ИНГИБИТОР КЛЕТОЧНОГО БЕЛКА Р53 ПОЗИТИВНО

ɂɇȽɂȻɂɌɈɊ ɄɅȿɌɈɑɇɈȽɈ ȻȿɅɄȺ Ɋ53…
ɈɊɂȽɂɇȺɅɖɇɕȿ ɋɌȺɌɖɂ
25
ɍȾɄ 616-006.831-085.2/.3:578.826:576.533
ɂ.ȼ. ɍɥɚɫɨɜ1, ɇ.ȼ. Ʉɚɜɟɪɢɧɚ2, Ⱥ.ɘ. Ȼɚɪɵɲɧɢɤɨɜ.2
ɂɇȽɂȻɂɌɈɊ ɄɅȿɌɈɑɇɈȽɈ ȻȿɅɄȺ Ɋ53
ɉɈɁɂɌɂȼɇɈ ɊȿȽɍɅɂɊɍȿɌ ɐɂɌɈɌɈɄɋɂɑɇɈɋɌɖ
ɈɇɄɈɅɂɌɂɑȿɋɄɈȽɈ ȼȿɄɌɈɊȺ ȼ ɄɅȿɌɄȺɏ ȽɅɂɈȻɅȺɋɌɈɆɕ
1
ɐɟɧɬɪ ɨɩɭɯɨɥɟɣ ɝɨɥɨɜɧɨɝɨ ɦɨɡɝɚ, ɒɜɟɞɫɤɢɣ ɦɟɞɢɰɢɧɫɤɢɣ ɰɟɧɬɪ, ɋɢɷɬɥ, ɋɒȺ
2
ɎȽȻɇɍ «Ɋɨɫɫɢɣɫɤɢɣ ɨɧɤɨɥɨɝɢɱɟɫɤɢɣ ɧɚɭɱɧɵɣ ɰɟɧɬɪ ɢɦ ɇ.ɇ. Ȼɥɨɯɢɧɚ», Ɇɨɫɤɜɚ
Ʉɨɧɬɚɤɬɧɚɹ ɢɧɮɨɪɦɚɰɢɹ
ɍɥɚɫɨɜ ɂɥɶɹ ȼɚɥɟɧɬɢɧɨɜɢɱ, ɤ.ɛ.ɧ., ɜɟɞɭɳɢɣ ɫɨɬɪɭɞɧɢɤ ɰɟɧɬɪɚ ɥɟɱɟɧɢɹ ɨɩɭɯɨɥɟɣ ɝɨɥɨɜɧɨɝɨ ɦɨɡɝɚ
ɚɞɪɟɫ: 550 17th Avenue, suite 570, Seattle, Wa, 98122, USA; ɬɟɥ. +1-206-991-2053
e-mail: ulasov75@yahoo.com
03.10.2014,
24.11.2014.
Ɋɟɡɸɦɟ
.
53 –
,
.
50%
53
,
.
Ɋ53,
.
,
,
53
,
,
-
U87
PFTa
.
,
.
Ʉɥɸɱɟɜɵɟ ɫɥɨɜɚ:
, P53,
,
.
I.V. Ulasov1;2, N.V. Kaverina2 and A.Yu. Baryshnikov2
SUPPRESSION OF CELLULAR P53
PROMOTES CYTOTOXICITY OF ONCOLYTIC VECTOR
AT THE MODELS OF HUMAN GLIOBLASTOMA
1
The Center for Advanced Brain Tumor Treatment, Swedish Medial Center, Seattle, USA
2
FSBSI «N.N. Blokhin Russian Cancer Research Center», Moscow, Russia
Abstract
Glioblastoma multiforme resistance requires a new approach for glioma therapy. Protein p53 is one of the main
cellular oncogene, overexpressed in 50% of brain tumor cases. Impact of p53 attenuation was evaluated in the presence
of oncolytic adenovirus and temodar, which exhibit anti-glioma effect using in vitro glioma models. Using U87 human
glioma cells we observed an additive effect between alkilated chemotherapeutic agent temodar and oncolytic adenovirus which results into p53 inhibition. It occurs that attenuation of p53 using PFTa inhibitor, significantly prolongs cell
death type II- autophagy and, therefore improves effect mediated by autophagy induced agents. In conclusion, combination of PFTa and temodar might represent a powerful therapeutic combination which sensibilises glioma cells to the
infection with oncolytic vector.
Key words: adenovirus, p53, autophagy, proliferation.
ȼɜɟɞɟɧɢɟ
.
[6].
[1; 2; 4].
.
,
Galvin-Burgess,
,
,
,
53.
,
,
,
-
53
53.
,
-
,
,
-
-
,
.
.
53
,
-
[7].
,
–
,
.
,
-
.
,
,
1/
14/2015
-
ɂɇȽɂȻɂɌɈɊ ɄɅȿɌɈɑɇɈȽɈ ȻȿɅɄȺ Ɋ53…
ɈɊɂȽɂɇȺɅɖɇɕȿ ɋɌȺɌɖɂ
26
-
[8; 9].
,
CRAd-s-pK7,
-
.
+37 °C,
CO2
4
5 %
0,1
-
,
,
.
.
540
.
ɐɟɥɶɸ ɧɚɫɬɨɹɳɟɣ ɪɚɛɨɬɵ
-
,
53.
Ɇɚɬɟɪɢɚɥɵ ɢ ɦɟɬɨɞɵ
Ʉɥɟɬɤɢ ɢ ɤɥɟɬɨɱɧɵɟ ɥɢɧɢɢ
HEK293,
N10 U87,
A549
-
(Tokyo,
(Fisher
Scientific
Ⱥɧɚɥɢɡ ɷɤɫɩɪɟɫɫɢɢ ɛɟɥɤɨɜ
ɫ ɩɨɦɨɳɶɸ ɜɟɫɬɟɪɧ ɛɥɨɬɬɢɧɝɚ
,
6–
(ATCC, Manassas, VA).
5%
Ɋɟɚɝɟɧɬɵ
(Temozolomide),
53 (PFTalpha), 2 %PI, 1
/
Aldrich (
).
53
100μ
PFTa (1000×
AO
Sigma-
[10]
20
-
, [11]).
ȼɢɪɭɫɧɵɟ ɜɟɤɬɨɪɚ
CRAd-S-pK7
,
CRAd-S-pK7 (5
IU/ ),
–
PFTa.
72
,
50 M HEPES; 0,15
M NaCl, 0,5 %Nonidet P-40
.
10 %–
(PVDF, BioRad,
).
P53 (
DO-1), E1A
(
M58)
(
-2),
Santa Cruz Biotechnology (
,
).
,
Santa Cruz
Biotechnology (
,
).
,
(Bio-
Rad, Hercules,
SpeI,
,
7
,
PI.
,
CRAd-S-pK7 (5 IU/ ),
PFTa
HEK293
.
CRAd-S-pK7–
A549
Adeno Titer X kit
(Clontech, Mountain View, Ca, USA).
Ɍɟɫɬ ɞɥɹ ɨɩɪɟɞɟɥɟɧɢɹ ɩɪɨɥɢɮɟɪɚɰɢɢ
ɤɥɟɬɨɤ (MTT-ɬɟɫɬ)
+37 °
5%
72
.
-
PI (50
mg/
-
,
.
(«Costar»,
24
.
CRAd-S-pK7–
.
200
14/2015
-
10 %
).
,
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
1/
,
70 %)
4 .
.
2
L-
965 000 – 6 000
)
).
1-pSurvivin
.
,
2
(«Atlanta Bio», Atlanta,
,
Ɉɩɪɟɞɟɥɟɧɢɟ ɤɥɟɬɨɱɧɨɝɨ ɞɟɥɟɧɢɹ
ɢ ɚɭɬɨɮɚɝɢɢ
ɫ ɩɨɦɨɳɶɸ ɢɦɦɭɧɨɮɥɭɨɰɢɬɨɦɟɬɪɢɢ
-
,
2
).
ɐɢɬɨɥɨɝɢɱɟɫɤɢɣ ɦɟɬɨɞ
ɨɤɪɚɲɢɜɚɧɢɹ ɤɥɟɬɨɤ,
ɢɦɟɸɳɢɯ ɮɪɚɝɦɟɧɬɚɰɢɸ ȾɇɄ
TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick end labeling)
Roche
(
)
In situ cell death Detection kit.
)
(DMEM; Life Technologies),
10 %
(
) ;GIBCO-Life Technologies,
), 2 mM La, 100 IU/
a, 50 IU/
a (Invitrogen,
).
+37 °
2.
.
10 000
.
ɋɬɚɬɢɫɬɢɱɟɫɤɢɣ ɚɧɚɥɢɡ
±
,
(SPSS 13.0
).
.
-
.
,
0,05
,
-
ɂɇȽɂȻɂɌɈɊ ɄɅȿɌɈɑɇɈȽɈ ȻȿɅɄȺ Ɋ53…
ɈɊɂȽɂɇȺɅɖɇɕȿ ɋɌȺɌɖɂ
Ɋɢɫ. 1.
P53
U87
Ɋɢɫ. 2.
P53
:
N10
–
U87,
.
27
,
,
+
E1A
,
-
, P53
M58).
(
Ⱥ
ȼ
Ȼ
Ɋɢɫ. 3.
P53
-
Ƚ
:
–
P53;
–
P53
, “ # “ “##” p<0,05;
-
–
53
+
–
53,
“**”, “***” p<0,05
;
, “#”
1/
14/2015
“##”
. “*”,
.
ɂɇȽɂȻɂɌɈɊ ɄɅȿɌɈɑɇɈȽɈ ȻȿɅɄȺ Ɋ53…
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28
Ɋɟɡɭɥɶɬɚɬɵ ɢ ɨɛɫɭɠɞɟɧɢɟ
P53
Ɏɭɧɤɰɢɨɧɚɥɶɧɚɹ ɯɚɪɚɤɬɟɪɢɫɬɢɤɚ
ɤɥɟɬɨɱɧɨɝɨ P53
P53
20
,
P53
[3; 12]
U87.
0–
[3; 12].
24
.
.1
,
N10,
[3]
P53
U87.
"
"
.
,
P53
U87
N10,
P53.
-
Ɋ53
,
Ɋ53
21,
P53
.
.
,
-
P53
55,8 %),
( 18,2
24,1 63,8 %, p<0,05;
2–3
,
49,4 %)
. 3, ).
.
"
pK7,
72
CRAd-s-pK7–
E1A (
.2 ;
+
.
,
-
,
-
.
,
+
P53
( . 3, ).
(
P53
CRAd-S).
TUNEL
-
).
,
. 3, ).
U87
G2/M
.
Ɂɚɤɥɸɱɟɧɢɟ
,
,
,
-
P53
,
-
.
P53
,
P53
. 2, ).
,
PFTa
-
,
.
(
-
.
,
a
,
( 25,2
(
,
P53
,
,
P53
CRAd-S-pK7Ɋ53
U87
PFTalpha.
,
",
,
(
+
,
,
CRAd-S-pK7
,
,
-
P53
P53
[10].
-
,
[5].
Ʉɨɦɛɢɧɚɰɢɹ
ɬɟɪɚɩɟɜɬɢɱɟɫɤɢɯ ɩɨɞɯɨɞɨɜ
ɜɵɡɵɜɚɟɬ ɚɤɬɢɜɚɰɢɸ ɤɥɟɬɨɱɧɨɝɨ P53
,
ɂɧɝɢɛɢɪɨɜɚɧɢɟ P53
ɩɨɜɵɲɚɟɬ ɚɤɬɢɜɧɨɫɬɶ ɈɇȼɂɊ
ɢ ɬɟɦɨɞɚɥɚ
,
P53
U87 ( . 3, ).
,
P53
+
,
G2– M–
( . 3, ).
,
48
P53
-
,
-
.
Ʌɢɬɟɪɚɬɭɪɚ
ɍɥɚɫɨɜ ɂ.ȼ., Ʉɚɜɟɪɢɧɚ ɇ.ȼ., Ʉɚɞɚɝɢɞɡɟ Ɂ.Ƚ., Ȼɚɪɵɲɧɢɤɨɜ Ⱥ.ɘ.
:
,
//
. – 2014. – . 13, 2. – . 11–8.
2.
ɍɥɚɫɨɜ ɂ.ȼ., Ɍɭɫɨɧ Ⱥ., Ȼɚɪɵɲɧɢɤɨɜ Ⱥ.ɘ.
//
. – 2014. – . 13, 2. – . 51–5.
3.
Badie B., Goh C.S., Klaver J., Herweijer H., Boothman D.A. Combined radiation and p53 gene therapy of malignant glioma cells //Cancer Gene
Ther. – 1999. – 6. – . 155–62.
4.
Bao S., Wu Q., McLendon R.E. et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response //
Nature. – 2006. – 444. – . 756–60.
5.
el-Deiry W.S., Tokino T., Velculescu V.E. et al. WAF1 a potential mediator of p53 tumor suppression // Cell. – 1993. – 75. – . 817–25.
6.
Galvin-Burgess K.E., Travis E.D., Pierson K.E., Vivian J.L. TGF-beta-superfamily signaling regulates embryonic stem cell heterogeneity: selfrenewal as a dynamic and regulated equilibrium // Stem Cells. – 2013. – 31. – . 48–58.
7.
Kremenetskaya O.S., Logacheva N.P., Baryshnikov A.Yu. et al. Distinct effects of various p53 mutants on differentiation and viability of human
K562 leukemia cells // Oncology Research. – 1997. – 9. – P. 155–66
8.
Ulasov I.V., Tyler M.A., Rivera A.A. et al. Evaluation of E1A double mutant oncolytic... // J Med Virol. – 2008. – 80. – P. 1595–603.
9.
Ulasov I.V., Zhu Z.B., Tyler M.A. et al. Survivin-driven and fiber-modified oncolytic adenovirus exhibits potent antitumor activity in established
intracranial glioma // Hum Gene Ther. – 2007. – 18. – P. 589–602.
10. Ulasov I.V., Sonabend A.M., Nandi S. et al. Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant
glioma through autophagic and apoptotic cell death in vivo // Br J Cancer. – 2009. – 100. – P. 1154–64.
11. Xu G.W., Mymryk J.S., Cairncross J.G. Pharmaceutical-mediated inactivation of p53 sensitizes U87MG glioma cells to BCNU and
temozolomide // Int J Cancer. – 2005. – 116. – P. 187–92.
12. Yamashita K., Nakashima S., You F. et al. Overexpression of immediate early gene X-1 (IEX-1) enhances gamma-radiation-induced apoptosis
of human glioma cell line, U87-MG. // Neuropathology. – 2009. – 29. – P. 20–4.
1.
1/
14/2015