Molecular cloning and functional analysis of the murine bax gene promoter.

Bcl-2-associated X protein (Bax) is a proapoptotic protein and is suggested to have an important role in carcinogenesis. To investigate the mechanism of bax gene transcriptional regulation, we isolated and sequenced the genomic DNA fragment of the 5' flanking region of the murine bax gene, and subcloned its promoter region into a luciferase reporter construction. The murine bax promoter is TATA-less, and the sequence is only partially homologous to that of the human bax promoter. Transient transfection into NIH 3T3 cells using unidirectionally deleted promoters and mutants of Sp1 sites revealed that two Sp1 sites were partially responsible for the basal activity. The murine bax promoter was not responsive to exogenous p53, suggesting that the p53-responsive element may not exist in the region used in our current experiments.

Molecular cloning and functional characterization of the upstream promoter region of the human p73 gene.

The p73 gene encodes a protein that shares structural and functional homologies with the p53 tumor suppressor protein. To investigate the mechanism of transcriptional regulation of the p73 gene, we isolated a genomic DNA fragment spanning the 5' upstream region of the human p73 gene and characterized the promoter region. Unlike the p53 gene promoter, the human p73 gene promoter contained a putative TATA-box, and did not exhibit any extended homology to the p53 gene. Two CpG islands were located in the 5' upstream region. Transient transfection assays using progressive truncations of the p73 promoter showed that deletion from -119 to +19 relative to exon 1 resulted in a 13- to 20-fold reduction in the p73 promoter activity, suggesting that the elements for basal promoter activity exist in this region, where putative Sp1, AP-2 and Egr-1, 2, 3 sites are located and CpG dinucleotides are especially concentrated.

Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter.

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha and beta subunits. The alpha subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-beta/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2alphaC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Calpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-beta/BMP signaling.

The ubiquitous transcription factor NF-Y positively regulates the transcription of human p27Kip1 through a CCAAT box located in the 5-upstream region of the p27Kip1 gene.

Abnormally low levels of the cyclin-dependent kinase inhibitor p27Kip1 are found frequently in human carcinomas, and these levels correlate directly with both histological aggressiveness and patient mortality. p27Kip1 is haplo-insufficient for tumor suppression. Thus, p27Kip1 may be a useful molecule for the development of cancer therapies. To know the possible mechanisms underlying transcriptional control, we previously cloned the promoter region of human p27Kip1 gene. We report here the characterization of the 5'-regulatory region of the human p27Kip1 gene. Promoter analysis using 5'-deletion mutants revealed that a 39-bp region between -549 and -511 was required for maximal promoter activity. Point mutation analysis revealed that a CCAAT box within this region was essential for promoter activity. Gel shift assays and cotransfection experiments using a dominant negative form of the NF-Y transcription factor showed that NF-Y directly regulates p27Kip1 transcription through this CCAAT box. This finding might provide a clue to approach the mechanism of tumorigenesis.

Promoter activation and following induction of the p21/WAF1 gene by flavone is involved in G1 phase arrest in A549 lung adenocarcinoma cells.

Flavonoids are present in many plants including edible fruits and vegetables. Recently, many of the biological activities of flavonoids have been elucidated. Flavone is a well known flavonoid, and many of its derivatives have been shown to have anti-proliferative effects on several cancer cells. We report here that flavone can effectively inhibit the cell growth of human lung adenocarcinoma A549 cells in a dose-dependent manner, and 100 microM flavone causes cell cycle arrest at the G1 phase. As a mechanism underlying the cell cycle arrest, flavone markedly increases the mRNA and protein levels of a universal inhibitor of cyclin-dependent kinase, p21/WAF1, and inhibits phosphorylation of retinoblastoma (RB) protein. Although A549 cells possess wild-type p53, flavone does not induce the p53 protein, suggesting that p21/WAF1 induction is p53-independent. In addition, 100 microM flavone significantly increases the promoter activity of the p21/WAF1 gene by 5-fold. These results suggest that the G1 phase arrest by flavone is due to p53-independent transcriptional induction of the p21/WAF1 gene and the subsequent dephosphorylation of RB protein.

p53-Independent activation of the gadd45 promoter by Delta12-prostaglandin J2.

A p53-inducible gene, the growth arrest and DNA damage-inducible gene 45 (gadd45), is associated with cell growth inhibition, DNA damage response and DNA repair. Here we report that Delta12-prostaglandin J2 (Delta12-PGJ2), an in vivo metabolite of arachidonic acid, which inhibits cell proliferation, induces gadd45 mRNA in HeLa cells. Because the p53 protein in HeLa cells is inactivated by papilloma virus E6, this type of gadd45 induction appears to be p53-independent. The induction was dose-dependent, and the maximum induction was observed at a concentration of 7.5 microgram/ml. In a time course study, gadd45 mRNA was induced 3 h after the addition of 7.5 microgram/ml Delta12-PGJ2. To investigate the transcriptional mechanism of gadd45 mRNA induction, we cloned a human genomic DNA fragment containing the gadd45 promoter region, and investigated the effect of Delta12-PGJ2 on the gadd45 promoter activity. In HeLa cells, 7.5 microgram/ml Delta12-PGJ2 markedly stimulated the gadd45 gene promoter about 20-fold or more. On the other hand, Delta12-PGJ2 did not stimulate the promoter activity of a reporter plasmid containing only p53 binding sites in HeLa cells, indicating that the gadd45 promoter activation by Delta12-PGJ2 was not mediated by p53. These results suggest that one of the mechanisms of cell growth arrest by Delta12-PGJ2 is mediated through a p53-independent induction of gadd45.

Cell cycle-dependent modulation of promoter activities of RB and WAF1/Cip1 genes.

A universal inhibitor of cyclin-dependent kinases, WAF1/Cip1 can dephosphorylate the RB gene product to arrest the cell cycle at the G1 phase. Here we show that the mRNA level and the promoter activities of the RB and WAF1/Cip1 genes exhibit cell cycle-dependent change when cells are released from either serum-starvation or the confluent cell state with serum. RB expression and promoter activity are elevated at middle to late G1. In contrast, the mRNA and promoter activity of the WAF1/Cip1 gene increase at early G1. These results suggest that the RB and WAF1/Cip1 expression and promoter activities depend not only on serum, but also on the cell cycle progression itself. Moreover, we identified the responsive region for serum-released cell cycle progression in the RB promoter and mapped it to the region between -4 and -182 relative to the initiating codon of the RB gene. The region in the WAF1/Cip1 promoter responsible for the serum-released cell cycle progression mapped not to the p53 binding site, but to the 374 base-pair region between -1770 and -1396 from the transcription start site.

p53-independent induction of WAF1/Cip1 is correlated with osteoblastic differentiation by vitamin D3.

1Alpha,25(OH)2 vitamin D3 (1,25(OH)2D3) can induce differentiation of osteoblastic cells by arresting the cell cycle at G1. The p53-inducible gene, WAF1/Cip1, is one of the inhibitors of cyclin-dependent kinases and can inhibit the phosphorylation of retinoblastoma protein (pRB), thereby keeping pRB functionally active. Here we show that in a p53-null human osteoblastic osteosarcoma MG-63 cell line, 10 nM of 1,25(OH)2D3 completely inhibits cell growth and increases alkaline phosphatase activity, which suggests the induction of osteoblastic differentiation. We also found a p53-independent increase of WAF1/Cip1 mRNA and promoter activation by 1,25(OH)2D3. On the other hand, the expression and the promoter activity of the RB gene decreased after treatment with 1,25(OH)2D3 during the differentiation of MG-63 cells. Our results suggest that the p53-independent WAF1/Cip1 induction by 1,25(OH)2D3 is important for osteoblastic differentiation of MG-63 cells.

Hypermethylation in the retinoblastoma gene is associated with unilateral, sporadic retinoblastoma.

We previously reported 9 unilateral, sporadic retinoblastomas with hypermethylation in the 5' region of the RB gene, and we found that CpG methylation in the RB promoter inhibits the binding of the retinoblastoma binding factor 1 (RBF-1) and the activating transcription factor (ATF)-like factors, thereby resulting in a considerable reduction in RB promoter activity. In this study, we screened for hypermethylation in 121 additional cases of retinoblastoma, and found 5 tumors with hypermethylation, including 4 unilateral, sporadic tumors, and one hereditary tumor. The hereditary tumor had a germline deletion of one allele, and the hypermethylation was an acquired, epigenetic change in the other allele. Another tumor had hypermethylation restricted to approximately 800 base pairs in the RB promoter region including the essential RBF-1 and ATF sites. The frequency of hypermethylation in unilateral, sporadic tumors was 9.3% combining our previous and present examinations (13 among 140), whereas the frequency was 1.0% in bilateral hereditary tumors (one among 101). The statistical analyses using the chi-square test indicated significant correlation between hypermethylation and unilateral, sporadic tumors (p < 0.05). These results suggest that hypermethylation in the RB gene is always an acquired, epigenetic change and causes about 9% of unilateral, sporadic tumors.

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 of WAF1/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.

Molecular cloning and characterization of the human p27Kip1 gene promoter.

p27Kip1 is an inhibitor of multiple cyclin-dependent kinases (cdk), and can arrest the cell-cycle progression by inhibiting the phosphorylation of the retinoblastoma gene family products. Tumor formation in p27Kip1 knockout mice clearly shows that p27Kip1 plays an important role in inhibiting tumor formation and progression. To investigate the mechanism of transcriptional p27Kip1 gene expression, we isolated the genomic DNA fragment of the 5' flanking region of the human p27Kip1 gene and characterized its promoter region. The human p27Kip1 promoter is TATA-less, and the sequence is highly homologous to the murine p27Kip1 promoter sequence. In the promoter assay, deletion from -774 to -435 relative to the initiating codon resulted in a 15-20-fold reduction of the p27Kip1 promoter activity, suggesting that the elements for basal promoter activity exist in this highly conserved 340 bp region, where putative CTF and ATF sites are conserved.

Monochloramine-induced cell growth inhibition and apoptosis in a rat gastric mucosal cell line.

Recent studies have indicated that monochloramine (NH2Cl), a reaction product of NH3 and hypochlorous acid, is involved in the pathogenesis of Helicobacter pylori-associated gastric mucosal damage, but how NH2Cl contributes to lesions is unclear. In the present study, the effects of NH2Cl on mucosal cell growth and the cell cycle were evaluated in vitro using a normal rat gastric mucosal cell line RGM-1. Cell viability was assessed by the Trypan Blue dye exclusion test and cell cycle patterns were determined by DNA labeling with propidium iodide and flow cytometric quantification. NH2Cl inhibited the growth of RGM-1 cells in a concentration-dependent manner. Exposure of cells to NH2Cl caused a time- and dose-dependent loss of G1-phase cells with accumulation of G2/M-phase cells, and produced a fraction of subdiploid cells with oligonucleosomal DNA degradation characteristic of apoptosis. NH2Cl-induced apoptosis was confirmed by fluorescent microscopy with Hoechst 33342 and propidium iodide. These results suggest that NH2Cl inhibits gastric mucosal cell growth and induces apoptosis in RGM-1 cells, events that may be important in gastric mucosal damage or atrophy induced by H. pylori infection.

Identification of an RB-responsive region in the 5' untranslated region of the RB gene.

To investigate the ability of the retinoblastoma gene product (RB protein, pRB) to regulate its own expression, cotransfection assays using human RB promoter-luciferase fusion plasmids and a human pRB expression plasmid were employed. In B104, a rat neuroblastoma cell line, pRB stimulated luciferase activity about 2-fold from the wild-type promoter, and about 4-fold from a mutant promoter with a mutation in the retinoblastoma binding factor 1 (RBF-1) site. The RB-responsive region was mapped to a novel 44 bp sequence in the 5' untranslated region in both wild-type and mutant promoters. When apparent stimulation of luciferase activity by pRB was observed, the luciferase mRNA level did not increase, suggesting that through this 44 bp region, pRB could post-transcriptionally regulate its own expression.

A silencer element in the retinoblastoma tumor-suppressor gene.

We have previously identified two positive regulatory elements in the retinoblastoma gene (RB) promoter. One is an ATF site and the other we have called a retinoblastoma binding factor 1 site. In addition, a consensus E2F site is located directly 3' to the ATF site. Here we demonstrate with gel shift assays that E2F and E2F-dependent complexes can bind in vitro to the E2F site of the RB promoter (RB-E2F site). Moreover, we demonstrate that deletion of the RB-E2F site results in stimulation of RB promoter activity in transient transfection assays in several cell lines. Specific point mutations in the E2F site that inhibit the binding of E2F and its complexes also stimulate the RB promoter activity, suggesting that a factor(s) that can bind to the RB-E2F site could be a silencer. RB promoter activity was also stimulated two- to four-fold by mutation of the E2F site in the RB-negative cell lines, J82 and HTB9. Taken together, our results show that the E2F site in the promoter of the RB tumor-suppressor gene acts as a silencer element and that the silencer effect of the E2F site is not necessarily dependent on the presence of the RB protein.

Identification of a p53 binding site in the human retinoblastoma susceptibility gene promoter.

p53 is a tumor suppressor gene found to be mutated in a wide variety of tumors. The encoded p53 protein has properties of a classical transcription factor, but the promoter targets for its regulation are largely unknown. We have investigated the ability of p53 to regulate activity of the human retinoblastoma susceptibility gene (Rb) promoter using a cotransfection assay in CCL-64 and Saos-2 cells. p53 was able to stimulate transcription from the Rb promoter at low input doses of p53 expression plasmid, whereas transcription was repressed at high input doses. The stimulatory effect of p53 on Rb promoter activity mapped to a region between 4 and 92 base pairs upstream from the start site of translation, whereas the region controlling repression by p53 mapped to the basal transcriptional control region of the promoter between -207 and -185. Moreover, an oligonucleotide containing Rb promoter sequences between -63 and -88 was sufficient to confer stimulation by p53 when inserted upstream from a minimal heterologous promoter. Gel mobility shift analysis was used to demonstrate that p53 can bind to a sequence within the -63 to -88 oligonucleotide with homology to a p53 binding site. The presence of a functional p53 binding site in the human retinoblastoma tumor suppressor gene promoter suggests that p53 can regulate Rb promoter activity.

Effect of tumor suppressors on cell cycle-regulatory genes: RB suppresses p34cdc2 expression and normal p53 suppresses cyclin A expression.

We show that expression of the p34cdc2 and cyclin A genes is induced by interleukin-2 in normal human T cells and present evidence to support the idea that these genes are deregulated in leukemic T cells. Our DNA sequencing data indicate that the promoter region of the p34cdc2 gene contains putative E2F-like binding sites which are recognized by Rb and binding sites for c-myb, Sp1, and ATF, and that the promoter region of the cyclin A gene contains binding sites for p53, Sp1, and ATF. In this study we focus on the effect of p53 and Rb on these cell cycle-regulatory genes. Cotransfection of Y79 human retinoblastoma cells with a p34cdc2 promoter-luciferase expression vector and a plasmid expressing the retinoblastoma gene (RB) indicated that RB suppresses p34cdc2 expression. Cotransfection of B104 rat neuroblastoma cells with a cyclin A promoter-luciferase expression vector and a plasmid expressing the normal or mutant p53 indicated that only the normal p53 suppresses cyclin A expression. In normal T cells, PHA stimulation reduces the amount of complexes in the p34cdc2 promoter between the E2F-like binding site and the RB gene product. These complexes were not detected in leukemic T cells. Our data support the idea that tumor suppressors modulate the expression of cell cycle-regulatory genes: RB regulates p34cdc2 expression and p53 regulates cyclin A expression.

CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene.

Cytosine methylation of CpG sites in the promoter region of eucaryotic genes is involved in the inactivation of expression of certain genes. Given that methylation can lead to reduced transcription, it is possible that expression of tumor-suppressor genes is also inactivated by hypermethylation, thereby contributing to the etiology of cancer. Recently we found five sporadic retinoblastoma tumors (16% of all unilateral cases) with hypermethylation of the 5' end of the retinoblastoma gene without detecting any structural abnormalities. However, it is unclear whether the promoter of the retinoblastoma gene is actually inactivated by its hypermethylation. Here we show that specific hypermethylation in the promoter region of the retinoblastoma gene reduces its expression to only 8% of the unmethylated control. Furthermore, we have found that two transcription factors important for the promoter activity, an activating transcription factor (ATF)-like factor and the retinoblastoma binding factor 1, do not bind when their recognition sequences are CpG methylated. These results in vitro strongly support the hypothesis that CpG methylation of the human tumor-suppressor gene can result in the inactivation of the gene and thus lead to oncogenesis.