1. THE INFLUENCE OF THE P53STATUS FOR BIOLOGICAL EFFECTS OF THE GLIOBLASTMA CELLS FOLLOWING BORON NEUTRON CAPTURE THERAPY Keiko Seki, Yuko Kinashi, Sentaro Takahashi and Koji Ono Kyoto University Research Reactor Institute

2. ☑ T HE AUTHOR HAS NO CONFLICT OF INTEREST TO DISCLOSE WITH RESPECT TO THIS PRESENTATION.

3. To investigate the relationship of p53 tumor suppressor gene with biological effects of BNCT in two types of human glioblastoma cells; A172 expressing wild type (wt) of p53 and T98G cells expressing mutant type (mu) of p53. Top view of the KUR reactor PURPOSE

4. Cells Human glioblastoma cells A172 expressing wild type (wt) of p53 and T98G cells expressing mutant type (mu) of p53 Boron compound Cells were trypsinized and cell suspensions were incubated with 20 μg/ml BPA at 1hour prior to the neutron irradiation. Neutron irradiation The Heavy Water Facility of the Kyoto University Research Reactor (KUR) was used for mixed beam irradiation (total doses: approximately 2Gy/50min (1MW) Neutron fluence was measured by radio-activation of gold foil and gamma-ray doses by TLD. MATERIALS AND METHODS BPA:borono phenyl alanine 10

5. MATERIALS AND METHODS 1. Cell survival assay Survival of human glioma cells were determined by colony formation. 2. Evaluation of 53BP1 foci After irradiation, the cells were fixed with 4% formalin and stained immunochemically with 53BP1 antibody. The number of DNA-DSBs was determined by counting the 53BP1 foci. For image processing and automated foci counting, Image J (National Institutes of Health, Bethesda, Maryland, USA) was used. 3. Apoptosis detection TUNEL method to apoptosis detection was used. Irradiated cells were seeded on to 22x22mm coverslips and were detected apoptosis cells with In situ Cell Death Detection Kit, TMR red (Roche). Biological effects was studied by three methods.

6. RESULT1-1 SURVIVAL CURVES OF A172 AND T98G GLIOMA CELLS FOLLOWING NEUTRON IRRADIATION ○ No BPA ● With BPA No BPA With BPA T98G(mu p53) was more resistant than A172(wt p53). The difference of the radio sensitivity became small when BPA existed.

7. Under no BPA existence, RBE of A172 is 2.3 times lager than that of T98G. Under 20 ppm of BPA, RBE of A172 is slightly smaller than that of T98. A172T98G D 10 (Gy) BNCR no BPAWith BPA (20ppm) no BPAWith BPA (20ppm) 1.60.825.21.1 D 10 (Gy) Co 60 gamma ray4.87.0 RBE (Relative Radiological Effectiveness) = BNCR D10/gamma D10 3.05.91.36.4 RESULT1-2 RBE VALUES CALCULATED BY D10 VALUES OF THE SURVIVAL CURVES

8. 8 A172 60min after NT 0.665Gy with BPA T98G 60min after NT 0.665Gy withBPA RESULT2 53BP1 FOCI STUDY 53BP1 foci was increased depend on the neutron irradiation dose. There was little difference of foci number between the A172 and T98G under 1.0 Gy. There was little difference of foci number with or without BPA.

9. RESULT3 APOPTOSIS DETECTION BY TUNEL METHOD 9 A172(wt p53 ） A172 + BPA T98G(mu p53)T98G + BPA 5hours after neutron irradiation (1.015 Gy) by KUR Red stained nuclei are the apoptotic cells. Intact nuclei are stained blue by DAPI. A172(wt p53) cells were induced apoptosis with or without BPA. T98G (mu p53) cells were induced apoptosis with BPA.

10. 10 Result3 Induction Rates of Apoptosis Detection A172(wt p53) cells were induced apoptosis with or without BPA. T98G (mu p53) cells were induced apoptosis with BPA. The apparent difference of apoptosis induction was shown between A172 cells and T98G cells following neutron irradiation.

11.  A172(wt P53) cells were more sensitive than T98G(mt P53) cells following neutron irradiation. The difference of cell killing effect of neutron between two cell lines was reduced under the existence of BPA.  There were no difference between the initial damage of the DSBs foci following neutron irradiation in A172 and T98G cells.  The apparent difference of apoptosis induction betweA172 cells and T98G cells following neutron irradiation. SUMMARY OF THE RESULTS

12. CONCLUSION This study revealed that BNCT caused cell death in the glioblastoma cells, regardless of mutant p53 status. In the T98G cells(mutant p53 ), cell killing and apoptosis were occurred effectively following BNCT, which may be due to p53- independent apoptosis or other mechanism KUR reactor

13. Thank you for your attention.