Modulation ofp53 by antisense phosphorothioate oligonucleotides

The p53 protein functions in the cellular response to DNA damage through its involvement in the cell cycle checkpoint pathway at G1-phase of the cell cycle. Inhibition of this cell cycle checkpoint has been shown to increase the sensitivity of cells to DNA-damage. Therefore, the studies presented here tested the hypothesis that phosphorothioate oligonucleotides (PTOs) with sequence complementary to the p53 mRNA will attenuate p53 expression resulting in an increase in cellular sensitivity to DNA damage. The antisense to p53 PTO, p53t, caused a decrease in p53 protein levels in NIH3T3 cells which increased the sensitivity of these cells to DNA-damage. The p53t PTO also decreased the percentage of cells in G1 phase of the cell cycle. Antisense to p53 PTOs had similar effects in Chang human liver cells. The effect of an antisense to p53 PTO, OL(1)p53, on the sensitivity of acute myelogenous leukemia (AML) cells to DNA-damage was also investigated. OL(1)p53 caused an increase in the sensitivity of AML cells to DNA-damaging agents. This effect was independent of changes in p53, though as OL(1)p53 had no effect on the expression of p53 protein. Investigation of alternate mechanisms, led to the isolation of a tumor-inhibitory factor (TIF), which was shown to be primarily composed of histones. It was shown that this factor was present in OMA-AML-1 cells, inhibitory for HL-60 cell growth, and was induced by OL(1)p53. Therefore, OL(1)p53 inhibited the growth and increased the sensitivity of AML cells to DNA-damage through possible induction of the tumor-inhibitory factor. Next, considering the clinical importance of PTO compounds, the ability of PTOs to interact with a reactive metabolite of acetaminophen, NAPQI, was investigated. These studies showed that both phosphorothioate nucleotides and PTOs were reactive with NAPQI. Next, it was determined that this interaction was not mutagenic, but may have inhibited the antisense effect of the PTOs. In conclusion, antisense PTOs directed against p53 mRNA could increase the sensitivity of cells to DNA-damaging agents; although, in some cell lines this effect may be independent of changes in p53.