The function of wtp53 is depressed by http://www.selleckchem.com/products/baricitinib-ly3009104.html mp53 in a way that mp53 forms heterogeneous tetramer with wtp53. This ef fect of mp53 is the dominant negative effect. From this, one possibility is that glycerol may depress the dom inant 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 transfected with mp53 gene, when the cells were pre treated with glyc erol. WAF1 expression was not induced even after com bined 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 ex pression by heat in glycerol treated cells. These results strongly support that the conformation of mp53 was re stored to normal type of p53 by glycerol.
Furthermore, to confirm the conformational change of mp53 to wtp53 suggested by Western blot analysis, the DNA binding activity of p53 for p53CON was measured in nuclear proteins extracted from A 172/neo or A 172/ mp53/248 cells using the gel mobility shift assay. It is known that wtp53 can bind to p53CON homologous to a specific DNA sequence located upstream of the bax gene which positively controls apoptosis. In agreement of this, the binding activity of wtp53 significantly increased in A 172/neo cells treated with heat or combination of heat and glycerol. In contrast, a slight increase in the DNA binding activity of the nuclear proteins was ob served when A 172/mp53/248 cells were treated with heat.
The in crease may be due to endogenous wtp53 in A 172/mp53/ 248 cells. The defective DNA binding ability of p53 from heat treated A 172/mp53/248 cells may be due to the dominant negative nature of mp53 protein. It is known that mp53 has no ability to induce apoptosis, be cause most mp53 can not bind to a specific DNA sequence. On the other hand, when A 172/mp53/248 cells were heated in the presence of glycerol before heating, they showed clear increased DNA binding activi ty of p53 to p53CON. This result shows that mp53 underwent conformational change to wtp53. The enhanced heat sensitivity observed in glycerol pretreated A 172/mp53/143 cells might be induced through bax gene expression up regulat ed by the activated mp53.
No binding activity of p53 was observed when whole cell proteins extracted from intact A 172/neo or A 172/mp53/248 cells were treated with heat, heat plus glycerol or heat plus glycerol plus wort mannin. Thus, the acquisition of binding activity of wtp53 or glycerol treated Dacomitinib mp53 is likely to demand cellular signal transduc tion as described above. Furthermore, the increased DNA binding activity of nuclear proteins from A 172/neo or A 172/mp53/248 cell was suppressed by wortmannin.