P53 INTERFERENCE AND GROWTH-INHIBITION IN P53-MUTANT AND OVEREXPRESSING ENDOMETRIAL CANCER CELL-LINES

Background. The presence of p53 mutations and associated mutant p53 ov erexpression has been demonstrated in many cancer systems. Whether the overexpression of mutant p53 represents cause or effect, and whether p53 mutation contributes actively to the malignant phenotype is a matt er of controversy. We examined the growth effects of oligonucleotides designed to interfere with p53 expression and/or activity in p53-mutan t/overexpressing endometrial cancer cell lines. Methods. Phosphorothio ate oligonucleotides were used to target p53-related sequences in two p53-mutant/overexpressing endometrial cancer cell lines (KLE and RL95- 2) and a normal fibroblast control. The ATP cell viability assay was u sed to measure growth effects after 6-day treatments with 27-mer and 1 4-mer sense (S) or antisense (AS) phosphorothioate oligodeoxyribonucle otides (oligos) targeting the promoter/ATG region of p53 and/or the p5 3 consensus (CON) DNA binding sequence. These sequences were designed to interfere with p53 expression and activity, respectively. Random se quences of the p53 27- and 14-mer were used as controls for nonspecifi c oligo effects, and a normal fibroblast cell line was used to compare oligo effects and serve as a negative p53 immunostaining control. Res ults. Mean +/- SE IC50 (50% growth inhibition) of the S, AS p53, and p 53 CON oligos were 4.2 +/- 1.3, 4.7 +/- 0.9, and 7.6 +/- 1.4 mu M, res pectively, for the two endometrial cell lines combined, The AS and S p 53 oligos demonstrated dose-dependent inhibitory effects in both cell lines, while p53 CON produced variable effects alone and in combinatio n with p53 AS. In KLE, a uniform inhibitory dose response was seen wit h p53 CON oligos. In RL95-2, the approximate IC50 for p53 CON was 0.5- 1.0 mu M, but at increasing doses above this, an inverse dose response was consistently observed. Combinations of p53 AS and p53 CON oligos produced predominantly synergistic growth inhibition. Although combina tions of p53 AS and p53 CON in KLE were synergistic at low doses, anta gonistic effects occurred at higher concentrations. Oligos had little effect on normal fibroblast growth, with calculated IC50 > 16 mu M. Eq uimolar combinations of p53 S and AS were antagonistic, indicating tha t antiproliferative effects were sequence-specific. Random oligos demo nstrated some nonspecific inhibitory effects, with >25% growth inhibit ion at 16 mu M and beyond. Immunoperoxidase staining for mutant p53 af ter exposure to 16 mu M concentrations of p53 AS oligos demonstrated r eductions in p53 staining but persistent overexpression relative to wi ld-type (fibroblast) cells. Conclusion. Phosphorothioate oligos direct ed against p53 sequences in two p53-mutant endometrial cancer cell lin es demonstrated antiproliferative effects. Combined anti-p53 and anti- p53 binding site oligos resulted in predominantly synergistic antiprol iferative effects. The activity of sense oligos, the variable response s to p53 CON, and the persistent overexpression of mutant p53 at high concentrations of growth-inhibiting anti-p53 oligos suggest that, whil e promising, the antineoplastic effects of these oligos occur through complex and incompletely understood mechanisms. (C) 1997 Academic Pres s.