J Korean Cancer Assoc.
1999 Oct;31(5):1065-1073.
Survival, Differentiation and ATM Phenotype of PC-12 Cells by Down - regulation of AT Gene
- Affiliations
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- 1Department of Pediatrics, Chonbuk National University Medical School, Chonju, Korea.
- 2Department of Institute for Medical Sciences, Chonbuk National University Medical School, Chonju, Korea.
- 3Department of Pediatrics, Seonam University Medical School, Namwon, Korea.
Abstract
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PURPOSE: Ataxia Telangiectasia (AT) is a hereditary multi-systemic disease resulting from mutations of AT gene and is characterized by progressive neurodegeneration, cancer, immune system defects, and hypersensitivity to ionizing radiation. AT gene has a homologue sequence of PI3-kinase. The activity and cellular function of PI3-kinase in AT gene remains unclear. This study was undertaken to evaluate the function of AT gene through the effect on cell survival and differentiation by the inhibition of AT gene expression.
MATERIALS AND METHODS
NH2-terminal portion of AT gene was isolated from MCF-7 cells by RT-PCR. The isolated DNA fragment was ligated in reverse orientation in pcDNA3. This antisense ATM expression vector was transfected to PC-12 cells by calcium phosphate method, and the transformed cells were selected using G418 and immunohisto- chemistry. To analyze the cell survival and differentiation, cells were cultured in serum free medium supplemented with/without NGF. We performed the immunoprecipitation for the p53 induction of cells after ionizing radiation, and the FACS for the apoptosis of cells after the exposure of wortmanin.
RESULTS
PC-12 cells which down-regulated AT gene (like ATM, AT mutated) showed decreased survival and ceased differentiation with NGF. Also, PC-12 (ATM) cells showed increased apoptosis with wortmanin and reduced or delayed p53 induction after ionizingradiation.
CONCLUSION
Results obtained from these studies suggest that AT gene regulates survival and differentiation of PC-12 cells through PI3-kinase activity. It seems that apoptosis is induced by the inhibition of AT gene expression.
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