Interaction of amifostine and ionizing radiation on transcriptional patterns of apoptotic genes expressed in human microvascular endothelial cells (HMEC).
OBJECTIVE
Amifostine is a prodrug that requires dephosphorylation by alkaline phosphatase to become activated. This process occurs rapidly within the bloodstream after its i.v. administration to patients undergoing cancer treatment with selected radiation and chemotherapies. Vascular endothelial cells will, therefore, represent a normal cell system that is among the first to experience the radioprotective effects of this agent. Amifostine's active free thiol WR-1065 was investigated to determine its effect on radiation-induced changes in transcriptional patterns and subsequent apoptosis in human microvascular endothelial cells (HMEC) growing in vitro.


METHODS
Human microvascular endothelial cells were grown to confluency and then exposed to WR-1065 at a concentration of 4 mM for 30 min, radiation doses that ranged from 0 to 6 Gy, and WR-1065 at a concentration of 4 mM for 30 min before exposure to ionizing radiation. Cell survival was assessed by clonogenic assay, cell cycle phase was analyzed by flow cytometry, apoptosis was also assessed by flow cytometry in which Anexin V staining and sub-G1 fraction analysis were applied, and gene expression was analyzed by the Clontech Atlas Human cDNA array to identify synergistic and antagonistic effects as a function of amifostine and radiation exposure conditions with a focus on apoptotic-related factors.


RESULTS
Exposure of HMEC to 4 mM WR-1065 30 min before irradiation resulted in a protection enhancement factor of 2.0; that is, D(O-IRR) of 1.25 Gy and D(O-IRR+WR) of 2.56 Gy. Expression profiling revealed 29 genes that were synergistically activated by the combined action of WR-1065 and ionizing radiation, and an additional 12 genes were synergistically or additively suppressed. In particular, a subset of apoptosis-related genes that included caspases 2, 4, and 9 and different members of the bcl family, along with apoptosis-related receptors, were identified as being significantly affected by the combined treatment of WR-1065 and radiation exposure. In addition, a number of cell cycle-related genes that express cyclins A, G1, G2, and D3 and DNA damage/check point proteins ATM, DNA-PK and RAD23B were also found to be significantly affected. Functional assays of apoptosis were also performed that demonstrated the ability of WR-1065 to protect against radiation-induced apoptosis.


CONCLUSIONS
WR-1065, the active thiol form of amifostine, is an effective radioprotector of HMEC as determined by use of clonogenic and apoptotic assays for cell survival. Expression profiling successfully defined the transcriptional response of HMEC to both WR-1065 and ionizing radiation exposure, either alone or in combination, and demonstrated both synergistic and antagonistic effects on the expression of different cellular genes, along with corresponding functional responses. The radioprotective effects of amifostine are not limited to its well-characterized physiochemical properties, which include free-radical scavenging, auto-oxidation leading to intracellular hypoxia, and chemical repair by hydrogen atom donation, but include its ability to modulate the complex transcriptional regulation of genes that are involved in apoptosis, cell cycle, and DNA repair.
Khodarev
Nikolai N
NN
Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, USA.
Kataoka
Yasushi
Y
Murley
Jeffrey S
JS
Weichselbaum
Ralph R
RR
Grdina
David J
DJ
D004999:Amifostine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D042783:Endothelial Cells / Q000187:drug effects / Q000528:radiation effects; D004730:Endothelium, Vascular / Q000166:cytology; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D008624:Mercaptoethylamines / Q000494:pharmacology*; D012372:ROC Curve; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects*
D008624:Mercaptoethylamines; D011837:Radiation-Protective Agents; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane; D004999:Amifostine