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: [email protected] 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… ɈɊɂȽɂɇȺɅɖɇɕȿ ɋɌȺɌɖɂ 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 - , - . 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