Mechanism of Folate Deficiency as a Co-Factor for HPV16-induced Carcinogenesis

叶酸缺乏作为 HPV16 诱发癌变的辅助因素的机制

基本信息

批准号：
8774199
负责人：
Asok Antony
金额：
--
依托单位：
RLR VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8774199
关键词：
Affinity Applications Grants Benign Binding Capsid Capsid Proteins Cells Clone Cells DNA DNA Fragmentation DNA Integration DNA Maintenance Dependovirus Developing Countries Diet Dose Elderly Ensure Exposure to Folic Acid Folic Acid Deficiency Frequencies Gene Expression Generations Genes Genome Genomic DNA Genomics HIV HPV-High Risk Health Healthcare Heterogeneous-Nuclear Ribonucleoproteins Human Papillomavirus Human papillomavirus 16 Immunodeficient Mouse Implant In Vitro Individual Investigation L2 viral capsid protein Lead Leucovorin Malignant Neoplasms Measures Methotrexate Minor Modeling Molecular Mus Normal Cell Oncogenes Operon Papillomavirus Patients Physiological Positioning Attribute RNA RNA-Protein Interaction Risk Series Staging Testing Time Tissues Transcript Uracil Veterans Viral Viral Load result Vitamin B 12 Deficiency carcinogenesis co-infection ds-DNA feeding implantation in vivo keratinocyte monolayer mutant novel nutrition permissiveness prevent subcutaneous

项目摘要

DESCRIPTION (provided by applicant): We have recently identified that homocysteinylation of heterogeneous nuclear ribonucleoprotein-E1 (hnRNP-E1) during physiological folate deficiency, activates a nutrition-sensitive posttranscriptional RNA operon that also includes an important interaction with two loci in Human Papillomavirus type-16 (HPV16) RNA. This HPV16 RNA-protein interaction with homocysteinylated-hnRNP-E1 led to a profound perturbation in the generation of both HPV16 major (L1) and minor (L2) viral capsid proteins in vitro; in HPV16-harboring keratinocytes that were propagated as monolayers; as well as when these HPV16-keratinocytes were developed into organotypic rafts in physiologic low-folate medium. Despite a similar HPV16 DNA viral load in HPV16-high folate- and low folate-organotypic rafts, the latter contained a high-level of integration of HPV16 DNA into genomic DNA. Subcutaneous implantation of 18-day HPV16-low folate-organotypic rafts in Beige Nude XID immunodeficient mice led to an aggressive HPV16-induced cancer within 12 weeks. Thus, we have developed a new model of HPV16-induced carcinogenesis within a time frame of less than 4 months. Because folate deficiency can induce single-strand nicks in genomic DNA and also double-strand DNA fragmentation, our overarching hypothesis is that the unstable genomic DNA in HPV16-low folate-organotypic rafts is primarily responsible for the high-level integration of HPV16 DNA into genomic DNA and transformation of this benign tissue to cancer. So we will test various aspects of this hypothesis using three specific aims: In Specific Aim #1, we will characterize the time-course relationship, extent, and consequence of integration of HPV16 DNA into the genomic DNA of HPV16-organotypic rafts in vitro and in vivo, and assess the frequency of transformation of benign HPV16-organotypic rafts into cancer in immunodeficient mice. In Specific Aim #2 we will assess the potential of amplified 'capsid-less' HPV16 DNA to integrate into unperturbed and intact [stable] genomic DNA after transduction and expression of novel genes encoding various strengths of molecular mimics of homocysteinylated-hnRNP-E1 into HPV16-harboring keratinocytes that are subsequently developed into organotypic rafts under high-folate conditions. In Specific Aim #3, we will assess the permissiveness of unperturbed and transiently perturbed genomic DNA to integrate HPV16 DNA in AAV2-transduced [high folate] HPV16-organotypic rafts that either do or do not contain an abundance of 'capsid-less' HPV16 DNA, and then evaluate the potential for such genomic HPV16 DNA integration to induce carcinogenesis within implants of rafts in Beige Nude XID mice. Such investigations will ultimately provide a better understanding of the mechanism of transformation of HPV16-infected tissues to cancer and benefit HPV-infected elderly Veterans with poor nutrition; Veterans with HPV16 and human immunodeficiency virus (HIV); and those in developing countries where the combination of poor nutrition, and co-infection with HIV and HPV16 places individuals at high risk for HPV-induced cancers

描述（由申请人提供）：我们最近发现，在生理性叶酸缺乏期间，异质核核糖核蛋白-E1 (hnRNP-E1) 的同型半胱氨酸化会激活营养敏感的转录后 RNA 操纵子，该操纵子还包括与人乳头瘤病毒 16 型 (HPV16) RNA 中两个基因座的重要相互作用。 HPV16 RNA 蛋白与同型半胱氨酸化 hnRNP-E1 的相互作用导致体外 HPV16 主要 (L1) 和次要 (L2) 病毒衣壳蛋白的产生产生深刻的干扰；在以单层形式繁殖的携带 HPV16 的角质形成细胞中；以及当这些 HPV16 角质形成细胞在生理低叶酸培养基中发育成器官型筏时。尽管 HPV16 高叶酸和低叶酸器官型筏中的 HPV16 DNA 病毒载量相似，但后者包含 HPV16 DNA 与基因组 DNA 的高水平整合。在 Beige Nude XID 免疫缺陷小鼠皮下植入 18 天的 HPV16 低叶酸器官型筏，在 12 周内导致了侵袭性 HPV16 诱导的癌症。因此，我们在不到 4 个月的时间内开发了 HPV16 诱导癌变的新模型。由于叶酸缺乏会导致基因组 DNA 中出现单链切口以及双链 DNA 断裂，因此我们的总体假设是，HPV16 低叶酸器官型筏中不稳定的基因组 DNA 是导致 HPV16 DNA 高度整合到基因组 DNA 以及良性组织向癌症的转化。所以我们将测试这个假设的各个方面使用三个具体目标：在具体目标#1中，我们将描述时间进程关系，体外和体内 HPV16 DNA 整合到 HPV16 器官型筏基因组 DNA 中的程度和后果，并评估良性 HPV16 器官型筏在免疫缺陷小鼠中转化为癌症的频率。在具体目标#2中，我们将评估在编码不同强度的同型半胱氨酸化-hnRNP-E1分子模拟物的新基因转导和表达后，扩增的“无衣壳”HPV16 DNA整合到未受干扰和完整[稳定]基因组DNA中的潜力。携带 HPV16 的角质形成细胞随后在高叶酸条件下发育成器官型筏。在具体目标#3中，我们将评估未受干扰和短暂受干扰的基因组 DNA 将 HPV16 DNA 整合到 AAV2 转导的[高叶酸] HPV16 器官型筏中的允许性，该器官型筏包含或不包含大量“无衣壳”HPV16 DNA，然后评估这种基因组 HPV16 DNA 整合在 Beige Nude 的筏植入物中诱导癌变的潜力XID 小鼠。此类研究最终将有助于更好地了解 HPV16 感染组织转化为癌症的机制，并使营养不良的 HPV 感染老年退伍军人受益；患有 HPV16 和人类免疫缺陷病毒 (HIV) 的退伍军人；以及发展中国家的人们，营养不良以及同时感染 HIV 和 HPV16 使个人面临 HPV 诱发癌症的高风险