CELL CYCLE CONTROL AND TUMOR SUPPRESSORS

G1-S和G2-M细胞周期检查点在真核生物中响应遗传毒性应激维持基因组稳定性。1999年，我们报道了人类和小鼠细胞中Gadd 45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Gadd 45的中心区域（残基50-76）在其家族成员包括Gadd 45 β和Gadd 45 γ中是保守的，并且介导G2/M停滞。该区域也是Cdc 2、PCNA或p21 Waf 1的结合位点。当第62-67位的酸性残基变为丙氨酸时，该突变体丧失了诱导正常人成纤维细胞G2/M期阻滞的能力，但仍能与Cdc 2、PCNA或p21 Waf 1结合。我们目前正在研究这些GADD 45突变体是否调节Cdc 2/cyclin B1的酶活性。我们已经克隆了ING家族基因（p33 ING 2、p47 ING 3和p29 ING 4）。ING家族基因具有PHD-finger基序。PHD指是在核蛋白中发现的C4 HC 3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p33 ING 1、p33 ING 2或p47 ING 3在野生型p53癌细胞系RKO中抑制细胞生长并诱导G1期细胞周期停滞，但在用HPV-E6转染的p53缺陷型RKO细胞中不诱导。两者合计，我们的研究结果表明，ING基因家族与p53在细胞增殖的调节合作。这些基因在细胞凋亡中的作用目前正在研究中，我们正在进行定位克隆项目，以确定染色体3p12.2或3q25.3上的一个假定的肿瘤抑制基因，这些区域是先前通过等位基因缺失和细胞遗传学分析确定的，被认为含有候选肿瘤抑制基因。Koji Sasajima，前LHC研究员，发现了一个结肠直肠癌患者，患有弥漫性消化道息肉病和3号染色体的生殖系异常;（3）（p12.2q25.3）。因此，我们提出了这样的假设，即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。通过定位克隆策略分离了该患者的3号染色体断裂点。我们和我们的合作者在显微镜下解剖了断裂点周围的染色体片段。使用从切割片段分离的DNA探针，我们制作了YAC重叠群和含有3 p中的断裂点的YAC克隆，其通过FISH（荧光原位杂交）鉴定。从这些YAC中制备粘粒重叠群，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。该患者在3p12.2有一个2bp的缺失，在3q25.3有一个约300 kb的缺失。目前正在通过两种方法对染色体倒位破坏的基因进行广泛的研究：（1）确定断裂点周围的核苷酸序列-3 p中的120 kb和3q中的130 kb-已经确定;（2）最近开发的远程外显子捕获方法。候选转录序列将通过调查它们在癌细胞中的异常来研究。到目前为止，我们已经获得了两个可能的转录序列簇，这两个簇都与未知功能的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，液体，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.