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Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53.

Ohnishi T, Ohnishi K, Takahashi A - BMC Biotechnol. (2002)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. tohnishi@nmu-gw.naramed-u.ac.jp

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

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a, Clonogenic survival rates of cells after heating or combination treatments with glycerol. Two duplicate flasks were used per experiment, and two or more independent experiments were repeated for each survival point. Open square, A-172 neo contol cells; closed square, neo control cells pretreated with 0.6 M glycerol; open triangle, A-172/mp53/143 cells; closed circle, A-172/mp53/143 cells pretreated with 0.6 M glycerol. b, Restoration by glycerol of heat-induced Bax accumulation in A-172/mp53/143 cells. Western blotting samples were prepared from cells 10 h after heating at 44°C for 30 min. The glycerol was added 48 h before heating at a final concentration of 0.6 M. Glycerol was present in the medium during culture after heating.
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Figure 1: a, Clonogenic survival rates of cells after heating or combination treatments with glycerol. Two duplicate flasks were used per experiment, and two or more independent experiments were repeated for each survival point. Open square, A-172 neo contol cells; closed square, neo control cells pretreated with 0.6 M glycerol; open triangle, A-172/mp53/143 cells; closed circle, A-172/mp53/143 cells pretreated with 0.6 M glycerol. b, Restoration by glycerol of heat-induced Bax accumulation in A-172/mp53/143 cells. Western blotting samples were prepared from cells 10 h after heating at 44°C for 30 min. The glycerol was added 48 h before heating at a final concentration of 0.6 M. Glycerol was present in the medium during culture after heating.

Mentions: To elucidate the effect of glycerol on the heat sensitivity of transformed A-172 cells, the clonogenic surviving fractions by heat after pre-treatment with or without glycerol (0.6 M) were measured. As shown in Fig. 1a, A-172 cells tranfected with mp53 (val to ala at codan 143) (A-172/mp53/143 cells) were more resistant to heat than the A-172/neo cells. By glycerol treatment, A-172/mp53/143 cells became about 1.5 times more heat-sensitive at D10 dose, whereas A-172/neo cells showed no enhanced heat sensitivity. In addition, A-172/mp53/143 and A-172/neo cells treated with glycerol alone (0.6 M) showed about 80% survival fractions, and thus the concentration of glycerol appeared to cause no serious cell damage regardless of p53 status. These results suggest that glycerol affects the heat sensitivity of those cells in a way that it enhances heat sensitivity in mp53 cells.


Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53.

Ohnishi T, Ohnishi K, Takahashi A - BMC Biotechnol. (2002)

a, Clonogenic survival rates of cells after heating or combination treatments with glycerol. Two duplicate flasks were used per experiment, and two or more independent experiments were repeated for each survival point. Open square, A-172 neo contol cells; closed square, neo control cells pretreated with 0.6 M glycerol; open triangle, A-172/mp53/143 cells; closed circle, A-172/mp53/143 cells pretreated with 0.6 M glycerol. b, Restoration by glycerol of heat-induced Bax accumulation in A-172/mp53/143 cells. Western blotting samples were prepared from cells 10 h after heating at 44°C for 30 min. The glycerol was added 48 h before heating at a final concentration of 0.6 M. Glycerol was present in the medium during culture after heating.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC111188&req=5

Figure 1: a, Clonogenic survival rates of cells after heating or combination treatments with glycerol. Two duplicate flasks were used per experiment, and two or more independent experiments were repeated for each survival point. Open square, A-172 neo contol cells; closed square, neo control cells pretreated with 0.6 M glycerol; open triangle, A-172/mp53/143 cells; closed circle, A-172/mp53/143 cells pretreated with 0.6 M glycerol. b, Restoration by glycerol of heat-induced Bax accumulation in A-172/mp53/143 cells. Western blotting samples were prepared from cells 10 h after heating at 44°C for 30 min. The glycerol was added 48 h before heating at a final concentration of 0.6 M. Glycerol was present in the medium during culture after heating.
Mentions: To elucidate the effect of glycerol on the heat sensitivity of transformed A-172 cells, the clonogenic surviving fractions by heat after pre-treatment with or without glycerol (0.6 M) were measured. As shown in Fig. 1a, A-172 cells tranfected with mp53 (val to ala at codan 143) (A-172/mp53/143 cells) were more resistant to heat than the A-172/neo cells. By glycerol treatment, A-172/mp53/143 cells became about 1.5 times more heat-sensitive at D10 dose, whereas A-172/neo cells showed no enhanced heat sensitivity. In addition, A-172/mp53/143 and A-172/neo cells treated with glycerol alone (0.6 M) showed about 80% survival fractions, and thus the concentration of glycerol appeared to cause no serious cell damage regardless of p53 status. These results suggest that glycerol affects the heat sensitivity of those cells in a way that it enhances heat sensitivity in mp53 cells.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. tohnishi@nmu-gw.naramed-u.ac.jp

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus