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：在聚腺苷核糖聚合酶（PARP）催化下，聚腺苷5′-二磷酸（ADP）核糖基化反应将聚腺苷核糖从烟酰胺腺嘌呤二核苷酸（NAD）转移到受体蛋白上。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过程。因此，本研究的目的是评估聚adp核糖基化，并比较PARP在人类结直肠癌（crc）和邻近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中获得肿瘤组织和邻近非肿瘤组织，然后均质成细胞质和膜质部分。每个匀浆用[腺苷酸-^<32> P] - nad检测adp -核糖基化。用电泳和放射自显影法分离放射性标记蛋白。用阴离子交换柱层析法纯化adp -核糖基化蛋白，并用飞行时间质谱（TOF-MS）和免疫沉淀法分析。Western blotting检测PARP的表达。结果：若干蛋白在人结直肠癌组织中发生adp核糖基化。值得注意的是，在crc的细胞质组分中发现了显著的113-kDa蛋白的放射性标记，并且在13/15的受试者中（87%）比邻近的非肿瘤粘膜中更高。抑制实验显示该反应为酶促聚adp核糖基化反应。通过TOF-MS和免疫沉淀鉴定，113-kDa蛋白为肿瘤排斥抗原，糖蛋白（gp） 96。PARP在CRCs中的表达明显高于相邻的非肿瘤粘膜，而gp96在CRCs中的表达与正常粘膜相同。结论：与癌旁非肿瘤粘膜组织相比，人CRC组织中gp96的聚adp核糖基化和PARP的表达增加，可能参与了人CRC的癌变或发展。

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.