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 effects extend over a wide concentration range making amifostine a strong 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 differential display RT-PCR to assess the effects of both the thiol (SH), i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of amifostine on gene expression in CHO-AA8 cells. Cells were exposed to either 40 microM or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in corresponding untreated control cells. One band that showed a differential response was sequenced and was found to have 78% homology with a segment of the human pHL-1 cDNA clone contained in GenBank. This clone contains a COX III mitochondrial DNA insert and two exons of human c-myc. Northern blot analyses were performed by using the cloned human c-myc exon 1 probe to confirm whether c-myc gene expression was affected. Repression of c-myc expression was observed under all of the conditions evaluated. An exposure of cells to 40 microM of the disulfide form of amifostine was the most effective in repressing c-myc, i.e. 27% of control 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 microM concentrations reducing c-myc gene expression to 65% and 46% of control levels, respectively.
Liu
S C
SC
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, IL 60439, USA.
Murley
J S
JS
Woloschak
G
G
Grdina
D J
DJ
D004999:Amifostine / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides; D015536:Down-Regulation; D005786:Gene Expression Regulation / Q000187:drug effects; D016259:Genes, myc; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics; D013438:Sulfhydryl Compounds
D004220:Disulfides; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D004999:Amifostine