Human fibroblasts transfected with an ATM antisense vector respond abnormally to ionizing radiation.
ATM, the gene mutated in ataxia-telangiectasia (A-T), mediates multiple cellular responses to DNA damage. A-T homozygotes have a high risk of cancer and exhibit spontaneous chromosomal instability, and cultured A-T cells react abnormally to ionizing radiation. We have developed an ATM antisense vector that confers an A-T phenotype on normal cells. An episomal antisense vector was created that contained a 1.3 kb segment of the ATM cDNA, and was transfected into normal human fibroblasts. Intracellular levels of ATM protein were typically reduced 10-fold in antisense-expressing (GM639-46alpha) clones. GM639-46alpha clones exhibited the low threshold for radiation-induced apoptosis, low clonogenic survival, and cell cycle defects normally seen in A-T cells. Transfection with the corresponding ATM sense strand vector had no effect on the behavior of normal cells, and neither vector affected the behavior of A-T cells. Our results demonstrate that interference with ATM gene expression recreates the A-T phenotype in normal cells, and provide functional evidence linking the ATM gene to cellular DNA damage responses. The ATM antisense vector should prove a useful tool for studying ATM function in a variety of normal, mutant, and malignant cell lines.
Uhrhammer
N
N
Laboratoire d'Oncologie Moleculaire, Centre Jean Perrin, Clermont-Ferrand 63011, France.
Fritz
E
E
Boyden
L
L
Meyn
M S
MS
D017209:Apoptosis; D001260:Ataxia Telangiectasia / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000528:radiation effects; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics*; D011836:Radiation Tolerance / Q000235:genetics; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D017931:DNA Primers; D016373:DNA, Antisense; D004268:DNA-Binding Proteins; D011506:Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases