REPRESSION OF C-MYC GENE-EXPRESSION BY THE THIOL AND DISULFIDE FORMS OF THE CYTOPROTECTOR AMIFOSTINE

The clinically approved cytoprotector amifostine, designated WR-2721, [S-2-(3-aminopropylamino)ethylphosphorothioic acid], protects against both radiation and drug-induced mutagenesis in animal systems, These e ffects extend over a wide concentration range making amifostine a stro ng candidate for evaluation as a possible cancer chemopreventive agent , To better identify and develop potential intermediate biomarkers for chemoprevention at the molecular level we applied the technique of di fferential display RT-PCR to assess the effects of both the thiol (SK) , i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of am ifostine on gene expression in CHO-AAS cells, Cells were exposed to ei ther 40 mu M or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in cor responding untreated control cells, One band that showed a differentia l response was sequenced and was found to have 78% homology with a seg ment of the human pHL-1 cDNA clone contained in GenBank, This clone co ntains a COX III mitochondrial DNA insert and two exons of human c-myc , Northern blot analyses were performed by using the cloned human c-my c exon 1 probe to confirm whether c-myc gene expression was affected, Repression of c-myc expression was observed under all of the condition s evaluated, An exposure of cells to 40 CIM of the disulfide form of a mifostine was the most effective in repressing c-myc, i.e. 27% of cont rol level, A concentration of 4 mM of the disulfide form reduced gene expression to 45% of the control level, while the thiol form was less effective, with 4 mM and 40 mu M concentrations reducing c-myc gene ex pression to 65% and 56% of control levels, respectively.