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.
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