Therapeutic/Mechanistic Role of Ape1 in Germ Cell Tumors

描述(申请人提供)：播散性生殖细胞肿瘤(GCT)的治疗已经成功，70%-80%的患者通过一线化疗治愈。然而，对于那些患有性腺外原发疾病或难治性疾病的20-30%的患者，治疗反应很差，只有3-30%的患者在二线药物治疗后无病存活。一种治疗耐药疾病的方法是开发策略，以增加化疗药物，这些药物在大多数GCT患者中非常成功。人们对DNA修复系统在GCT中的作用知之甚少，除了有效的修复似乎会使肿瘤细胞对治疗产生抵抗力。我们观察到，与正常组织相比，GCT组织表达高水平的APE1/REF-1。APE1/REF-1是一种多功能蛋白，具有DNA碱基切除修复(BER)活性和氧化还原活性，是激活Fos、Jun、NFkappaB、HIF-1α(缺氧诱导因子)、P53和PAX5等特定转录因子所必需的。这种新的功能组合将ape1/ref-1与许多治疗药物(博莱霉素、顺铂、放射和VP-16)的耐药性联系在一起，这些药物通过作为误码率的直接底物或通过ape1/ref-L调节的转录因子改变信号间接地治疗GCT。基于这一信息，我们假设：APE1/REF-1在GCT中的高水平表达是疾病的功能标志物，1)预测高危疾病，2)可被操纵以获得治疗优势。这项建议的主要目的是描述GCT中APE1/REF-1的分子生物学特征，因为它与患者的临床病程和GCT细胞对治疗药物的反应有关。为了达到这一目标，我们制定了四个具体目标：特异性目标1：确定APE1/REF-1在预后良好和高危GCT中的相对表达。这利用了印第安纳大学丰富的临床GCT材料。具体目标2：APE1/REF-1在GCT进展和发育中的作用是什么，包括细胞生长、凋亡、细胞周期和分化？这就是APE1/REF-1的高水平表达，包括修复、氧化还原和核定位结构域突变的过度表达是如何独立影响GCT细胞作为癌细胞生长的能力的。具体目标3：APE1/REF-1氧化还原状态的变化如何影响修复功能？利用定点突变体，确定哪些半胱氨酸残基特异性控制APE1/REF-L的修复功能？具体目标4：APE1/REF-1修复和氧化还原功能的改变如何影响GCT细胞对治疗剂的反应？利用我们在AIMS 2和3中学到的知识，我们如何改变GCT细胞对治疗剂的抵抗力。通过这些分析，我们希望确定APE1/REF-1功能与睾丸癌进展相关的潜在机制。如果任何突变体如预期的那样使GCT细胞系对化疗/IR药物敏感，这将为未来的基因治疗方法使GCT对治疗增敏奠定基础。

DESCRIPTION (provided by applicant): Therapy for disseminated germ cell tumors (GCT) has been successful with 70-80% of patients being cured with front line chemotherapy. However, for those 20-30% of patients with extra-gonadal primaries or refractory disease, the response to therapy is poor with only 3-30% surviving disease-free after second line agents. One approach to treating resistant disease is the development of strategies to augment the chemotherapeutic agents that have been so successful in the majority of GCT patients. Little is known about the role of DNA repair systems in GCT's except that efficient repair appears to make tumor cells resistant to therapy. We have observed that GCT's express high levels of Ape1/ref-1 compared to normal tissues. Ape1/ref-1 is a multifunctional protein with DNA base excision repair (BER) activity and redox activity required for activation of specific transcription factors including Fos, Jun, NFkappaB, HIF-1alpha (hypoxia inducible factor), p53, and PAX5. This novel combination of functions links Ape1/ref-1 with resistance to many of the therapeutic agents (bleomycin, cisplatin, radiation, and VP-16) used to treat GCT's by acting as direct substrates for BER or indirectly by altering signaling through transcription factors regulated by Ape1/ref-l. Based on this information, we hypothesize: High level expression of Ape1/ref-1 in GCT's is a functional marker of disease which 1) is predictive of high risk disease and 2) can be manipulated to gain a therapeutic advantage. The major thrust of this proposal is to characterize the molecular biology of Ape1/ref-1 in GCT's as it relates to the clinical course of patients and the response of GCT cells to therapeutic agents. To approach this goal, we have developed four Specific Aims: Specific Aim1: Determine the relative expression of, Ape1/ref-1in good prognosis and high-risk GCT's. This takes advantage of the wealth of clinical GCT material available at Indiana University. Specific Aim 2: What is the role of Ape1/ref-1 in GCT progression and development, including cell growth, apoptosis, cell cycle, and differentiation? That is how does the high level expression of Ape1/ref-1, including over-expression of repair, redox, and nuclear localization domain mutants independently affect the ability of GCT cells to grow as cancer cells. Specific Aim 3: How do changes in the redox status of Ape1/ref-1 affect repair function? Using site-specific mutants, determine which cysteine residues specifically control repair function of Ape1/ref-l? Specific Aim 4: How do alterations in the repair and redox functioning of Ape1/ref-1 affect the response of GCT cells to therapeutic agents? Using what we learn in Aims 2 & 3, how can we alter the resistance of GCT cells to therapeutic agents. Through these analyses, we hope to determine the underlying mechanisms by which Ape1/ref-1 function is linked to the progression of testicular cancer. If any mutants are shown to sensitize the GCT cell lines to chemo-/IR agents, as expected, this will set the stage for future gene therapy approaches to sensitize GCT's to therapy.