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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
☑ THE AUTHOR HAS NO CONFLICT OF INTEREST TO DISCLOSE WITH RESPECT TO THIS PRESENTATION.
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 p53and T98G cells expressing mutant type (mu) of p53.
Top view of the KUR reactor
PURPOSE
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
BHO
C
H
COOHHO
CH2
NH2
10
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.
RESULT1-1 SURVIVAL CURVES OF A172 AND T98G GLIOMA CELLS FOLLOWING NEUTRON IRRADIATION
○No BPA●With BPA
No BPAWith BPA
T98G(mu p53) was more resistant than A172(wt p53).The difference of the radio sensitivity became small when BPA existed.
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.
A172 T98G
D10 (Gy)
BNCR
no BPA With BPA
(20ppm)
no BPA With BPA
(20ppm)
1.6 0.82 5.2 1.1
D10 (Gy)
Co60 gamma ray4.8 7.0
RBE(Relative Radiological Effectiveness)= BNCR D10/gamma D10
3.0 5.9 1.3 6.4
RESULT1-2 RBE VALUES CALCULATED BY D10 VALUES OF THE SURVIVAL CURVES
8A172 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.
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
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.
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
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
Thank you for your attention.