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，在生理性叶酸缺乏期间，异质性核糖核蛋白-E1(hnRNP-E1)的同型半胱氨酸化激活了一个营养敏感的转录后RNA操纵子，该操纵子还包括与人乳头瘤病毒16型(HPV16)RNA中的两个位点的重要相互作用。这种HPV16RNA-蛋白质与同型半胱氨酸化的hnRNP-E1的相互作用导致在体外产生HPV16主要(L1)和次要(L2)病毒衣壳蛋白；在携带HPV16的角质形成细胞中作为单层繁殖；以及当这些HPV16角质形成细胞在生理低叶酸介质中发育成器官型木筏时。尽管HPV16-高叶酸和低叶酸-器官型木筏中的HPV16DNA载量相似，但后者包含了HPV16DNA与基因组DNA的高水平整合。将18天的HPV16-低叶酸器官型筏植入米色裸鼠XID免疫缺陷小鼠的皮下，在12周内导致了HPV16诱导的侵袭性癌症。因此，我们开发了一种新的HPV16诱导致癌模型，在不到4个月的时间框架内。由于叶酸缺乏可导致基因组DNA的单链缺口和双链DNA断裂，我们的主要假设是HPV16-低叶酸器官型筏中不稳定的基因组DNA主要是导致HPV16DNA高水平整合到基因组DNA中并使这种良性组织转化为癌症的主要原因。因此，我们将测试这一假设的各个方面 使用三个具体目标：在具体目标#1中，我们将描述时间-过程关系， HPV16DNA整合到HPV16型排筏基因组DNA中的程度和后果，并评估良性HPV16型排架在免疫缺陷小鼠中转化为癌症的频率。在特定目标#2中，我们将评估扩增的‘无衣壳’HPV16DNA在转导和表达编码不同强度的同型半胱氨酸化的hnRNP-E1分子模拟物的新基因到含有HPV16的角质形成细胞后整合到未受干扰和完整[稳定]的基因组DNA的可能性，这些角质形成细胞随后在高叶酸条件下发育为器官型筏。在特定的目标#3中，我们将评估未受干扰和短暂受扰的基因组DNA在AAV2转导的[高叶酸]HPV16器官型排泄物中整合HPV16DNA的渗透性，这些排泄物无论是否包含丰富的‘无衣壳’HPV16DNA，然后评估这种基因组HPV16DNA整合在Beige裸鼠排泄物中诱导癌变的可能性。这些研究最终将使人们更好地了解HPV16感染的组织转化为癌症的机制，并使营养不良的HPV感染的老年退伍军人、携带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受益，在这些国家，营养不良以及与HIV和HPV16的混合感染使个人面临HPV诱发癌症的高风险。

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