In vitro studies of cellular response to DNA damage induced by BNCT.

PERONA, M; PONTIGGIA, O; CARPANO, M; THOMASZ, L; THOP, S; POZZI, E; SIMIAN, M; KAHL, S; JUVENAL, G; PISAREV , M; DAGROSA, MA

Buenos Aires

Congreso; 14th International Congress on Neutron Capture Therapy; 2010

CNEA, UNSAM, ROFFO

Little is known about the mechanisms of cellular response to double-strand DNA damage induced by BNCT. Cell cycle regulation and repair mechanisms such as homologous recombination (HR) and non homologous end-joining (NHEJ) are important determinants of ionizing radiation sensitivity. The p53 protein plays a role in the activation of multiple genes that can induce: temporary G1-S cell cycle arrest, DNA repair, irreversible growth arrest and apoptosis. On the other hand, Ku70 protein is involved in the repair of DNA double strand breaks via NHEJ pathway. In the present work we analyzed the survival fraction, the expression of p53 and Ku70 involved in cell cycle regulation and DNA repair. We also evaluated the mechanisms of death induced by BNCT and compared them with the conventional gamma radiation. Materials and Methods: Exponentially growing cells of follicular thyroid carcinoma (WRO) were distributed into the following groups: 1) BPA (10 µg10B/mL) + neutrons; 2) BOPP 2,4-bis (a,b-dihydroxyethyl)-deutero-porphyrin IX) (10 µg10B/mL) + neutrons; 3) neutrons alone; 4) gamma-rays.  A control group without irradiation for each treatment was added. The cells were irradiated in the thermal neutron beam of the RA-3 (flux= 7.5 109 n/cm2 sec) or with 60Co (1Gy/min) in order to obtain a total physical dose of 3 Gy (±10%). After 24 and 48 hr post irradiation, the protein expression of p53 and Ku70 was analyzed by Western blotting. The frequency of cell death (apoptosis and necrosis) was measured using fluorescence microscope. Cell cycle analysis was performed by flow cytometry. Results and Discussion: The neutron irradiation of the cells showed a decrease in the cell viability. This effect was greater in the cells incubated with both boron compounds (BPA and BOPP). An increase of p53 at 24 hr for the neutrons alone and gamma groups compared to the control group (without irradiation and boron compound) was observed. On the other hand an up regulation of Ku 70 was observed in the group irradiated with neutrons alone at 24 hr. At this time the BOPP + neutrons group showed a decrease in the expression of Ku70 of approximately 1.6 times compared to its control group and an increase of 1.6 times for BPA + neutrons group. At 48 hr it was observed an increase of Ku70 in the BOPP +neutrons group of 1.13 and 1.3 times for gamma group.  Accumulation of cells in G2/M was observed at both time points for all the irradiated groups (1, 2, 3 and 4). The study of mechanisms of death showed that at 24 hr the frequency of apoptosis and necrosis increased in all the groups irradiated with neutrons (Groups 1, 2 and 3) respect to gamma rays and control groups. On the other hand at 48 hr an increase in the cell death was observed in the BNCT group irradiated with BOPP (around 5% apoptosis and 20% necrosis). These preliminary results suggest different cellular response for all the irradiated groups. All the groups irradiated with gamma or neutrons seem to induce the repair system NHEJ by increase of Ku70 at different times. But the decrease of Ku in the BNCT (neutrons + BOPP) group could play a role in the increase of sensitization to radiation of follicular thyroid cells.