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5 '-二磷酸（ADP）-核糖基化由聚（ADP-核糖）聚合酶（PARP）催化，将聚ADP-核糖部分从烟酰胺腺嘌呤二核苷酸（NAD）转移到受体蛋白。它是一种重要的核蛋白翻译后修饰，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聚ADP-核糖基化和比较PARP在人结直肠癌（CRCs）和邻近的非肿瘤mucosae.Methods的表达：肿瘤和邻近的非肿瘤组织，从手术切除的结直肠癌患者15例，然后被匀浆成细胞溶质和膜组分。用[腺苷酸-β P] -NAD测定每个匀浆的ADP-核糖基化<32>。通过电泳和放射自显影分离放射性标记的蛋白质。用阴离子交换柱层析法对ADP核糖基化蛋白进行部分纯化，并通过飞行时间质谱（TOF-MS）和免疫沉淀法进行分析。结果：在结直肠癌组织中，有几种蛋白被ADP核糖基化。值得注意的是，在CRC的胞质组分中发现了113-kDa蛋白质的显著放射性标记，并且在13/15例受试者（87%）中大于邻近非肿瘤粘膜中的放射性标记。抑制试验表明，该反应是酶促聚ADP核糖基化。通过飞行时间质谱和免疫沉淀，113 kDa的蛋白被发现是肿瘤排斥抗原，糖蛋白（gp）96。结直肠癌组织中PARP的表达明显高于癌旁组织，而gp 96的表达与癌旁组织无明显差异。结论：结直肠癌组织中gp 96的多聚ADP核糖基化和PARP的表达高于癌旁组织，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.