Robertson K A, Bullock H A, Xu Y, Tritt R, Zimmerman E, Ulbright T M, Foster R S, Einhorn L H, Kelley M R
Herman B. Wells Center for Pediatric Research, Department of Pediatrics, James Whircomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis 46202, USA.
Cancer Res. 2001 Mar 1;61(5):2220-5.
The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions including oxidative signaling, transcription factor regulation, and cell cycle control. It acts on mutagenic AP (baseless) sites in DNA as a critical member of the DNA BER repair pathway. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1alpha, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2-3-fold increase in protection against bleomycin. Lesser protection was observed with gamma-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents.
人类脱嘌呤嘧啶内切核酸酶（Ape1或ref-1）是一种DNA碱基切除修复（BER）酶，是一种多功能蛋白质，对多种重要的细胞功能有影响，包括氧化信号传导、转录因子调节和细胞周期控制。作为DNA BER修复途径的关键成员，它作用于DNA中的诱变脱嘌呤嘧啶（无碱基）位点。此外，Ape1/ref-1通过氧化还原机制刺激转录因子（Fos-Jun、核因子-κB、Myb、ATF/环磷酸腺苷反应元件结合蛋白家族、HIF-1α、HLF、PAX和p53）的DNA结合活性，因此代表了调节真核基因表达的信号转导过程的一个新组分。Ape1/ref-1也已被证明与硫氧还蛋白相关的细胞凋亡密切相关，并且在一些癌症中发现Ape1/ref-1水平发生改变。在一项初步研究中，我们通过免疫组织化学检查了10例不同组织学类型（包括精原细胞瘤、卵黄囊瘤和恶性畸胎瘤）睾丸癌患者的生殖细胞肿瘤（GCT）切片中Ape1/ref-1的表达。几乎所有切片的肿瘤细胞中Ape1/ref-1都呈相对高水平表达。我们推测Ape1/ref-1表达升高部分导致了对治疗药物的耐药性。为了验证这一推测，我们使用载体LAPESN通过逆转录病毒基因转导在GCT细胞系NT2/D1中过表达Ape1/ref-1 cDNA。分别使用寡核苷酸切割试验和免疫组织化学来评估Ape1/ref-1的修复活性和表达，我们发现GCT细胞系中的修复活性与Ape1/ref-1相对表达直接相关。用Ape1/ref-1转导的NT2/D1细胞在寡核苷酸切割试验中表现出高2倍的AP内切核酸酶活性，这反映在对博来霉素的抗性增加了2至3倍。γ射线照射后的保护作用较小。我们得出结论：（a）Ape1/ref-1在一些GCT中呈相对高水平表达；（b）睾丸癌细胞系中Ape1/ref-1表达升高导致对某些治疗药物产生耐药性；（c）通过免疫组织化学和修复活性试验确定的GCT细胞系中Ape1/ref-1表达与对博来霉素的保护水平平行。我们进一步推测，在人类睾丸癌中观察到的Ape1/ref-1水平升高可能与其对治疗的相对耐药性有关，并且可能作为难治性疾病的诊断标志物。据我们所知，这是在哺乳动物系统中过表达Ape1/ref-1导致对DNA损伤剂的保护增强的首个实例。
