Cell Cycle Control and Tumor Suppressors

细胞周期控制和肿瘤抑制剂

• 批准号: 6433067
• 负责人: CURTIS HARRIS
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433067
• 关键词: adenomatous polyps; artificial chromosomes; cell cycle; cell growth regulation; chromosome inversion; colorectal neoplasms; cytogenetics; fluorescent in situ hybridization; gene expression; gene mutation; genetic library; genetic mapping; human genetic material tag; human tissue; molecular cloning; neoplasm /cancer genetics; nucleic acid probes; nucleic acid repetitive sequence; tissue /cell culture; tumor suppressor genes

We have cloned the ING family genes (p33ING2, p47ING3 and p29ING4). ING family genes have a PHD-finger motif, which is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation. The function of this domain in not yet known, but in analogy with the LIM domain, it could be involved in protein-protein interaction and necessary for the assembly or activity of multicomponent complexes involved in transcriptional activation or repression. We have found that p33ING1, p33ING2 or p47ING3 inhibit cell growth and induce G1 cell cycle arrest in the wild-type p53 cancer cell line, RKO, but not in p53-deficient RKO cells transfected with HPV-E6. ING2 expression is induced by inhibitors of topoisomerase 1 and 2. In contrast, ING1 is not inducible by DNA damage and apparently is a housekeeping gene. Taken together, our results indicate that the ING gene family cooperates with p53 in the regulation of cell proliferation. Currently, we are investigating the role of these genes in apoptosis. We are conducting a positional cloning project to identity a putative tumor suppressor gene(s) on chromosomes 3p12.2 or 3q25.3, which are regions previously identified by allelic deletion and cytogenetic analysis, thought to harbor candidate tumor suppressor genes. Koji Sasajima, a former LHC fellow, has identified a colorectal cancer patient with diffuse digestive tract polyposis and a germline abnormality in chromosome 3;(3)(p12.2q25.3). Therefore, we proposed the hypothesis that the gene(s) affected by the chromosome inversion, predisposed the patient to diffuse polyposis and malignancy. The chromosomal 3 breakpoints of this patient have been isolated by positional cloning strategy. We and our collaborator microscopically dissected the chromosome fragments around the breakpoints. Using a DNA probe isolated from the dissected fragments, we made YAC contigs and YAC clones containing the breakpoint in 3p that were identified by FISH (fluorescent in situ hybridization). The cosmid contigs were made from these YACs, cosmid clones spanning the breakpoint were identified by FISH, and the nucleotide sequence at and around the breakpoint in both 3p12.2 and 3q25.3 were determined. The patient was found to have a 2bp deletion in 3p12.2 and about a 300kb deletion in 3q25.3. An extensive search for the gene(s) disrupted by the chromosome inversion is in progress by two ways: (1) determination of the nucleotide sequence around the breakpoints - 120 kb in 3p and 130 kb in 3q - have been determined; and (2) long-range exon trapping which is a recently developed procedure. The candidate transcribed sequences are to be studied by investigating their abnormalities in cancer cells. Thus far, we have obtained two clusters of possible transcribed sequences, both of which have significant homology with the EST(expressed sequence tag) clones of unknown function.

我们克隆了 ING 家族基因（p33ING2、p47ING3 和 p29ING4）。 ING 家族基因具有 PHD 指基序，这是一种在核蛋白中发现的 C4HC3 锌指样基序，被认为参与染色质介导的转录调控。 该结构域的功能尚不清楚，但与 LIM 结构域类似，它可能参与蛋白质-蛋白质相互作用，并且对于参与转录激活或抑制的多组分复合物的组装或活性是必需的。 我们发现p33ING1、p33ING2或p47ING3在野生型p53癌细胞系RKO中抑制细胞生长并诱导G1细胞周期停滞，但在转染HPV-E6的p53缺陷型RKO细胞中则不然。 ING2 表达由拓扑异构酶 1 和 2 抑制剂诱导。相比之下，ING1 不受 DNA 损伤诱导，显然是一个管家基因。 综上所述，我们的结果表明 ING 基因家族与 p53 合作调节细胞增殖。 目前，我们正在研究这些基因在细胞凋亡中的作用。 我们正在进行一个定位克隆项目，以鉴定染色体 3p12.2 或 3q25.3 上的假定肿瘤抑制基因，这些区域是先前通过等位基因删除和细胞遗传学分析鉴定的区域，被认为含有候选肿瘤抑制基因。前 LHC 研究员 Koji Sasajima 发现一名结直肠癌患者患有弥漫性消化道息肉病和 3 号染色体种系异常；(3)(p12.2q25.3)。因此，我们提出这样的假设：受染色体倒位影响的基因使患者易患弥漫性息肉病和恶性肿瘤。该患者的3号染色体断点已通过定位克隆策略分离出来。我们和我们的合作者用显微镜解剖了断点周围的染色体片段。使用从解剖片段中分离的 DNA 探针，我们制备了包含 3p 断点的 YAC 重叠群和 YAC 克隆，并通过 FISH（荧光原位杂交）进行鉴定。由这些 YAC 制备粘粒重叠群，通过 FISH 鉴定跨越断点的粘粒克隆，并确定 3p12.2 和 3q25.3 中断点处及其周围的核苷酸序列。发现该患者的3p12.2有2bp缺失，3q25.3有约300kb缺失。目前正在通过两种方式广泛寻找被染色体倒位破坏的基因：(1) 确定断点周围的核苷酸序列——3p 中的 120 kb 和 3q 中的 130 kb——已经确定； (2)长程外显子捕获，这是最近开发的程序。通过研究候选转录序列在癌细胞中的异常情况来研究它们。到目前为止，我们已经获得了两簇可能的转录序列，它们都与未知功能的EST（表达序列标签）克隆具有显着的同源性。