Tome Yasunori, Uehara Fuminari, Miwa Shinji, Yano Shuya, Mii Sumiyuki, Efimova Elena V, Bouvet Michael, Kimura Hiroaki, Tsuchiya Hiroyuki, Kanaya Fuminori, Hoffman Robert M
AntiCancer, Inc., San Diego, CA, U.S.A. Department of Surgery, University of California, San Diego, CA, U.S.A. Department of Orthopedic Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
AntiCancer, Inc., San Diego, CA, U.S.A. Department of Surgery, University of California, San Diego, CA, U.S.A. Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan.
Anticancer Res. 2016 Aug;36(8):3821-6.
The ability to image DNA repair in cancer cells after irradiation, as well as its inhibition by potential therapeutic agents, is important for the further development of effective cancer therapy. 53BP1 is a DNA repair protein that is overexpressed and forms foci when double-stranded DNA breaks occur in DNA.
The re-localization of green fluorescent protein (GFP) fused to the chromatin-binding domain of 53BP1 to form foci was imaged after UVC irradiation of breast and pancreatic cancer cells expressing 53BP1-GFP using confocal microscopy.
During live-cell imaging, 53BP1-GFP focus formation was observed within 10 minutes after UVC irradiation. Most 53BP1 foci resolved by 100 minutes. To block UVC-induced double-strand break repair in cancer cells, poly(ADP-ribose) polymerase (PARP) was targeted with ABT-888 (veliparib). PARP inhibition markedly enhanced UVC-irradiation-induced persistence of 53BP1-foci, even beyond 100 minutes after UVC irradiation, and reduced proliferation of breast and pancreatic cancer cells.
Confocal microscopy of 53BP1-GFP is a powerful method for imaging UVC-induced DNA damage and repair, as well as inhibition of repair.
对癌细胞受辐射后的DNA修复进行成像，以及评估潜在治疗药物对其的抑制作用，对于有效癌症治疗的进一步发展至关重要。53BP1是一种DNA修复蛋白，当DNA中发生双链断裂时，它会过度表达并形成病灶。
使用共聚焦显微镜对表达53BP1-GFP的乳腺癌和胰腺癌细胞进行紫外线C（UVC）照射后，对与53BP1染色质结合域融合的绿色荧光蛋白（GFP）重新定位形成病灶的情况进行成像。
在活细胞成像过程中，UVC照射后10分钟内观察到53BP1-GFP病灶形成。大多数53BP1病灶在100分钟时消失。为了阻断癌细胞中UVC诱导的双链断裂修复，使用ABT-888（维利帕尼）靶向聚（ADP-核糖）聚合酶（PARP）。PARP抑制显著增强了UVC照射诱导的53BP1病灶的持续性，甚至在UVC照射后100分钟以上仍存在，并降低了乳腺癌和胰腺癌细胞的增殖。
对53BP1-GFP进行共聚焦显微镜检查是一种强大的方法，可用于对UVC诱导的DNA损伤和修复以及修复抑制进行成像。
