Glycerol alone treatment may not be suf ficient 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 selleck Regorafenib the possible involvement of os motic stress induced signal transduction in the p53 path way. 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 stabi lize 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.

Glycerol treatment alone was insufficient to induce Bax accumulation. This result led us to assume that some initial signals evoked by heating are required to ef fectively induce signals leading to apoptosis. Thus, we next examined the activation of mp53 through phospho rylation after heat and glycerol treatments. It has been re ported that the phosphorylation of serine 15 of p53 by the PI3 K family induces p53 activation. Therefore, we estimated the activation of mp53 based on the induc tion of WAF1 which is one of downstream factors regulat ed by p53. As shown in Fig. 2, the phosphorylation of serine 15 of wtp53 was observed 6 h after heating without or with glycerol in A 172/neo cells, whereas it was not observed after glycerol treatment alone.

The phosphorylation of serine 15 after heating with glycerol was suppressed by an inhibitor of wortman nin for PI3 K family. WAF1 was accumulated in relation to the level of serine 15 phosphorylation. These results suggest that the PI3 K family such as ATM, ATR or DNA PK contributes to heat induced acti vation of p53 at serine 15 as reported in radiation induced p53 activation. In contrast to A 172/neo cells, A 172/mp53/248 cells did not show any significant increase of WAF1 6 h after heating, although the serine 15 was phosphorylated. When A 172/mp53/ 248 cells were heated in the presence of glycerol, WAF1 was accumulated with the phosphorylation of the serine 15. Wortmannin suppressed WAF1 accumulation and serine 15 phosphorylation after heat ing in the presence of glycerol.

These results mean that the phosphorylation of serine 15 of p53 is not sufficient for the heat induced mp53 activation. It is suggested that the activation of mp53 demands both the reconstruction process by glycerol and the heat induced phosphorylation process by the PI3 K family. In addition, it Entinostat is suggested that the glycerol enhanced heat sensitivity of mp53 cells is dependent on p53 dependent signal transduction and mediated by p53 regulated Bax induction.