Delayed cytoprotection after enhancement of Sod2 (MnSOD) gene expression in SA-NH mouse sarcoma cells exposed to WR-1065, the active metabolite of amifostine.
SA-NH mouse sarcoma cells were grown to confluence and then exposed to either 40 microM or 4 mM of WR-1065, i.e. the active thiol form of amifostine, for 30 min and then washed. Total RNA and protein were isolated at various times up to 24 h after exposure. Both concentrations of WR-1065 were equally effective in affecting Sod2 (also known as MnSOD) gene expression and protein levels. Northern blot analysis using a mouse cDNA probe revealed three Sod2 transcripts of 1, 4 and 6 kb. Expression of both the 4- and 6-kb transcripts increased by 20 and 60%, respectively, and remained elevated over a period of 4 to 20 h. Sod2 protein levels, as determined by Western blot analysis, increased 15-fold over background control levels over the same interval. Sod2 protein was evaluated using activity gels and was found to be active. SA-NH cells were irradiated with X rays either in the presence of 40 microM or 4 mM WR-1065 or 24 h later after its removal, when Sod2 protein levels were most elevated. No protection was observed for cells irradiated in the presence of 40 microM WR-1065. In contrast, survival after a dose of 2 Gy was elevated 1.27-, 1.14- and 1.20-fold in SA-NH cells irradiated in the presence of 4 mM WR-1065 or 24 h after exposure of the cells to 40 microM and 4 mM WR-1065, respectively. The increased survival levels observed 24 h after exposure to WR-1065 represents a delayed radioprotective effect of WR-1065 and corresponds to the time at which Sod2 protein levels are most elevated. These data demonstrate a novel mechanism for radioprotection by WR-1065 and suggest a new potential concern regarding the issue of tumor protection.
Murley
Jeffrey S
JS
Department of Radiation and Cellular Oncology, The University of Chicago, 5841 S. Maryland Avenue (MC1105), Chicago, Illinois 60637,USA.
Kataoka
Yasushi
Y
Weydert
Christine J
CJ
Oberley
Larry W
LW
Grdina
David J
DJ
D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D008624:Mercaptoethylamines / Q000494:pharmacology*; D051379:Mice; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D012513:Sarcoma, Experimental / Q000201:enzymology* / Q000235:genetics; D013482:Superoxide Dismutase / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014965:X-Rays; D000099156:Superoxide Dismutase 2
D008624:Mercaptoethylamines; D011837:Radiation-Protective Agents; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane