CELL CYCLE CONTROL AND TUMOR SUPPRESSORS

G1-S和G2-M细胞周期检查点在基因毒性应激下维持真核生物基因组的稳定性。1999年，我们报道了人类和小鼠细胞中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的中心区域（残基50-76）在包括Gadd45beta和Gadd45gamma在内的家族成员中是保守的，并介导G2/M阻滞。该区域也是Cdc2、PCNA或p21Waf1的结合位点。当62-67之间的酸性残基转变为丙氨酸时，该突变体在正常的人成纤维细胞中失去了诱导G2/M阻滞的能力，但仍然可以结合Cdc2、PCNA或p21Waf1。我们目前正在确定这些GADD45突变体是否调节Cdc2/细胞周期蛋白B1的酶活性。我们克隆了ING家族基因（p33ING2， p47ING3和p29ING4）。ING家族基因有一个博士指基序。PHD指是在核蛋白中发现的C4HC3锌指状基序，被认为参与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-蛋白质相互作用，并且是参与转录激活或抑制的多组分复合物的组装或活性所必需的。我们发现p33ING1， p33ING2或p47ING3在wt p53癌细胞系RKO中抑制细胞生长并诱导G1细胞周期阻滞，但在转染HPV-E6的p53缺陷RKO细胞中没有。综上所述，我们的研究结果表明，ING基因家族与p53共同调控细胞增殖。目前正在研究这些基因在细胞凋亡中的作用。我们正在进行一项定位克隆项目，以鉴定染色体3p12.2或3q25.3上的推定肿瘤抑制基因，这些区域先前通过等位基因缺失和细胞遗传学分析确定，被认为含有候选肿瘤抑制基因。Koji Sasajima，前LHC研究员，发现了一名患有弥漫性消化道息肉病和3号染色体种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，即受染色体倒置影响的基因使患者易患弥漫性息肉病和恶性肿瘤。采用定位克隆策略分离了该患者的3号染色体断点。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，我们制作了含有3p断点的YAC contigs和YAC克隆，并通过FISH（荧光原位杂交）进行了鉴定。cosmid序列由这些YACs组成，通过FISH鉴定了跨越断点的cosmid克隆，并确定了3p12.2和3q25.3中断点处和周围的核苷酸序列。发现该患者在3p12.2中有2bp的缺失，在3q25.3中有大约300kb的缺失。对被染色体倒位破坏的基因的广泛搜索正在通过两种方式进行：(1)已经确定了断点周围的核苷酸序列- 3p中的120 kb和3q中的130 kb；(2)远程外显子捕获，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，我们已经获得了两个可能的转录序列簇，它们都与未知功能的EST（表达序列标签）克隆具有显著的同源性。-细胞周期，肿瘤抑制基因，-人体组织，体液，细胞等

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.