An antisense construct of full-length human RAD50 cDNA confers sensitivity to ionizing radiation and alkylating agents on human cell lines.
In Saccharomyces cerevisiae, Rad50 is reported to participate in the repair of double-stranded DNA breaks, and most rad50 mutants are unable to repair gamma-ray-induced DNA damage. In this study, we examined whether human RAD50 is involved in the repair of DNA damage induced by gamma radiation, radiomimetic alkylating agents, or UVB radiation in cultured human cells. Because homozygous null RAD50 mutant cells could not be isolated, human 293 embryonic kidney cells and A431 epithelial tumor cells were transfected with antisense RAD50 cDNA to obtain viable cell lines which expressed reduced RAD50. Selected individual clones were subjected to PCR-Southern and Western blot analyses to confirm the integrity of the antisense RAD50 construct and the reduced RAD50 expression levels. The cells engineered to express reduced RAD50 levels showed significantly increased sensitivity to gamma radiation, mitomycin C and methylmethane sulfonate compared with control cells that were transfected with the vector alone. However, there were no differences in viability of cells with reduced RAD50 levels and control cells treated with UVB radiation. These results indicate that human RAD50 is involved in the repair of DNA damage induced by gamma radiation and alkylating agents in mammalian cells and suggest the possible application of antisense RAD50 cDNA transfection as a radiation sensitizer in radiation oncology.
Kim
Young Chul
YC
Department of Diagnostic Radiology, Chosun University Hospital, Kwangju 501-759, South Korea.
Koh
Jeong Tae
JT
Shin
Boo Ahn
BA
Ahn
Kyu Youn
KY
Choi
Bong Kyu
BK
Kim
Chang Guhn
CG
Kim
Kyung Keun
KK
D017766:Acid Anhydride Hydrolases; D000477:Alkylating Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D002460:Cell Line / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D018076:DNA, Complementary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005720:Gamma Rays / Q000009:adverse effects*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008113:Liver Neoplasms / Q000473:pathology; D000076228:MRE11 Homologue Protein; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016685:Mitomycin / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics* / Q000494:pharmacology; D011836:Radiation Tolerance / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects
D000477:Alkylating Agents; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D020319:Oligodeoxyribonucleotides, Antisense; D016685:Mitomycin; D008741:Methyl Methanesulfonate; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes