Hitoshi Nomura

Butyrate Activates the WAF1/Cip1 Gene Promoter through Sp1 Sites in a p53-negative Human Colon Cancer Cell Line

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mm butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at −82 and −69 relative to the transcription start site. We also found that a TATA element at −46 and two overlapping consensus Sp1 sites at −60 and −55 are essential for the basal promoter activity ofWAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.

Inactivation of Tumor Suppressor p53 by Mot-2, a hsp70 Family Member

The mortalin genes, mot-1 andmot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection ofmot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21WAF-1 andmdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent protein-taggedmot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.

Histone Deacetylase Inhibitor Activates the p21/WAF1/Cip1 Gene Promoter through the Sp1 Sites

Trichostatin A (TSA), a specific histone deacetylase inhibitor, induces histone hyperacetylation and modulates the expression of some genes. We examined the effects of TSA on MG63 cells. TSA induced growth arrest and expression of the p21/WAF1/Cip1 protein. A close correlation between the level of histone acetylation and induction of the p21/WAF1/Cip1 protein was detected. Using several mutant p21/WAF1/Cip1 promoter fragments, mutation of either of two Sp1 sites at -82 or -69 of the p21/WAF1/Cip1 promoter reduced the responsiveness to TSA. This finding indicates that TSA activates the p21/WAF1/Cip1 promoter through the Sp1 sites in a p53-independent manner.

Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan

By immunoscreening with an antibody raised against a plasma membrane protein, we have cloned a growth suppressor gene, Gros1 and assigned it to short arm of human chromosome 1. Two alternatively spliced forms of the gene encoding 84- and 41-kDa (carboxy-terminus truncated) proteins were cloned. The two transcripts, 4.4 and 2.7u2009kb, were expressed weakly in most of the human tissues, with a high expression of the smaller transcript in placenta, ovary and testis. Normal human fibroblasts in culture showed two transcripts, with a higher level of expression of the 4.4u2009kb transcript. Transformed cells on the other hand showed predominant expression of the 2.7u2009kb transcript. Two Gros1 transcripts were also detected in most of the mouse tissues. Stable transfection of the mouse cDNA encoding the 85-kDa protein into NIH3T3 cells resulted in their slow growth and reduced colony-forming efficiency. Stable clones expressing antisense RNA on the other hand exhibited higher colony forming efficiency. While our data implied that Gros1 is a novel growth suppressor gene on human chromosome 1, an independent study has recently characterized its rat-homolog as a leucine proline-enriched novel basement membrane-associated proteoglycan leprecan. We describe here cloning, expression and biological activity analysis implying that this novel proteoglycan is a potential growth suppressor on chromosome 1p31, frequently altered in many malignancies.

ATF site of human RB gene promoter is a responsive element of myogenic differentiation.

RB mRNA increases during terminal differentiation of C2 myoblasts. We demonstrate that RB promoter activity increases about 4‐fold during differentiation. The increase of RB promoter activity was reduced when a point mutation was designed in the ATF site. In a gel shift assay of the ATF site, two specific bands were observed. One of them, with the lower mobility, disappeared during differentiation. This band reacted with an antibody against ATF‐1. We cotransfected an RB promoter‐luciferase plasmid with the TREB36/ATF‐1 plasmid. ATF‐1 suppressed the activity of the wild‐type RB promoter but not of that with a point mutation at the ATF site. These results suggest that the ATF site of the RB promoter is a responsive element during myogenic differentiation of C2 cells. We hypothesize that RB promoter activity is stimulated partially due to the dissociation of ATF‐1, which suppresses the promoter activity through the ATF site in C2 myoblasts.

Cloning and characterization of a novel gene, striamin, that interacts with the tumor suppressor protein p53.

Expression analysis of a novel cDNA isolated from immortal murine fibroblasts revealed a single transcript of 3.0 kilobase pairs that was highly expressed in mouse and humanstriated muscle and in mouse heart. The gene has therefore been named striamin. Its expression was confined to skeletal muscle types with a fast glycolytic (2B) contractile phenotype. It was also detected in C2C12 mouse myoblasts and was down-regulated during in vitro myogenesis. The cDNA has a single open reading frame encoding a predicted 16.8-kDa protein of 149 amino acids with no homology to known proteins. Microinjection and transfection of green fluorescence protein-taggedstriamin demonstrated that it localizes to the nucleus. Coimmunoprecipitations revealed that it can interact with p53 (a positive marker for myoblast differentiation) in vivo andin vitro. Furthermore, it repressed p53 activity in p53-mediated reporter assays. Fluorescence in situhybridization with a mouse P1 genomic clone localized the gene to chromosome 12C3, which is syntenic to human chromosome 14q21–22.