PTHRP GENE EXPRESSION IN SQUAMOUS CARCINOMAS

摘要恶性体液高钙血症是一种常见的副肿瘤。 通常与鳞状细胞癌有关的综合征。这个 综合症似乎是由一种由肿瘤产生的新的 甲状旁腺激素相关肽(PTHrP)。这种多肽是一种天然的- 正常角质形成细胞的局部产物，但当由 恶性肿瘤，甲状旁腺素受体进入体循环，并与 骨骼和肾脏中的经典甲状旁腺素受体，导致原型 HHM的生化特征。 我们已经描述了一个由12个人的鳞片组成的实验系统 肿瘤株，这将使我们能够详细研究分子 调节PTHrP基因表达的机制，目的是识别 为什么有些鳞状肿瘤会引起HHM，而另一些则不会。使用这个系统， 我们观察到鳞状细胞瘤株普遍分泌PTHrP， 但在一个广泛的频谱上这样做，这是稳定性的直接函数- 甲状旁腺素受体基因的表达水平。这些观察向我们表明，一个给定的 鳞状细胞瘤引起HHM的能力可能与其 PTHrP基因的定量表达能力，而不是简单的 基因表达的质的或“开-关”缺陷。这似乎是 例如，当这些肿瘤系在裸鼠体内生长时， 循环中的钙和甲状旁腺素受体与血浆 PTHrP在肿瘤组织中的表达，证实了 关系预测。此外，我们提出的证据表明 甲状旁腺素受体基因表达差异的机制 在这些肿瘤中看到的是转录性质的，并以反式方式运作。 到目前为止，我们已经确定了PTHrP启动子的一个500bp的区域 似乎包含控制这些差异的调节序列 转录速度。我们建议进一步研究这一机制。 PTHrP基因在本系统中的表达：L)通过比较详细地定位 PTHrP差异率的调控序列 在这些不同的鳞状肿瘤系中转录，2)通过定义 蛋白质-DNA相互作用对甲状旁腺素受体的这些不同速率很重要 基因转录和3)通过纯化和鉴定转录 甲状旁腺激素相关蛋白表达的差异调节因子 这些鳞状肿瘤株，如果是新的，就克隆它们的cDNA。 首席研究员是一位内分泌学家，对 血管内皮细胞异常分泌相关基因调控机制的研究 恶性肿瘤带来的荷尔蒙。该提案提供了一种理想的培训工具 对于有这些兴趣的临床医生和研究人员来说，这涉及到 广泛使用与人类免疫相关的各种分子技术 针对一种常见临床研究的基因表达研究 综合症。这个项目结合了丰富和多样化的 在耶鲁大学的研究，将最大限度地增加我作为 独立的年轻调查员。

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome that is often associated with squamous cell carcinomas. The syndrome appears to be mediated by the production by tumors of a novel parathyroid hormone-related peptide (PTHrP). This peptide is a naturally- occurring, local product of normal keratinocytes, but when produced by malignant tumors, PTHrP enters the systemic circulation and interacts with classical PTH receptors in bone and kidney, resulting in the prototypical biochemical features of HHM. We have characterized an experimental system of twelve human squamous tumor lines which will allow us to study in detail the molecular mechanisms regulating PTHrP gene expression, with the aim of identifying why some squamous tumors cause HHM and others do not. Using this system, we have observed that squamous cell tumor lines universally secrete PTHrP, but do so over a wide spectrum that is a direct function of the steady- state level of PTHrP mRNA. These observations suggested to us that a given squamous cell tumor's ability to cause HHM might be related to its quantitative ability to express the PTHrP gene, as opposed to a simple qualitative or "on-off" defect in gene expression. This appears to be the case, for when these tumor lines were grown in athymic mice, the levels of calcium and PTHrP in the circulation correlated directly with the level of PTHrP mRNA expression in tumor tissue, confirming the quantitative relationship predicted. Furthermore, we present evidence that the mechanisms underlying the differential levels of PTHrP gene expression seen in these tumors are transcriptional in nature and operate in trans. We have thus far identified a 500 bp region of the PTHrP promoter that appears to contain regulator sequences that govern these differential rates of transcription. We propose to study further the mechanisms of PTHrP gene expression in this system: l) by mapping in some detail the regulator sequences responsible for the differential rates of PTHrP transcription in these different squamous tumor lines, 2) by defining the protein-DNA interactions important to these differential rates of PTHrP gene transcriptions and 3) by purifying and identifying transcription factors which appear able to differentially regulate PTHrP expression in these squamous tumor lines and, if they are novel, to clone their cDNAs. The principal investigator is an endocrinologist with an interest in studying mechanisms of gene regulation related to abnormal secretion of hormones by malignancies. The proposal offers an ideal training vehicle for a clinician-investigator with these interests, for it involves extensive use of a wide range of molecular techniques pertinent to the studs of gene expression directed to the study of a common clinical syndrome. This project combined with the richness and diversity of research at Yale, will maximize my opportunities for growth as an independent young investigator.