INVESTIGATING THE MECHANISMS OF PRB TUMOR SUPPRESSION

视网膜母细胞瘤抑癌基因是肿瘤抑制基因的原型。它的蛋白质pRb是一种在绝大多数人类肿瘤中都被破坏的不可或缺的蛋白质。因此，了解PRB肿瘤抑制的潜在机制是至关重要的。PRb作用的分子机制是什么？在各种实验环境中，pRB可以介导细胞周期停滞，促进细胞分化，抑制细胞凋亡，抑制某些转录因子(E2F)的转录，并激活与其他转录因子(myoD和C/EBP)的转录。这些活性主要是在人工测定系统中研究的，目前还不清楚这些pRb特性中哪些是体内最重要的。众所周知，PRB的一个很大的领域，即口袋，对于PRB的大多数活动来说是必要的，也是足够的。对嵌合小鼠髓中Rb-/-细胞的研究表明，pRb的丢失并不会消除细胞周期控制的大部分方面。在这样的小鼠中，RB-/-细胞对大多数组织都有贡献，并且具有正常的组织学。我们假设，pRb通常不是细胞周期控制所必需的，但它一定与肿瘤的发生有非常特殊的作用。戴森实验室的初步数据表明，细胞基因组完整性的维持可能取决于pRB诱导细胞周期停滞的能力，以应对DNA损伤。最近的研究结果还表明，pRb促进分化的能力与肿瘤的致瘤性有关。这项建议将研究pRb通路在体内的意义，导致细胞周期停滞，以响应DNA损伤和细胞分化在pRb肿瘤抑制中。具体目标1：确定pRb的重要结构特征，以诱导细胞周期停滞以响应DNA损伤并促进细胞分化。对这两条途径至关重要的PRB相关蛋白将被识别出来。具体目标2：通过使用目标1中产生的Rb突变体来禁用这些特定途径，确定pRb阻止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起到重要作用。具体目标3：确定E2F依赖转录的解除调控是否与体内RB-/-细胞的致瘤潜力相关。

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.