PTHRP GENE EXPRESSION IN SQUAMOUS CARCINOMAS

恶性体液性高钙血症（HHM）是一种常见的副肿瘤 通常与鳞状细胞癌相关的综合征。的 综合征似乎是由肿瘤产生的一种新的 甲状旁腺相关肽（PTHrP）。这种肽是天然的- 发生，正常角质形成细胞的局部产物，但当由 在恶性肿瘤中，PTHrP进入体循环并与 骨和肾脏中的经典PTH受体，导致原型 HHM的生化特征 我们已经描述了一个实验系统的12个人类鳞状细胞癌， 肿瘤细胞系，这将使我们能够详细研究 调节PTHrP基因表达的机制，目的是鉴定 为什么有些鳞状肿瘤会引起HHM而有些不会。使用这个系统， 我们已经观察到鳞状细胞肿瘤系普遍分泌PTHrP， 而是在一个很宽的范围内这样做，这是一个稳定的直接函数， PTHrP mRNA水平。这些观察向我们表明， 鳞状细胞肿瘤引起HHM的能力可能与其 定量表达PTHrP基因的能力，而不是简单的 基因表达中的定性或“开关”缺陷。这似乎是 例，当这些肿瘤细胞系在无胸腺小鼠中生长时， 循环中的钙和PTHrP与 PTHrP mRNA在肿瘤组织中的表达，证实了肿瘤组织中PTHrP mRNA的定量表达。 关系预测此外，我们提出的证据表明， PTHrP基因表达水平差异的机制 在这些肿瘤中看到的是转录的性质和反式操作。 迄今为止，我们已经鉴定了PTHrP启动子的500 bp区域， 似乎包含调控这些差异的调节序列， 转录率。我们建议进一步研究 在该系统中PTHrP基因表达：1）通过对PTHrP基因的一些细节作图， 负责PTHrP差异速率的调节序列 在这些不同的鳞状肿瘤细胞系中转录，2）通过定义 蛋白质-DNA相互作用对PTHrP的这些差异速率很重要 基因转录产物的纯化和鉴定 这些因素似乎能够差异调节PTHrP的表达， 这些鳞状肿瘤细胞系，如果它们是新的，克隆它们的cDNA。 主要研究者是一名内分泌学家， 研究与异常分泌相关的基因调控机制， 激素的恶性肿瘤。该提案提供了一个理想的培训工具 对于一个有这些兴趣的临床研究者来说， 广泛使用各种分子技术， 研究基因表达，以研究常见的临床 综合征这个项目结合了丰富多样的 在耶鲁大学的研究，将最大限度地提高我的成长机会， 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.