The Influence of Telomerase on Induction and Repair of Targeted and Non-targeted Radiation Effects

The main aim of the project is to investigate the role of the telomere/telomerase system in the bystander effect. Pilot experiments were carried out on broad field X-ray and y-ray-induced bystander effect in normal BJ and immortalized BJ1-hTERT human foreskin fibroblasts. This work led to finding increased clonogenic inactivation and chromosomal damage in cells directly hit by ionizing radiation and provided direct evidence for medium-mediated bystander responses (micronuclei and cell inactivation) in fibroblasts irradiated with low LET radiation. Later, this work on targeted and non-targeted effects of radiation was extended as a number of different responses appeared which suggested expanding the scope of the studies. Therefore different cytogenetic responses and their relationship were studies. Connections between bystander effects and other non-targeted effects of radiation, such as low-dose hypersensitivity/increased radio-resistance phenomenon were considered. There was an indication that the bystander effect may play a role in cell inactivation at low radiation doses. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named y-H2AX, forms foci at DSB sites. Although y-H2AX foci were observed in exponentially growing cells containing media conditioned on X-irradiated cells, it is not clear if X-irradiation leads to double strand breaks in bystander cells. The data in this study suggests that lesions other than DNA double strand breaks are involved in the bystander effect and that different mechanisms are responsible for the production of y-H2AX foci in direct irradiated and bystander cells. No induction of foci of y-H2AX in bystander confluent cells were observed and this result is discussed. Our findings rule out any major involvement of the telomerase in the bystander effect and propose that telomerase may have other physiological functions associated with the protection of chromosomes from breakage

Correlation between DNA damage responses of skin to a test dose of radiation and late adverse effects of earlier breast radiotherapy

Aim: To correlate residual double strand breaks (DSB) 24 h after 4 Gy test doses to skin in vivo and to lymphocytes in vitro with adverse effects of earlier breast radiotherapy (RT). Patients and methods: Patients given whole breast RT P5 years earlier were identified on the basis of moderate/marked or minimal/no adverse effects despite the absence (‘RT-Sensitive’, RT-S) or presence (‘RT-Resistant’, RT-R) of variables predisposing to late adverse effects. Residual DSB were quantified in skin 24 h after a 4 Gy test dose in 20 RT-S and 15 RT-R patients. Residual DSB were quantified in lymphocytes irradiated with 4 Gy in vitro in 30/35 patients. Results: Mean foci per dermal fibroblast were 3.29 (RT-S) vs 2.80 (RT-R) (p = 0.137); 3.28 (RT-S) vs 2.60 (RT-R) in endothelium (p = 0.158); 2.50 (RT-S) vs 2.41 (RT-R) in suprabasal keratinocytes (p = 0.633); 2.70 (RT-S) vs 2.35 (RT-R) in basal epidermis (p = 0.419); 12.1 (RT-S) vs 10.3 (RT-R) in lymphocytes (p = 0.0052). Conclusions: Residual DSB in skin following a 4 Gy dose were not significantly associated with risk of late adverse effects of breast radiotherapy, although exploratory analyses suggested an association in severely affected individuals. By contrast, a significant association was detected based on the in vitro response of lymphocytes

Investigating the impact of long term exposure to chemical agents on the chromosomal radiosensitivity using human lymphoblastoid GM1899A cells

Abstract This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising radiation. Human lymphoblastoid GM1899A cells were cultured in the presence of 4-nitroquinoline N-oxide (4NQO), N-nitroso-N-methylurea (MNU) and hydrogen peroxide (H2O2) for up to 6 months at the highest non-(geno)toxic concentration identified in pilot experiments. Acute challenge doses of 1 Gy X-rays were given and chromosome damage (dicentrics, acentric fragments, micronuclei, chromatid gaps/breaks) was scored. Chronic exposure to 20 ng/ml 4NQO, 0.25 μg/ml MNU or 10 μM H2O2 hardly induced dicentrics and did not significantly alter the yield of X-ray-induced dicentrics. Significant levels of acentric fragments were induced by all chemicals, which did not change during long-term exposure. Fragment data in combined treatment samples compared to single treatments were consistent with an additive effect of chemical and radiation exposure. Low level exposure to 4NQO induced micronuclei, the yields of which did not change throughout the 6 month exposure period. As for fragments, micronuclei yields for combined treatments were consistent with an additive effect of chemical and radiation. These results suggest that cellular radiation responses are not affected by long-term low-level chemical exposure