SENESCENCE IN EPITHELIAL NEOPLASTIC PROGRESSION

上皮肿瘤进展中的衰老

• 批准号: 6197809
• 负责人: JAMES RHEINWALD
• 金额: $ 25.21万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6197809
• 关键词: athymic mouse; cell line; cell senescence; gene mutation; human tissue; keratinocyte; neoplasm /cancer genetics; neoplastic growth; neoplastic transformation; p53 gene /protein; preneoplastic state; squamous cell carcinoma; telomerase; tissue /cell culture; tumor suppressor genes

In renewing tissues such as stratified squamous epithelia (e.g., epidermis, oral mucosa) cell replication occurs continually, with the concomitant potential to acquire new mutations. Thus the probability increases with time and age that a constituent cell (keratinocyte) will acquire a combination of heritable alterations that permit it to evade normal restraints and become a cancer cell (squamous cell carcinoma (SCC)). Limiting the probability that a cell might progressively accumulate oncogenic mutations is an internal mechanism, activated by progressive telomere shortening, that imposes a state of replicative senescence on a cell after it has undergone a certain number of divisions. Earlier studies identified p53 as an essential enforcer of this telomere length-sensitive growth arrest mechanism. Recently we and others have found that another cell cycle inhibitor, INK4a (p16), can enforce a senescence arrest on keratinocytes and other cell types. p16 expression is activated by a sensor that apparently detects some aspect of cell age or physiological status unrelated to telomere length. Keratinocytes engineered to express telomerase, such that p53-dependent arrest is not triggered, cannot divide rapidly as immortalized cells unless they undergo mutation to evade the p16-enforced arrest system. Potentially intermediate stages of neoplastic progression are identifiable in epithelia as lesions of abnormal appearing cells (dysplasias), which are often found to have acquired some p16 and/or p53 mutations. At present there are no reliable ways to predict which lesions are premalignant (i.e., which will progress rapidly to SCC) and which will remain indolent or regress. We hypothesize that keratinocytes become premalignant when they have acquired mutations permitting them to bypass both p16- and p53-dependent senescence mechanisms and become extended lifespan, yet still non-immortal, cells. We have cultured cells having these characteristics from a dysplastic oral lesion and we have constructed an experimental model cell line from normal human keratinocytes by stably expressing proteins that block the activity of endogenous p16 and p53. We propose to test these cell lines, and others we generate using a combination of gene transduction and spontaneous mutant selection strategies, for susceptibility to induction of telomerase expression by transcriptional transactivators such as myc, for their ability to generate spontaneous immortalized variants after dividing to crisis, and for their tumorigenic behavior in athymic mice. We will also begin to identify and characterize other types of mutations that can result in inactivation of the p16- dependent mechanism in keratinocytes.

在更新的组织中，如分层的鳞状上皮（例如表皮，口服粘膜）细胞复制不断发生，并具有获取新突变的伴随潜力。 因此，成分细胞（角质形成细胞）将获得可遗传改变的概率增加的概率，使其可以逃避正常的约束并成为癌细胞（鳞状细胞癌（SCC））。 限制细胞可能逐渐积累致癌突变的概率是一种内部机制，它通过进行性端粒缩短而激活，该机制在细胞经历了一定数量的划分后将复制性衰老状态施加在细胞上。 较早的研究将p53确定为对这种端粒长度敏感生长停滞机制的必不可少的执行者。 最近，我们和其他人发现另一种细胞周期抑制剂Ink4a（P16）可以在角质形成细胞和其他细胞类型上强制强制衰老停滞。 P16表达被一种传感器激活，该传感器显然检测到与端粒长度无关的细胞年龄或生理状态的某些方面。 旨在表达端粒酶的角质形成细胞，使得p53依赖性停滞不会触发，除非经历突变以逃避P16增强的骤停系统，否则不能迅速将其分为永生的细胞。 肿瘤进展的潜在中间阶段在上皮中可以识别为异常出现细胞（发育不良）的病变，通常发现这些病变已获得了一些p16和/或p53突变。 目前，没有可靠的方法可以预测哪些病变是预先验证的（即，将迅速发展为SCC），并且将保持懒惰或退化。 我们假设角质形成细胞在获得突变使其允许它们绕过p16-和p53依赖性衰老机制并变得延长的寿命，但仍不屈服的细胞时，它们会变得过敏反应。 我们具有从异型性口服病变中具有这些特征的培养细胞，并且通过稳定表达蛋白质来阻断内源性P16和p53的活性，从正常人的人角质形成细胞中构建了实验模型细胞系。 我们建议测试这些细胞系，其他我们使用基因转导和自发突变选择策略的组合来实现的，以使转录反式反式激活器（如MyC）诱导端粒酶表达的敏感性，以产生自发性不朽变体，从，以及它们在无胸腺小鼠中的致瘤行为。 我们还将开始识别并表征其他类型的突变，这些突变可能导致角质形成细胞中的p16依赖机制失活。