American Ginseng Transcriptionally Activates p21 mRNA in Breast Cancer Cell Lines

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p< or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p< or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

Interferon-gamma upregulates the stromelysin-1 gene expression by human skin fibroblasts in culture

The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-gamma (IFN-gamma) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-gamma treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-gamma stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-beta (TGF-beta) showed the antagonistic action to the effects of IFN-gamma in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-gamma. These data suggest that IFN-gamma is an up-regulator and TGF-beta is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.

Genomic structure of the regulatory region of the voltage-gated calcium channel alpha 1D

In excitable and endocrine organs, calcium influxes through the L-type voltage-gated calcium channel (VGCC) which is composed of four (alpha 1, alpha 2, beta, and gamma) subunits. Temporal and spatial expression of calcium channel activity is regulated by the transcription of alpha 1 subunit. To elucidate the genomic organization of the VGCC alpha 1D subunit gene, a genomic clone was isolated from the human genomic library and its sequence was analyzed. A 12 kb genomic clone contained the 5'-flanking regulatory region and first two exons was selected and the initiation site for alpha 1D mRNA synthesis was examined by primer extension analysis. The major initiation site was found at the -523 NT position in the translation initiation site. The TATA box could not be found above the transcription initiation site. The CAT vector construct containing the 2.5 kb upstream region had high CAT activity on transfection to NG108-15 and PC12 cells, which confers the neuronal expression of the alpha 1D gene.

Cytosine methylation inhibits expression of hsp-CAT gene in Drosophila cells.

Chloramphenicol acetyl transferase gene under control of hsp promoter (hsp-CAT gene) was introduced and expressed upon heat shock in Drosophila cells at 48 and 72 hr following transfection. Expression of CAT gene was remarkably reduced when DNA methylated at CpG sites was used although presence of methylated plasmid DNA could be demonstrated in cells at 48 and 72 hr. Thus, in Drosophila cells exogenously introduced methylated DNA is expressed differently from unmethylated DNA.