Elucidation of a potential role of poly-ADP-ribosylation in the carcinogenesis or development of human colorectal carcinoma and its application for the diagnosis or trteatment of human colorectal carcinoma

目的：聚腺苷核糖聚合酶(PARP)催化聚腺苷5‘-二磷酸(ADP)核糖化，将多聚腺苷核糖部分从烟酰胺腺嘌呤二核苷酸(NAD)转移到受体蛋白。它是核蛋白的一种重要的翻译后修饰，参与包括恶性转化在内的多种生物学过程。因此，本研究的目的是评估多聚ADP核糖化，并比较PARP在结直肠癌(CRC)和癌旁非肿瘤粘膜中的表达。方法：从15例结直肠癌患者手术切除的结直肠组织中获得肿瘤组织和邻近非肿瘤组织，然后将其匀浆为胞浆和膜部部分。每种匀浆均用[腺苷-32&gt；P]-NAD检测ADP核糖化。用凝胶电泳法和放射自显影技术分离标记的蛋白质。用阴离子交换柱层析对ADP核糖化蛋白进行部分纯化，并用飞行时间质谱仪(TOF-MS)和免疫沉淀进行分析。结果：在人结直肠癌组织中，有几种蛋白质被ADP核糖化。值得注意的是，在15名受试者中，有13名受试者(87%)在癌组织的胞浆组分中发现了113 kDa蛋白质的显著放射性标记，并且高于邻近非肿瘤粘膜中的标记。抑制实验表明，该反应为酶促多聚ADP核糖基化反应。经TOF-MS和免疫沉淀鉴定，该113 kDa蛋白为肿瘤排斥抗原糖蛋白(GP)96。PARP在癌组织中的表达明显高于癌旁非癌粘膜，而gp96在癌组织和正常粘膜中的表达无明显差异。结论：癌组织中gp 96的Poly-ADP核糖化和PARP的表达均高于癌旁非癌粘膜，可能与结直肠癌的发生发展有关。

Objective: Poly-adenosine 5'-diphosphate (ADP)-ribosylation, catalyzed by poly (ADP-ribose) polymerase (PARP), transfers poly-ADP-ribose moieties from nicotinamide adenine dinucleotide (NAD) to an acceptor protein. It is an important post-translational modification of nuclear proteins and is involved in various biological processes including malignant transformation. Thus, the aim of this study was to evaluate poly-ADP-ribosylation and to compare the expression of PARP in human colorectal carcinomas (CRCs) and adjacent non-tumorous mucosae.Methods: Tumorous and adjacent non-tumorous tissues were obtained from surgically removed colorectums of 15 patients with CRC, and then were homogenized into cytosolic and membranous fractions. Each homogenate was assayed for ADP-ribosylation with [adenylate-^<32> P] -NAD. The radiolabeled proteins were separated by electrophoresis and autoradiography. The ADP-ribosylated protein was partially purified by anion exchange column chromatography, and analyzed by time-of-flight mass spectrometry (TOF-MS) and immunoprecipitation. Expression of PARP was also evaluated by Western blotting.Results: Several proteins were ADP-ribosylated in human CRC tissues. Notably, the remarkable radiolabeling of a 113-kDa protein was found in the cytosolic fractions from CRCs, and greater than that in adjacent non-tumorous mucosae in 13/15 subjects (87 %). Inhibition assays revealed that the reaction was enzymatic poly-ADP-ribosylation. By TOF-MS and immunoprecipitation, the 113-kDa protein was found to be tumor rejection antigen, glycoprotein (gp) 96. The expression of PARP was significantly greater in CRCs than in adjacent non-tumorous mucosae, while the expression of gp96 in CRCs and normal mucosae was identical.Conclusion: Poly-ADP-ribosylation of gp 96 and PARP expression were increased in human CRCs compared with those in adjacent non-tumorous mucosae, and it may be implicated in the carcinogenesis or development of human CRC.