CELL CYCLE CONTROL AND TUMOR SUPPRESSORS

细胞周期控制和肿瘤抑制剂

• 批准号: 6289170
• 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/6289170
• 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

G1-S and G2-M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. In 1999, we reported both genetic and functional evidence of a Gadd45-mediated G2-M checkpoint in human and murine cells. In our ongoing studies, the results indicate that the central region (residues 50-76) of Gadd45, is conserved among its family members including Gadd45beta and Gadd45gamma, and mediates the G2/M arrest. This region also is the binding site for Cdc2, PCNA or p21Waf1. When the acidic residues between 62-67 were changed into alanine, this mutant lost its ability to induce G2/M arrest in normal human fibroblasts, but could still bind to Cdc2, PCNA or p21Waf1. We are currently determining if these GADD45 mutants modulate the enzymatic activity of Cdc2/cyclin B1.We have cloned ING family genes (p33ING2, p47ING3 and p29ING4). ING family genes have a PHD-finger motif. The PHD finger 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 wt p53 cancer cell line, RKO, but not in p53-deficient RKO cells transfected with HPV-E6. Taken together, our results indicate that the ING gene family cooperates with p53 in the regulation of cell proliferation. The role of these genes in apoptosis is currently being investigated.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. We have so far obtained two clusters of possible transcribed sequences, both of which have significant homology with the EST(expressed sequence tag) clones of unknown function. - Cell Cycle, Tumor suppressor genes, - Human Tissues, Fluids, Cells, etc.

在真核生物中，G1-S和G2-M细胞周期检查点维持基因组的稳定性，以响应遗传毒性应激。1999年，我们报道了在人类和小鼠细胞中存在GADD45介导的G2-M检查点的遗传和功能证据。在我们正在进行的研究中，结果表明GADD45的中心区(残基50-76)在包括Gadd45beta和Gadd45Gamma在内的家族成员中保守，并介导了G2/M期的停滞。该区域也是cdc2、增殖细胞核抗原或p21Waf1的结合部位。当62-67之间的酸性残基转变为丙氨酸时，该突变体失去了诱导正常人成纤维细胞G2/M期停滞的能力，但仍能与CDc2、增殖细胞核抗原或p21Waf1结合。我们目前正在确定这些GADD45突变体是否调节CDC2/Cyclin B1的酶活性。我们已经克隆了ING家族基因(p33ING2、p47ING3和p29ING4)。ING家族的基因有一个博士-手指模体。PhD Finger是在核蛋白中发现的一个C4HC3类锌指基序，被认为参与了染色质介导的转录调节。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-蛋白质的相互作用，并对参与转录激活或抑制的多组分复合体的组装或活性是必需的。我们发现p33ING1、p33ING2或p47ING3抑制了p53癌细胞株RKO的生长并诱导其细胞周期停滞，但对转HPV-E6的p53缺陷型RKO细胞无此作用。综上所述，我们的结果表明，ING基因家族与P53在细胞增殖调控中具有协同作用。这些基因在细胞凋亡中的作用目前正在研究中。我们正在进行一项定位克隆计划，以确定染色体3p12.2或3q25.3上的一个可能的肿瘤抑制基因(S)，这是先前通过等位基因缺失和细胞遗传学分析确定的区域，被认为包含候选肿瘤抑制基因。前LHC研究员Koji Sasajima发现了一名患有弥漫性消化道息肉和3号染色体生殖系异常的结直肠癌患者；(3)(p12.2q25.3)。因此，我们提出了一个假设，即该基因(S)受染色体倒位的影响，使患者容易发生弥漫性息肉病和恶性病变。采用定位克隆的方法分离了该患者的3个染色体断裂点。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，我们制备了含有3P断裂点的YAC重叠群和YAC克隆，并通过FISH(荧光原位杂交)进行了鉴定。从这些YAC中制备粘粒重叠群，通过FISH鉴定跨越断裂点的粘粒克隆，并测定了3p12.2和3q25.3断裂点及其附近的核苷酸序列。该患者在3p12.2和3q25.3分别有2bp和300kb的缺失。对被染色体倒位破坏的基因(S)的广泛搜索正在通过两种方式进行：(1)断裂点附近的核苷酸序列的测定-3p中的120kb和3q中的130kb-已经确定；(2)长程外显子捕获，这是最近发展起来的一种方法。候选转录序列将通过研究它们在癌细胞中的异常来进行研究。到目前为止，我们已经获得了两组可能的转录序列，它们都与功能未知的EST(表达序列标签)克隆有显著的同源性。-细胞周期、肿瘤抑制基因-人体组织、体液、细胞等