Unique proteomic signature for radiation sensitive patients; a comparative study between normo-sensitive and radiation sensitive breast cancer patients.

Radiation therapy is a cornerstone of modern cancer treatment. Understanding the mechanisms behind normal tissue sensitivity is essential in order to minimize adverse side effects and yet to prevent local cancer reoccurrence. The aim of this study was to identify biomarkers of radiation sensitivity to enable personalized cancer treatment. To investigate the mechanisms behind radiation sensitivity a pilot study was made where eight radiation-sensitive and nine normo-sensitive patients were selected from a cohort of 2914 breast cancer patients, based on acute tissue reactions after radiation therapy. Whole blood was sampled and irradiated in vitro with 0, 1, or 150 mGy followed by 3 h incubation at 37°C. The leukocytes of the two groups were isolated, pooled and protein expression profiles were investigated using isotope-coded protein labeling method (ICPL). First, leukocytes from the in vitro irradiated whole blood from normo-sensitive and extremely sensitive patients were compared to the non-irradiated controls. To validate this first study a second ICPL analysis comparing only the non-irradiated samples was conducted. Both approaches showed unique proteomic signatures separating the two groups at the basal level and after doses of 1 and 150 mGy. Pathway analyses of both proteomic approaches suggest that oxidative stress response, coagulation properties and acute phase response are hallmarks of radiation sensitivity supporting our previous study on oxidative stress response. This investigation provides unique characteristics of radiation sensitivity essential for individualized radiation therapy.

Radiation-induced stress response in peripheral blood of breast cancer patients differs between patients with severe acute skin reactions and patients with no side effects to radiotherapy.

The aim of the study was to compare the radiation-induced oxidative stress response in blood samples from breast cancer patients that developed severe acute skin reactions during the radiotherapy, with the response in blood samples from patients with no side effects. Peripheral blood was collected from 12 breast cancer patients showing no early skin reactions after radiotherapy (RTOG grade 0) and from 14 breast cancer patients who developed acute severe skin reactions (RTOG grade 3-4). Whole blood was irradiated with 0, 5 and 2000mGy γ-radiation and serum was isolated. The biomarker for oxidative stress, 8-oxo-dG, was analyzed in the serum by a modified ELISA. While a significant radiation-induced increase of serum 8-oxo-dG levels was observed in serum of the RTOG 0 patients, no increase was seen in serum of the RTOG 3-4 patients. The radiation induced increase in serum 8-oxo-dG levels after 5mGy did not differ significantly from the increase observed for 2000mGy in the RTOG 3-4 cohort, thus no dose response relation was observed. A receiver operating characteristic (ROC) value of 0.97 was obtained from the radiation-induced increase in 8-oxo-dG indicating that the assay could be used to identify patients with severe acute adverse reactions to radiotherapy. The results show that samples of whole blood from patients, classified as highly radiosensitive (RTOG 3-4) based on their skin reactions to radiotherapy, differ significantly in their oxidative stress response to ionizing radiation compared to samples of whole blood from patients with no skin reactions (RTOG 0). Extracellular 8-oxo-dG is primarily a biomarker of nucleotide damage and the results indicate that the patients with severe acute skin reactions differ in their cellular response to ionizing radiation at the level of induction of oxidative stress or at the level of repair or both.

Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA.

UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.