dermatology, Tohoku University, Graduate School of Medicine, 2-1 Seiryo-mach, Aoba-ku, Sendai, Japan 980-8574


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The tumor suppressor gene p53 plays a central role in determining cell fate in response to DNA damage; cells may undergo either senescence or apoptosis, depending on cell type. Phosphorylation of Serine 46 (Ser46) of p53 is considered to be a primary determinant for the induction of apoptosis, by selectively inducing transactivation of p53 target genes that have proapoptotic function. However, the generality of this mechanism of regulation of p53 remains a matter of debate. We investigated the role of p53 phosphorylation in adriamycin (ADR)-induced apoptosis. We found that Ser46 was phosphorylated in four different cell lines undergoing ADR-induced senescence, as well as in two different cell lines undergoing ADR-induced apoptosis. Using alanine and glutamic acid substitution mutants of p53 Ser46, we showed that Ser46 phosphorylation is not a prerequisite for induction of the proapoptotic gene AIP1. These results indicate that Ser46 phosphorylation of p53 is not required for ADR-induced apoptosis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The p53 tumor suppressor protein plays a central role in the determination of cell fate in response to DNA damage. In response to light damage, p53 induces temporary growth arrest to enable completion of DNA repair processes. In response to severe damage, it induces senescence to prevent expansion of potentially pre-cancerous cells with irreparable DNA damage or apoptosis to eliminate them (Lowe et al. 1993; Huang et al. 1996; Levine 1997; Vousden & Lu 2002). Thus, loss of functional p53 in these key cellular processes in response to DNA damage predisposes cells to undergo malignant transformation, making the gene the most frequently mutated in human cancers (Hollstein et al. 1991, 1994).

The tumor suppressor functions of p53 depend heavily on its transactivation function, hence cell fate determination by p53 is largely a consequence of its ability to selectively induce target genes (Chao et al. 2000; Jimenez et al. 2000). The selective induction of growth arrest and expression of proapoptotic genes induces senescence and apoptosis, respectively. In an effort to elucidate the mechanisms of cell fate determination by p53, numerous studies have examined phosphorylation of p53. The NH2-terminal domain of human p53 is phosphorylated in a sequential manner on residues Ser15, Ser20, Ser33, Ser37 and Ser46. Phosphorylation of some of these residues, such as Ser15 and Ser20, correlate well with the induction of growth arrest (Bode & Dong 2004). Ser15 and Ser20 are consistently phosphorylated earlier than of any other target residue in response to DNA damage. Although phosphorylation of these residues is not sufficient for full p53 activity, it is generally accepted that they are key phosphorylation sites, enabling basal activation and stabilization of p53. These two events are prerequisites for the induction of p53 target genes, including the growth arrest promoting gene p21, and for subsequent cellular responses (Shieh et al. 1997, 1999). In addition to early phosphorylation events, phosphorylation sites that are critical for the induction of later responses, particularly apoptosis, have also been identified. Among them, phosphorylation of Serine 46 (Ser46) induced in delayed kinetics that correlate well with the induction of apoptosis, and appears to be critical for the induction of proapoptotic genes, including AIP1 (Oda et al. 2000). Thus, Ser46 phosphorylation of p53 enables p53 to selectively transactivate proapoptotic genes and induce apoptosis in response to DNA damaging stimuli. Nevertheless, a number of reports have brought into question the requirement for Ser46 phosphorylation in cellular apoptosis (Ashcroft et al. 1999; Kim et al. 2003; Saito et al. 2003; Thompson et al. 2004). In the current study, we examined the role of Ser46 phosphorylation in the induction of proapoptotic genes and apoptosis in cells exposed to adriamycin (ADR), a drug that induces double-strand breaks (DSBs). Recently, we characterized the responses of several cell lines to ADR in an analysis of the role of p53 family genes (p53, p73 and p51/p63, reviewed in Melino et al. 2003; Harms et al. 2004) in ADR-induced apoptosis. We observed that some of these cell lines underwent apoptosis, while others underwent senescence in response to ADR exposure. Using these two groups of cells, we examined the role of p53 Ser46 phosphorylation in apoptosis.

Here we report that p53 Ser46 phosphorylation is not essential for the induction of apoptosis and expression of proapoptotic genes, including AIP1, in response to ADR. We also discuss the significance of phosphorylation in the induction of apoptosis by p53 family genes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Phosphorylation of Ser46 of p53 does not always lead to the induction of apoptosis

We examined the responses of the cell lines HCT116-53+/+, U2OS, HCT116-53–/–, TIG1, TIG3, A549 and MCF7 to exposure to 0.3 µg/mL of ADR. Apoptosis was assessed by Western blot detection of cleaved caspase 3 (Fig. 1A). Senescence was judged by microscopic observation of SA-ß-gal-positive staining (Fig. 1B). HCT116-53+/+ and U2OS cells, but not HCT116-53–/–, TIG1, TIG3, A549 and MCF7 cells, were induced to undergo apoptosis after 48 h of ADR exposure, whereas TIG1, TIG3, A549 and MCF7 cells, but not HCT116-53+/+ and U2OS cells, underwent senescence after 4 days of ADR exposure. Also note that 0.3 µg/mL (0.5 µM) of ADR is much lower than 3 µM ADR used by Oda et al. (2000), which induced cell death (not typical apoptotic death) to all cells regardless of p53 status in our hands.


Figure 1
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  		Figure 1  Cell sensitivity to ADR-induced apoptosis and ADR-induced senescence. (A) Apoptosis of HCT116-53+/+, U2OS, TIG1, TIG3, A549, HCT116-53–/– and MCF7 cells exposed to 0.3 µg/mL of ADR. Apoptosis was assessed by detection of cleaved caspase 3 after 48 h of exposure to ADR. U2OS and HCT116-53+/+ cells (apoptosis-sensitive cells), but not HCT116-53–/–, TIG1, TIG3, A549 and MCF7 cells underwent apoptosis. Arrows indicate molecular weights of cleaved caspase 3. (B) Senescence in TIG1, TIG3, A549, MCF7, U2OS, HCT116-53+/+ and HCT116-53–/– cells exposed to ADR was defined by the presence of SA-ß-gal-positive staining 4 days of ADR exposure. TIG1, TIG3, A549 and MCF7 cells underwent senescence, but not U2OS, HCT116-53+/+ and HCT116-53–/– cells.

 
We next examined p53 protein expression and phosphorylation of p53 in HCT116-53+/+ and U2OS cells (apoptosis-sensitive cells), and in TIG1, TIG3, A549 and MCF7 cells (senescence-sensitive cells) by Western blot analysis (Fig. 2). In all cells, the amount of p53 protein increased after 3–6 h of ADR exposure, and was maintained for up to 48 h (Fig. 2A–F). Phosphorylation of Ser15 and Ser20 was evident starting at 3–6 h, coinciding with the increased accumulation of p53 (Fig. 2A–F). Following these early phosphorylation events, delayed phosphorylation of Ser46 began to emerge in all cells after 12–36 h of ADR exposure, depending on the cell line. In the apoptosis-sensitive cells, Ser46 phosphorylation occurred prior to the induction of apoptosis, similar to other reports showing that Ser46 phosphorylation is critical for p53-dependent induction of apoptosis (Fig. 2A,B). The senescence-sensitive cells had a pattern of Ser46 phosphorylation that was similar to the apoptosis-sensitive cells, yet entered premature senescence, as opposed to apoptosis (Fig. 2C–F). These results indicated that in the four senescence-sensitive cell lines examined in this study, Ser46 phosphorylation of p53 in response to ADR exposure does not lead to apoptosis.


Figure 2
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  		Figure 2  Ser15, Ser20 and Ser46 phosphorylation of p53 in apoptosis-sensitive cells and senescence-sensitive cells after ADR exposure. Extracts of HCT116-53+/+ (A), U2OS (B), TIG1 (C), TIG3 (D), A549 (E) and MCF7 (F) were prepared after indicated hours of ADR exposure. They were subjected to immunoprecipitation with anti-p53 (Ab-1) monoclonal antibody, then analyzed by Western blot using antibodies specific for phosphorylated Ser15, Ser20 and Ser46. IgG (24 h): Negative controls immunoprecipitating the lysates prepared after 24 h of ADR exposure with nonspecific IgG instead of Ab-1 monoclonal antibody. The extracts were also probed directly using anti-p53 (Ab-6) monoclonal and anti-{alpha} tubulin (DM1A) antibodies. Ser15 and Ser20 phosphorylation following ADR exposure correlated with p53 accumulation in all cells. In contrast, initiation of Ser46 phosphorylation was significantly delayed compared to Ser15 and Ser20 phosphorylation in all cells. {alpha}-Tubulin was probed as a loading control.

 
Phosphorylation of Ser46 does not always lead to the induction of proapoptotic genes

Since the transactivation function of p53 is believed to be critical for p53-mediated cellular responses, we measured the transcript levels of p53 target genes in cells exposed to ADR, and examined whether they correlated with a specific cellular response. Total RNA was extracted from HCT116-53+/+, TIG3 and HCT116-53–/– cells at various time points after exposure to ADR, and analyzed by reverse transcriptase-real time polymerase chain reaction (RT-rtPCR) (Fig. 3). Strong induction of AIP1 was observed after 12–24 h of ADR exposure in HCT116-53+/+ cells, while induction was rather subtle in TIG3 cells, consistent with the responses of these cells to apoptosis and senescence, respectively (Fig. 3A,B). These results indicated that Ser46 phosphorylation does not always lead to the induction of AIP1 expression. In contrast, the level of the transcripts of p21, which induces cell cycle arrest and/or senescence, was similar in both HCT116-53+/+ and TIG3 cells. These observations suggest that the mechanism of AIP1 induction is distinct from that of p21 (Fig. 3D,E). Also note that expression of both AIP1 and p21 was dependent on the presence of p53, as none of these two was induced in HCT116-53–/– cells (Fig. 3C,F).


Figure 3
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  		Figure 3  Ser46 phosphorylation is not a prerequisite for induction of AIP1 expression. HCT116–53+/+ cells (A and D), TIG3 cells (B and E) and HCT116-53–/– cells (C and F) were exposed to ADR for the indicated lengths of time, and total RNA was used as the template for generating cDNAs from each cell line. RT-rtPCR was used to determine the levels of the transcripts of p53 target genes, AIP1 (A, B, C) and p21 (D, E, F). AIP1 was induced in HCT116-53+/+ cells, but not in TIG3 cells, after ADR exposure. The level of transcripts in each sample was normalized to that of GAPDH.

 
To further investigate the contribution of Ser46 phosphorylation to AIP1 transactivation, we introduced point mutations in p53 replacing Ser46 with Ala (p53-S46A) or Glu (p53-S46E) to mimic constitutively unphosphorylated or phosphorylated p53, respectively. HCT116-53–/– cells were infected with adenoviruses transducing the Ala and Glu mutants (Adp53-S46A and Adp53-S46E, respectively) or wild-type p53 (Adp53-WT), and then cultivated in the presence of ADR for 36 h. The expression level and apoptosis inducing capability of each of p53 protein were essentially identical (Fig. 4C,D). When levels of p53 target gene transcripts were monitored by RT-rtPCR, both of the mutants, as well as wild-type p53, strongly induced AIP1 and p21 expression (Fig. 4A,B). These results implied that in this experimental system, phosphorylation of Ser46 is not critical for the induction of AIP1 expression or apoptosis.


Figure 4
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  		Figure 4  Point mutations of Ser46 do not influence the ability of p53 to induce AIP1 expression. (A, B) Effects of Ser46 point mutations on the induction of p53 target genes, AIP1 (A) and p21 (B). HCT116-53–/– cells were infected with Adp53-WT, Adp53-S46A or Adp53-S46E, then exposed to ADR for 36 h. cDNAs prepared from these cells were analyzed by RT-rtPCR. There was no difference in the ability of the p53 substitution mutants and wild-type p53 to induce p21 or AIP1 expression. The level of transcripts in each sample was normalized to that of GAPDH. (C) Point mutations of Ser46 do not influence the ability of p53 to induce apoptosis. Degree of apoptosis in the cells described above was quantified as the fraction of the cell population in sub-G1, as monitored by FACS. (D) Immunoblot detection of p53 proteins. {alpha}-Tubulin was used as loading control.

 
Phosphorylation of p53 Ser46 does not influence p73-dependent ADR induced apoptosis

The results described above indicated that the mechanism of induction of AIP1 does not require p53 Ser46 phosphorylation. Previously, we observed that p73 was a good inducer of AIP1 (A. Kurihara et al., submitted), so we next investigated the effect of Ser46 phosphorylation on p73-dependent ADR-induced apoptosis. We assayed ADR-induced apoptosis of U2OS cells in the presence of siRNAs targeting p53 and p73 again by detection of cleaved caspase 3 (Fig. 5A). Transfection of U2OS cells with siRNAs specific for p53 or p73 significantly blocked ADR-induced apoptosis, implying that p73, as well as p53, substantially contribute to ADR-induced apoptosis. We then examined p53 phosphorylation in these cells by Western blot analysis (Fig. 5B). As expected, p53 siRNAs significantly reduced ADR-induced expression of p53, and hence Ser15,20,46 phosphorylation. In contrast, p73 siRNAs significantly reduced p73 protein, but did not affect either expression or Ser46 phosphorylation of p53. In addition, RT-rtPCR analyses revealed that this p73 reduction led to concomitant reduction in the induction of AIP1 transcript (A. Kurihara et al., submitted). These results indicated that reduced expression of p73 prevents U2OS cells from undergoing apoptosis, despite the fact that Ser46 phosphorylated p53 is abundant in these cells. Thus, p53 Ser46 phosphorylation cannot induce apoptosis when apoptosis was blocked by p73 siRNAs.


Figure 5
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  		Figure 5  Phosphorylation of p53 Ser46 does not influence p73-dependent ADR-induced apoptosis. (A) Both p53 and p73 contribute to ADR-induced apoptosis. siRNAs specific for green fluorescent protein (GFP, si-GFP), p53 (si-p53–1 and si-p53–2) or p73 (si-p73–1 and si-p73–2) were introduced into U2OS cells, then cells were treated with ADR for 48 h. Apoptosis was examined as described for Fig. 1A. Arrows indicate molecular weights of cleaved caspase 3. (B) Ser46 phosphorylated p53 was abundantly expressed in cells in which apoptosis was blocked by p73 siRNAs. Cell extracts from the indicated cells were prepared and analyzed as described for Fig. 2. The extracts were also probed directly using anti-p53 (Ab-6) monoclonal, anti-p73 (IMG246) monoclonal and anti-{alpha} tubulin (DM1A) antibodies. {alpha}-Tubulin was probed as a loading control. (C, D) Ser46 point mutations do not influence p73{alpha}-dependent transactivation. HCT116-53–/– cells were co-infected with Adp53-WT, Adp53-S46A or Adp53-S46E, and AdTAp73{alpha}, as indicated, then treated as described for Fig. 4. Point mutations of Ser46 of p53 did not influence p73{alpha}-dependent induction of AIP1 or p21. The level of transcripts in each sample was normalized to that of GAPDH. (E) Apoptosis induction in cells infected with various adenoviruses. Degree of apoptosis in the cells described above was quantified as Fig. 4C. (F, G) Immunoblot detection of p53 proteins (F) and p73 (G). {alpha}-Tubulin was used as loading control.

 
Similar to p53, transactivation of target genes is believed to be critical for p73-mediated cellular responses. We examined the effect of p53 Ser46 phosphorylation on p73-dependent transactivation of target genes. HCT116-53–/– cells were co-infected with Ad p53-WT, Adp53-S46A or Adp53-S46E in combination with an adenovirus expressing TAp73{alpha} (AdTAp73{alpha}), an isoform of p73. Infected cells were cultivated in the presence of ADR for 36 h, and then subjected to RT-rtPCR analysis (Fig. 5C). Interestingly, TAp73{alpha} was much more potent in transactivating AIP1 than p53. Furthermore, p53 substantially augmented transactivation of AIP1 by TAp73{alpha}, independent of Ser46. In contrast, the ability of each of the p53 substitution mutants to induce p21 was indistinguishable, and did not differ significantly with co-expression of exogenous TAp73{alpha} (Fig. 5D). These results implied that phosphorylation of p53 Ser46 does not influence AIP1 gene induction by TAp73{alpha}. In addition, induction of apoptosis did not differ significantly by simultaneous expression of TAp73{alpha} and each of p53 protein under such condition (Fig. 5E). We speculate that low expression level of p73 could not augment apoptosis induced by each of p53 protein, which is expressed at much higher level compared to p73 (Fig. 5F,G).

Collectively, our results suggested that p53 Ser46 phosphorylation does not influence p73-dependent ADR-induced apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we showed that the Ser46 phosphorylation of p53, which has been proposed as a critical event in p53-mediated apoptosis, does not lead to the induction of apoptosis in all cells.

The p53 tumor suppressor protein is stabilized and activated in response to various DNA damaging stimuli, and plays a central role in the determination of cell fate in response to DNA damage (Levine 1997; Vousden & Lu 2002). p53 is phosphorylated at multiple residues in the NH2-terminal transactivation domain, including Ser15, Ser20, Ser33, Ser37 and Ser46, as well as other residues in the COOH-terminal regulatory domain (Bode & Dong 2004). Phosphorylation of Ser46 in delayed kinetics prior to cellular apoptosis led many investigators to propose that post-translational modification of p53 elicits the critical signal for the execution of apoptosis (Oda et al. 2000). p53-responsive elements in many proapoptotic gene promoters, excluding PUMA, have weak affinity for p53, suggesting that efficient induction of proapoptotic genes requires co-factors other than p53, an extraordinarily large amount of p53, hyper-activation of p53 (Vousden & Lu 2002), or some combination of these. Accordingly, it was originally reported that the role of Ser46 phosphorylation is to hyper-activate p53.

We have shown here that Ser46 phosphorylation is not always sufficient to induce apoptosis, as four different cell lines were induced to undergo senescence in response to DNA damage, despite the presence of phosphorylated Ser46 in these cells. Nevertheless, a similar phosphorylation was observed in two different cell lines induced to apoptosis, which was assessed by cleaved caspase 3 detection and by the emergence of a subG1 population by flow cytometry (A. Kurihara et al., submitted). This apparent paradox may be reconciled with previous results if we hypothesize that the regulation of p53 occurs through multiple mechanisms, depending on cell type. Delayed induction of p73 and/or p51, in cooperation with sustained activation of p53, is necessary for the induction of cellular apoptosis in response to ADR exposure (Akira Kurihara and Shuntaro Ikawa, unpublished observation). Furthermore, p73 was an extremely efficient co-activator of p53 in the transactivation of proapoptotic gene promoters, particularly AIP1, but not of non-proapoptotic gene promoters (A. Kurihara and S. Ikawa, unpublished observation). In this report, we showed that in addition to Ser46 phosphorylation being dispensable for apoptosis, it did not influence the cooperation between p53 and p73 in the induction of apoptosis. Yet, Ser46 phosphorylation by p38 MAPK was reported to transactivate the proapoptotic gene PUMA (Li et al. 2005). Interestingly, a human polymorphism of Pro47/Ser47, adjacent to Ser46, affects the induction of PUMA, cancer risk and cancer progression (Li et al. 2005). Recently, it was reported that p53 Ser46 phosphorylation moderately enhances the induction of both apoptosis and senescence (Feng et al. 2006). We postulate that p53 Ser46 phosphorylation results in moderate activation of p53 following basal activation by Ser15 and Ser20 phosphorylation. Subsequently, the cooperation of p73 or p51 is necessary to fully activate p53 and induce apoptosis.

The existence of multiple mechanisms of regulation of p53 may also help explain the well-known observation that sensitivity to various DNA damaging stimuli and p53-dependent apoptosis differs considerably in different tissues (Gudkov & Komarova 2003). One of the sources of this variation may be the quality of stimuli; p73/p51 was induced in HCT116-53+/+ cells by DSB-inducing reagents, which cause the most fatal damage, but not by ultraviolet (UV) irradiation (data not shown). In contrast, UV irradiation, but not DSB-inducing ionizing radiation, induces phosphorylation of Ser46 by homeodomain-interacting protein kinase-2 (HIPK2) (D’Orazi et al. 2002; Hofmann et al. 2002). Adding further complexity is the observation that induction of apoptosis is mediated by direct translocation of p53 to mitochondria (Mihara et al. 2003; Erster et al. 2004; Chipuk et al. 2005). We speculate that multiple mechanisms of regulation of p53 are indispensable for tumor suppression in numerous types of tissue residing in extremely varied environments.

In summary, we showed that Ser46 phosphorylation of p53 is not a prerequisite for ADR-induced apoptosis and transactivation of proapoptotic genes. We speculate that this phosphorylation event strengthens the extent of p53 activation. Since the regulation of cellular apoptosis by p53 is very complex, further studies from a broad perspective of multiple pathways are certainly warranted.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and exposure to ADR

The normal human fibroblast cell lines TIG1 and TIG3, the human lung carcinoma cell line A549, and the human osteosarcoma cell line U2OS were cultured in DMEM; the human breast adenocarcinoma cell line MCF7 was cultured in RMPI-1640 supplemented with 10% fetal bovine serum in 5% CO2 at 37 °C. The human colorectal carcinoma cell lines HCT116-53 +/+  and HCT116-53–/– were kind gifts from Dr Bert Vogelstein (Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD, USA). Cells were exposed to ADR (Sigma-Aldrich, St. Louis, MO, USA) at the indicated concentrations for the indicated periods of time.

Recombinant adenovirus infection and antibodies

Recombinant adenoviruses transducing wild-type human p53, p53 point mutants and LacZ were constructed as previously described (Ishimoto et al. 2002). Antibodies and their sources were as follows: anti-p53 mouse monoclonal antibody (Ab-1, Ab-6) was from Oncogene Research Products (Boston, MA, USA), anti-Cleaved Caspase-3 rabbit monoclonal antibody (5A1), anti-phospho (p)-Ser-15 and p-Ser-46 antibodies were from Cell Signaling Biotechnology (Beverly, MA, USA); anti-p-Ser-20 antibody was from R&D systems (Minneapolis, MN, USA); anti-p73 mouse monoclonal antibody (IMG246) was from IMGENEX (San Diego, CA, USA); and anti-{alpha}-tubulin (DM1A) was from Sigma-Aldrich (St. Louis, MO, USA).

Senescence assay

Cellular senescence was determined by detection of senescence-associated ß-galactosidase (SA-ß-gal) activity, using the Senescence Assay kit (Biovision, Mountain View, CA, USA), according to the manufacture's instructions. Cells were stained for 24 h at 37 °C and observed using microscopy.

Small interfering RNA experiments

Small interfering RNA (siRNA) constructs specific for p53 (p53 Validated Stealth RNA interference), p51 and p73 (designed by the manufacturer) were purchased from Invitrogen (Tokyo, Japan). siRNAs were transfected into cells with Hi-Perfect transfection reagent (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. Cells were incubated for 24 h, then cultivated in fresh medium for 12 h, and then treated with ADR for the indicated lengths of time.

Immunoprecipitation and Western blot analysis

Immunoprecipitations were carried out using anti-p53 antibody (Ab-1, Oncogene Research Products, Boston, MA, USA) in lysates of cells exposed to ADR, as indicated. Immune complexes were resolved on SDS-polyacrylamide gels, transferred to Immobilon P membranes (Millipore, Billerica, MA, USA), and immunoreactive proteins were detected using ECL-plus (GE Healthcare, Uppsala, Sweden). ECL-Advance (GE Healthcare, Uppsala, Sweden) with enhanced sensitivity was used for the detection of p73, and phosphorylated Ser20 and Ser46 of p53.

Reverse transcriptase real time polymerase chain reaction (RT-rtPCR)

Total RNA was prepared from cells using the RNeasy mini kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. cDNA was synthesized using Super script II reverse transcriptase (Invitrogen, Tokyo, Japan), as recommended by the manufacturer, using 3 µg of total RNA as the template. Real-time PCR (rtPCR) analysis was done with the Quantitect SYBR Green PCR kit (QIAGEN, Hilden, Germany) and Opticon 2 (MJ Research, Tokyo, Japan), using primer pairs specific for p21 and AIP1. All values were normalized to values obtained from amplification of GAPDH. The following primer pairs were used: p21, sense 5'-ACTGTGATGCGCTAATGGC-3' and anti-sense 5'-ATGGTCTTCCTCTGCTGTCC-3'; AIP1, sense 5'-ATGGGATCTTCCTCTGAGGCG-3' and anti-sense 5'-CCCAGCCAGGTGTGTGTGTCTG-3'; GAPDH, sense 5'-CAAGAAGGTGGTGAAGCAGG-3' and anti-sense 5'-ACATGGCAACTGTGAGGAGG-3'.


   Acknowledgements
 
The authors are heavily indebted to Prof. Masanobu Satake for invaluable discussion and critical reading of the manuscript. This study was partly supported by a Grant in Aid from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: E-mail: sikawa{at}cir.tohoku.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 5 April 2007





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