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

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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 　

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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.

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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.