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

描述（由申请人提供）： 我们最近发现，在生理叶酸缺乏症的异质性核核糖核蛋白-E1（hnRNP-E1）的同型半胱氨酰化，激活营养敏感的转录后RNA操纵子，也包括一个重要的相互作用与两个位点的人乳头瘤病毒16型（HPV 16）RNA。 这种HPV 16 RNA-蛋白质与同型半胱氨酸化的hnRNP-E1的相互作用导致了HPV 16主要（L1）和次要（L2）病毒衣壳蛋白在体外产生的深刻扰动;在作为单层繁殖的携带HPV 16的角质形成细胞中;以及当这些HPV 16-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HPV 16-高叶酸-和低叶酸-器官型筏中的HPV 16 DNA病毒载量相似，但后者含有高水平的HPV 16 DNA整合到基因组DNA中。 在Beige Nude XID免疫缺陷小鼠中皮下植入18天的HPV 16低叶酸器官型筏，在12周内导致HPV 16诱导的侵袭性癌症。 因此，我们在不到4个月的时间内开发了一种新的HPV 16诱导的致癌模型。 由于叶酸缺乏可诱导基因组DNA中的单链切口以及双链DNA片段化，因此我们的总体假设是HPV 16低叶酸器官型筏中的不稳定基因组DNA主要负责HPV 16 DNA高水平整合到基因组DNA中并将该良性组织转化为癌症。 所以我们将测试这个假设的各个方面 有三个具体目标： 在具体目标1中，我们将描述时间-过程关系， HPV 16 DNA整合到HPV 16器官型筏的基因组DNA中的程度和结果，并评估免疫缺陷小鼠中良性HPV 16器官型筏转化为癌症的频率。 在具体目标#2中，我们将评估扩增的“无帽”HPV 16 DNA整合到未受干扰和完整[稳定]基因组DNA中的潜力，所述基因编码不同强度的同型半胱氨酸化hnRNP-E1的分子模拟物的新基因转导和表达到携带HPV 16的角质形成细胞中，所述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，我们将评估未扰动和瞬时扰动的基因组DNA将HPV 16 DNA整合到AAV 2转导的[高叶酸] HPV 16器官型筏中的容许性，所述筏含有或不含有丰富的“无帽”HPV 16 DNA，然后评估这种基因组HPV 16 DNA整合在Beige Nude XID小鼠中的筏植入物内诱导致癌作用的潜力。 这些研究最终将更好地了解HPV 16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; HPV 16和人类免疫缺陷病毒（HIV）退伍军人;以及营养不良和HIV和HPV 16合并感染使个体处于HPV诱导的癌症高风险的发展中国家的退伍军人受益

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