Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53
© Ohnishi et al; licensee BioMed Central Ltd. 2002
Received: 15 August 2001
Accepted: 19 April 2002
Published: 19 April 2002
We have previously reported that glycerol acts as a chemical chaperone to restore the expression of WAF1 in some human cancer cell lines bearing mutant p53. Since the expression of WAF1 is up-regulated by activated wildtype p53, glycerol appears to restore wtp53 function. The aim of the present study is to examine the restoration of heat-induced p53-dependent apoptosis by glycerol in human glioblastoma cells (A-172) transfected with a vector carrying a mutant p53 gene (A-172/mp53 cells) or neo control vector (A-172/neo cells).
A-172/mp53 cells showed heat resistance compared with A-172/neo cells but A-172/mp53 cells in turn became heat sensitive when pre-treated with glycerol before heat treatment. The accumulation of Bax in the A-172/mp53 cells was induced by heating with glycerol pre-treatment, but not without it, whereas the accumulation in the A-172/neo cells was induced in both cases. Furthermore, mp53 extracted from heated cells came to bind to the sequence specific region after heating combined with glycerol pre-treatment. The phosphorylation of mp53 at serine15 was suppressed by an inhibitor of the phosphatidylinositol 3-kinase (PI3-K) family.
These results suggest that glycerol is effective in inducing conformational change of phosphorylated p53 and restoring mp53 to wtp53 function, leading to enhanced heat sensitivity through the induction of apoptosis. This novel tool for enhancement of heat sensitivity in cancer cells bearing mp53 may be applicable for p53-targeted hyperthermia, because mutation or inactivation of p53 is observed in approximately 50% of human cancers.
It is known that p53 induces cell growth arrest [1, 2] or cell death  and the suppression of DNA replication  to suppress the initiation, progression or growth of tumor. p53 exhibits its function through the induction of downstream genes and/or protein interaction relating to tumor suppression. However, mutations in the p53 gene cause conformational alterations in p53 protein and the majority of mp53 can no longer induce expression of downstream genes [5, 6]due to sequence specific DNA binding inability. Heat up-regulates sequence specific DNA binding activity of wtp53  and induces the expression of p53-regulated gene . Thus, hyperthermia is regarded as a good tool for cancer therapy from the view of suppression of tumor growth. We have already reported that wtp53-transfected p53-knockout cells show higher incidence of apoptosis by heat compared with mp53-transfected p53-knockout cells . Furthermore, patients bearing wtp53 show a high survival rate after radiotherapy resulting from Bax and Bcl-2 regulation [10, 11]. Such hyperthermia/radiotherapy based on p53 status, however, seems to have some difficulties for the treatment of cancer cells bearing mp53. To overcome this problem, we present here a new strategy for cancer therapy. In recent years, glycerol has been reported to act as a chemical chaperone to correct the conformation of proteins, which cause human diseases [12, 13]. Consistent with this, we have reported that glycerol acts as a chemical chaperone to restore the expression of WAF1 in some human cancer cell lines bearing mp53 and to restore apoptosis in p53-knockout mouse fibroblast cells transfected with mp53. Since the expression of WAF1 is up-regulated by activated wtp53, glycerol appears to restore wtp53 function. In the present study, we further examined the effect of glycerol on p53-dependent apoptosis induction through bax expression and whether the heat sensitivity of cells bearing mp53 is enhanced by glycerol. To enable a discussion of the results on the basis of p53 status only, we transfected A-172 cells with the mp53 gene, which had identical genetic backgrounds except for p53 status, and demonstrated so-called "dominant negative effect" of mp53 protein [16, 17].
Recently, it was reported that the PI3-K family such as ATM, ATR and DNA-PK contributes to the activation of p53 through the phosphorylation of serine 15 of p53 [18–22]. In the present study, to gain further insight into the mechanism of restoring mp53 to wtp53, we examined mediation of the phosphorylation of p53 by the PI3-K family to conformational change of mp53.
Results and Discussion
The change of cellular contents of Bax after heating was analyzed in A-172/mp53/143 cells with Western blot. As shown in Fig. 1b, Bax was accumulated after heating in the presence of glycerol at 0.6 M, although Bax accumulation was not induced after heating alone or treatment with 0.6 M glycerol alone in the cells. It is possible that mp53 might function as a transcriptional factor in heat-induced Bax accumulation under the presence of glycerol. In addition, A-172/mp53/143 cells only heated accumulated large amounts of p53 but no significant Bax, suggesting that heat treatment induces accumulation of mp53 as is the case in human glioblastoma A-7 cells . The accumulation of mp53 is probably due to the elongation of its half-life by heat. Glycerol alone treatment may not be sufficient to convert mp53 into wtp53 because this treatment did not lead to accumulation of endogenous latent wtp53 and did not induce Bax accumulation in A-172/mp53/143 cells. This also excludes the possible involvement of osmotic stress-induced signal transduction in the p53 pathway. We assumed that denaturation by heat stress may disrupt the aberrant conformation of the p53 mutant, and glycerol may exert its effect during renaturation to stabilize the transient wtp53 conformation that is otherwise very unstable. Subsequently, the conformation-stabilized p53 could be activated as a transcriptional factor by heat-induced signal transduction. Thus, the inability to induce Bax accumulation by glycerol alone may be due to mutant conformation of p53.
The function of wtp53 is depressed by mp53 in a way that mp53 forms heterogeneous tetramer with wtp53. This effect of mp53 is the dominant negative effect [16, 17]. From this, one possibility is that glycerol may depress the dominant negative effect of mp53 and p53-centered signal transduction may be restored by glycerol. However, we have already reported that WAF1 expression after heating was induced in Saos-2 cells (p53-null) transfected with mp53 gene , when the cells were pre-treated with glycerol. WAF1 expression was not induced even after combined treatment with heat and glycerol in the cells transfected with neo vector alone without mp53 gene. At least, mp53 is necessary for induction of WAF1 gene expression by heat in glycerol-treated cells. These results strongly support that the conformation of mp53 was restored to normal type of p53 by glycerol.
New cancer therapies for patients with mp53-containing tumors are recently being developed. Especially, reports concerning to transfection of p53 gene into tumor [25–27] and molecules which activate latent p53 , change the conformation of mp53  or restore the function of mp53 [14, 15, 30, 31]are on the increase. We have recently reported that glycerol has an ability to restore normal function to mp53, leading to WAF1 induction . Thereafter, new compounds which rescue mp53 conformation and function have been reported by other laboratory . Among the reported molecules, glycerol is easy to be recognized as the most useful molecule for cancer therapy, because it is widely used as a convenient reagent in clinical course already. Furthermore, as reported in this paper, glycerol has an ability to enhance bax expression in mp53 cells as a chemical chaperone through phosphorylation of p53 at serine 15 by PI3-K family and conformational change of mp53. Thus, it is expected that the cancer therapy combined hyperthermia and glycerol is efficient for patients with mp53-containing tumors, in which p53-dependent bax expression is less frequently induced.
Materials and Methods
Human glioblastoma A-172 cells (provided by JCRB, Tokyo, Japan) were cultured at 37°C in Dulbecco's Modified Eagle medium containing 10% (v/v) fetal bovine serum, penicillin (50 U/ml), streptomycin (50 μg/ml) and kanamycin (50 μg/ml) (DMEM-10).
A-172 cells were transfected with the plasmids pC53-SCX3, pC53-248 (mp53, point mutation from Val to Ala at codon 143 or Arg and neomycin resistance marker) or pCMV-Neo-Bam (neomycin resistance marker alone). Before transfection, these plasmids were digested with HindIII and linearized (plasmids were provided by Dr. B. Vogelstein, Johns Hopkins Oncology Center, MD, USA). A-172 cells were electroporated three times at 600 V with linearized DNA (10 μg/10 μl of pC53-SCX3, pC53-248 or pCMV-Neo-Bam). The transfectants (A-172/mp53/143, transfected with pC53-SCX3; A-172/mp53/248, pC53-248; A-172/neo, pCMV-Neo-Bam) were selected by G418 (200–400 μg/ml, Sigma Chemical Co., St. Louis, MO) and incubated at 37°C through all experiments.
Cells were treated with glycerol (at final concentration of 0.6 M) 48 h before heating (44°C, 30 min) and then were incubated at 37°C for 6 or 10 h in the presence of glycerol until sampling. In the case of cell survival assay, the medium with glycerol was changed with glycerol free one after 10 h incubation and thereafter cells were incubated for ten to fourteen days at 37°C in glycerol free medium. In in vitro treatment, whole cell extracts from intact cells were treated with glycerol (at final concentration of 0.6 M) for 30 min at 37°C.
Cells were treated with wortmannin (Nacalai tesque, Inc., Kyoto, Japan, at final concentration of 20 μM) 2 h before heating (44°C, 30 min) and then were incubated at 37°C in the presence of wortmannin until sampling. In in vitro treatment, whole cell extracts from intact cells were treated with wortmannin (at final concentration of 20μM) during heating.
Cell survival assay
Cell survival after heating at 44°C for 0, 15, 30, 60, 90 or 120 min was quantitated by plating cells into 25 cm2 flask containing the medium. Ten to fourteen days later, cell colonies were rinsed with PBS, fixed with methanol, stained with 2% Giemsa solution (Merck, Woodbridge, NJ, USA). Colonies containing at least 50 cells were counted. The number of cells per colony was determined prior to experiment.
Western blotting analysis
Detailed procedure of Western blotting is described elsewhere(El-Deiry et al., 1994). Aliquots (20 μg) of whole cell extracts were used for Western blotting analysis of Bax and p53. After electrophoresis on 15% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS and electrophoretic transfer onto Poly Screen PVDF membranes (DuPont/NEN Research Products, Boston, MA), the proteins on each membrane were incubated with the anti-human Bax polyclonal antibody Ab-1 (Oncogene Science Inc., Uniondale, NY), anti-human p53 monoclonal antibody DO-1 (Oncogene Science Inc.), anti-human phosphorylated p53 polyclonal antibody Phospho-p53 (Ser15) or anti-human WAF1 monoclonal antibody EA10 (Oncogene Science Inc.). The bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-mouse IgG antibody (Zymed Labs. Inc., San Francisco, CA) and the BLAST®: Blotting Amplification System (DuPont/NEN Research, Boston, MA).
Preparation of nuclear or whole cell extracts for gel mobility-shift assay
Nuclear extracts were prepared from A-172 transformed cells 6 hr after heat treatment, heat and glycerol treatments or no treatments as in vivo treatment samples. As in vitro treatment samples, whole cell extracts were prepared from intact A-172 transformed cells suspended in extraction buffer (20 mM HEPES-KOH, pH 7.6, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA-NaOH, pH 8.0, 0.5 mM Dithiothreitol(DTT), 0.5 mM phenylmethyl-sulfanylfluoride (PMSF), 25% (v/v) glycerol, 1.2 μM spermidine) and were treated with glycerol (0.6 M), heat (44°C, 30 min) or combination of glycerol and heat, and subsequently incubated for 30 min at 37°C. The procedures of nuclear protein extraction are described previously. Shortly, the cells were washed with PBS and suspended in washing buffer (10 mM Tris-HCl pH 7.5, 130 mM NaCl, 5 mM KCl, 8 mM MgCl2) and then homogenized on ice in hypotonic buffer (20 mM HEPES-KOH, pH 7.6, 5 mM KCl, 0.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethyl-sulfanylfluoride (PMSF) with a hand-driven Dounce homogenizer. The homogenates were centrifuged to precipitate the nuclei, which were resuspended in extraction buffer (20 mM HEPES-KOH, pH 7.6, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA-NaOH, pH 8.0, 0.5 mM DTT, 0.5 mM PMSF, 25% (v/v) glycerol, 1.2 μM spermidine). The resulting nuclear suspensions were centrifuged to precipitate the chromatin and the nuclear extracts were collected and dialyzed against binding buffer (20 mM HEPES-KOH, pH 7.6, 0.5 mM EDTA-NaOH, pH 8.0, 50 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 10% (v/v)glycerol). The protein concentration of each extract was quantified using a BIO-RAD Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard.
Gel mobility-shift assay
The p53-p53CON binding activity was measured by a gel-shift assay using a synthetic double-stranded DNA fragment encoding the p53CON (5'-GGACATGCCCGGGCATGTCC-3') on the upstream of bax gene (Japan Bioservice, Niiza, Saitama, Japan) as a probe. Detailed procedure is described elsewhere . The probe was labeled with [γ-32P]ATP using Megalabel (Takara Shuzo Co., Ltd., Ohtsu, Shiga, Japan) and the required nuclear extract (5 μg as protein) was incubated at 25°C for 30 min with the labeled p53CON probe (1~3 × 105 cpm) and poly [dIdC]-poly [dIdC] (1 μg) (Pharmacia Biotech, Uppsala, Sweden) diluted with binding buffer to a final volume of 15 μl. After this incubation, the samples were electrophoresed on a 5% (w/v) polyacrylamide gel for 1 h at 150 V using Tris-acetate-EDTA buffer. Subsequently, the gel was dried and observed with Fujix BAS 1000 Imaging Analyzer (Fuji) and photographed on Pictrography 3000 (Fuji) connected to the BAS 1000.
This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
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