Altered apoptotic profiles in irradiated patients with increased toxicity

Abstract

Purpose: A retrospective study of radiation-induced apoptosis in CD4 and CD8 T-lymphocytes, from 12 cancer patients who displayed enhanced toxicity to radiation therapy and 9 ataxia telangiectasia patients, was performed to test for altered response compared to healthy blood-donors and normal cancer patients. Methods and Materials: Three milliliters of heparinized blood from each donor was sent via express post to the Paul Scherrer Institute (PSI) for subsequent examination. The blood was diluted 1:10 in RPMI medium, irradiated with 0-, 2-, or 9-Gy X-rays, and incubated for 48 h. CD4 and CD8 T-lymphocytes were then labeled using FITC-conjugated antibodies, erythrocytes were lysed, and the DNA stained with propidium iodide. Subsequently, cells were analyzed using a Becton Dickinson FACScan flow cytometer. Radiation-induced apoptosis was recognized in leukocytes as reduced DNA content attributed to apoptosis-associated changes in chromatin structure. Apoptosis was confirmed by light microscopy, electron microscopy, and by the use of commercially available apoptosis detection kits (in situ nick translation and Annexin V). Data from hypersensitive individuals were compared to a standard database of 105 healthy blood-donors, and a database of 48 cancer patient blood donors who displayed normal toxicity to radiation therapy. To integrate radiosensitivity results from CD4 and CD8 T-lymphocytes after 2 and 9 Gy, z-score analyses were performed. Results: A cohort of 12 hypersensitive patients was evaluated; 8 showed enhanced early toxicity, 3 showed enhanced late toxicity, and 1 showed both. The cohort displayed less radiation-induced apoptosis (؊1.8 ) than average age-matched donors. A cohort of 9 ataxia telangiectasia homozygotes displayed even less apoptosis (؊3.6 ). Conclusion: The leukocyte apoptosis assay appears to be a useful predictor of individuals likely to display increased toxicity to radiation therapy; however, validation of this requires a prospective study.

CD4 and CD8 T-Lymphocyte Apoptosis Can Predict Radiation-Induced Late Toxicity: A Prospective Study in 399 Patients

“…In several cell lines, high apoptotic frequency is correlated with increased radiosensitivity (20,29,30). Our previous data showed that lymphocytes from patients displaying increased radiation toxicity showed low, and not high, levels of radiation-induced apoptosis (10). Survival fraction at 2 Gy from the skin fibroblast clonogenic assays has been shown to be effective in predicting intrinsic radiosensitivity; however, it is a timeconsuming (ca.…”

Single Nucleotide Polymorphisms, Apoptosis, and the Development of Severe Late Adverse Effects After Radiotherapy

“…As described (4,5,8), heparinized whole blood collected before starting RT was diluted 1:10 in RPMI 1640 containing 20% fetal bovine serum, irradiated with 0 and 8 Gy, and incubated for 48 h. The cells were then labeled with FITC-conjugated anti-CD8 monoclonal antibodies, RBC were lysed, and the DNA of the remaining cells stained with propidium iodide. Samples were measured using a FACScan flow cytometer, and data analysis was done using CellQuest software (Becton Dickinson).…”

Toxicity from radiation therapy associated with abnormal transcriptional responses to DNA damage

“…Cultured skin fibroblasts from patients with acute radiation toxicity showed decreased survival after IR in some studies (13) but not others (14,15). Lymphocytes from patients with radiation toxicity showed a paradoxical decrease in IR-induced apoptosis (16). Lymphocytes from breast cancer patients with severe skin reactions developed abnormal numbers of chromosome aberrations after IR exposure (17).…”