Abstract
Objective: Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neurological and immunological diseases. AT is also characterized by cancer susceptibility including leukemia and lymphoma. In order to clarify the molecular basis for this cancer susceptibility in AT patients, DNA damage associated regulations of the cell cycle and apoptosis were investigated.
Materials: EBV-transformed cell lines (EB-LCLs) were established from two AT patients with homozygous ATM gene (AT-mutated gene) mutation, and two carriers with heterozygous ATM mutation. They were studied for DNA-damage associated with the cell cycle and apoptosis control, and were compared with the results in EB-LCLs obtained from healthy individuals.
Method: For the determination of acute phase apoptotic cell population, cells were fixed and stained by PI, and subjected to the calculation of subdiploid fraction by flow cytometry. The cell cycle status was studied according to the standard method by measuring DNA contents. For the reagents inducing DNA damage and/or apoptosis, X-irradiation, H2O2 and C2-ceramide were employed. Proteins involved in apoptosis and cell cycle regulation were analyzed by a standard western blotting. In vitro kinase assay was also used for the stress activated protein kinase/jun kinase (SAPK/JNK) activity involved in apoptosis induction. Radiation sensitivity of cells as determined by clonogenic cell survival activity was studied by the method described previously and compared with the results in apoptosis assay.
Results: LCLs from AT patients (AT- LCLs) showed a reduced activity to accumulate tumor suppressor p53 protein in response to X-irradiation, and the subsequent p21Cip1/wAF1transactivation was also defective. These results were in accord with the failure in the control of G 1 /S check point in AT-LCLs. AT-LCLs were also characterized by a failure in mitotic/spindle checkpoint control after X-irradiation, the precise mechanism for which may require further investigation. While the radiation induced clonogenic cell survival activity of AT-LCLs was poorer than the control LCLs, they were significantly more resistant to acute onset apoptosis.
Conclusion: Resistance to acute onset apoptosis with a low clonogenic activity and dysregulations of G1/S and mitotic checkpoints following DNA damage are the characteristic features of AT-LCLs. Further studies are needed to elucidate the molecular mechanisms of these features in regard to the physiological function of ATM protein which is mutated in AT patients.
