INVESTIGATING THE MECHANISMS OF PRB TUMOR SUPPRESSION

视网膜母细胞瘤肿瘤抑制基因是肿瘤抑制基因的原型。它的蛋白质pRB是一种完整的蛋白质，在绝大多数人类肿瘤中被破坏。因此，了解pRB抑制肿瘤的潜在机制至关重要。pRB作用的分子机制是什么？在各种实验环境中，pRB可以介导细胞周期阻滞，促进细胞分化，抑制凋亡，抑制某些转录因子（E2F）的转录，并激活其他转录因子（myoD和C/EBP）的转录。这些活性主要在人工分析系统中进行了研究，目前尚不清楚这些pRB的哪些特性在体内最重要。众所周知，一个很大的pRB领域，即口袋，对于大多数pRB活动是必要和充分的。对具有RB -/-细胞的嵌合小鼠的研究表明，pRB的丢失并不会消除细胞周期控制的大多数方面。在这些小鼠中，RB -/-细胞对大多数组织有贡献，并且具有正常的组织学。我们假设，pRB对细胞周期控制通常不是必需的，但它必须在肿瘤发生中具有非常特殊的作用。来自Dyson实验室的初步数据表明，细胞基因组完整性的维持可能依赖于pRB在DNA损伤时诱导细胞周期阻滞的能力。最近的研究结果还表明，pRB促进分化的能力与致瘤性有关。本研究将探讨pRB途径在DNA损伤和pRB肿瘤抑制中导致细胞周期阻滞和细胞分化的体内意义。具体目标1：确定pRB在DNA损伤时诱导细胞周期阻滞和促进细胞分化的重要结构特征。这两种途径所必需的prb相关蛋白将被确定。特异性目的2：利用目的1中产生的RB突变体来禁用这些特异性途径，以确定pRB阻滞细胞周期和促进细胞分化的能力是否对pRB在体内作为肿瘤抑制因子很重要。特异性目的3：确定E2F依赖性转录的失调是否与RB -/-细胞体内的致瘤潜能相关。

The retinoblastoma tumor suppressor gene is a prototype for tumor suppressor genes. Its protein, pRB, is an integral protein that is disrupted in a vast majority of human tumors. Understanding the underlying mechanisms of pRB tumor suppression is, therefore, of critical importance. What is the molecular mechanism of pRB action? In various experimental settings, pRB can be shown to mediate cell cycle arrest, promote cellular differentiation, suppress apoptosis, repress transcription of some transcription factors (E2F) and activate transcription with others (myoD and C/EBP). These activities have been studied primarily in artificial assay systems and currently it is unclear which of these pRB properties are most important in-vivo. It is known that a large domain of pRB, the pocket, is necessary and sufficient for most of pRB's activities. Studies of chimeric mice pith RB -/- cells demonstrate that loss of pRB does not remove most aspects of cell cycle control. In such mice, RB -/- cells contribute to most tissues and have normal histology. We hypothesize, that pRB is not generally essential for cell cycle control but that it must have very specific roles relevant to tumorigenesis. Preliminary data from the Dyson laboratory suggests that maintenance of the genomic integrity of cells may be dependent upon pRB's ability to induce a cell cycle arrest in response to DNA damage. Recent findings also suggest that pRB's ability to promote differentiation correlates with tumorigenicity. This proposal will investigate the in-vivo significance of pRB pathways leading to cell cycle arrest in response to DNA damage and to cellular differentiation in pRB tumor suppression. Specific Aim number 1: To identify the structural features important for pRB to induce cell cycle arrest in response to DNA damage and to promote cellular differentiation. The pRB-associated proteins essential to these two pathways will be identified. Specific Aim number 2: To determine whether the ability of pRB to arrest cell cycle and promote cellular differentiation is important for pRB to act as a tumor suppressor in-vivo using the RB mutants generated in aim number 1 to disable these specific pathways. Specific Aim number 3: To determine whether the deregulation of E2F dependent transcription is correlated with the tumorigenic potential of RB -/- cells in-vivo.