Transient Gene Expression Assays Research Articles

Expression of interleukin-6 is downregulated by 17-(allylamino)-17-demethoxygeldanamycin in human prostatic carcinoma cells

Interleukin-6 (IL-6) is a pleiotropic cytokine that is associated with tumor metastasis and prostate cancer. We evaluated the mechanism and effect of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), a novel inhibitor of heat shock protein 90 (Hsp90), on the IL-6 gene expression in human prostatic carcinoma (PC-3) cells. Quantitative IL-6 and IL-6 receptor (IL-6R) expressions were assessed using RT-PCR. The deregulation of 17AAG and phorbol 12-myristate 13-acetate (PMA) on the IL-6 gene was determined by ELISA and transient gene expression assays using an IL-6 reporter vector. Although the IL-6R is ubiquitously expressed by prostatic epithelium cells, the IL-6 expression is only found in advanced prostatic carcinoma cells, such as PC-3 and DU145. Further studies using RT-PCR indicated that 17AAG downregulated the gene expression of IL-6. ELISA and the transient gene expression assay revealed that 17AAG blocked the stimulation of PMA of IL-6 gene expression in PC-3 cells. The PMA-induced IL-6 gene expression is dependent on the NF-kappaB response element. However, the effect of 17AAG appears to be mediated via a region located at -149 to +8 bp upstream of the transcriptional starting site of the IL-6 gene, and might not be through the NF-kappaB signaling pathway. The present study reveals that IL-6 is transcriptionally downregulated in human prostatic carcinoma cells in response to 17AAG. This result suggests the presence of a novel Hsp90 mediation pathway that is involved in the deregulation on the transcription of the human IL-6 gene in human prostate cancer.

Differential regulation of zebrafish metallothionein-II (zMT-II) gene transcription in ZFL and SJD cell lines by metal ions

Two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5′-flanking region (1514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) and caudal fin (SJD) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn 2+ and that this Zn 2+ induction was dose-dependent. Further transient gene expression assay of the zMT-IIA gene promoter was carried out to study the effects of various metal ions (Zn 2+, Cd 2+, Cu 2+, Hg +, As 3+, As 5+, Cr 3+ and Cr 6+), and Zn 2+ and Cd 2+ were found to be the most efficient MT gene inducers of zMT-II. As 3+ was a weak inducer of zMT-II in the two cell lines, and Hg + caused significant induction only in the SJD cells. No significant induction was found in the other metal ion exposures. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn 2+ or Cd 2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.

Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin

As a result of natural hybridization and human selection over millennia, the skin colors of grapes have become greatly diversified. The color is determined by the quantity and composition of anthocyanins. Color-skinned cultivars accumulate anthocyanins in their skins, whereas white-skinned cultivars do not. Myb-related transcription-factor genes such as VvmybA1 regulate anthocyanin biosynthesis. VvMYBA2r, VlmybA1-1, VlmybA1-2, and VlmybA2, which are homologs of VvmybA1, also regulate anthocyanin biosynthesis. In this study, we isolated a novel Myb-related sequence, VlmybA1-3, from cultivars of Vitis labruscana (Vitis vinifera x Vitis labrusca) by means of inverse PCR, and confirmed by means of transient gene expression assay that the gene regulates anthocyanin biosynthesis in grape berry skin. Seedlings of V. labruscana with two functional haplotypes at a region of berry color loci accumulated more anthocyanins than seedlings with a single functional haplotype. In addition, we investigated the haplotypes at the region in 35 cultivars (both V. vinifera and V. labruscana), and found certain typical characteristics. These findings will contribute to the selection of seedlings with high anthocyanin quantities in breeding programs for wine and table grapes, and will help elucidate the origin and evolution of Vitis species.

Triiodothyronine modulates cell proliferation of human prostatic carcinoma cells by downregulation of the B‐Cell translocation gene 2

Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B-cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle. Effects of T3 on cell proliferation were determined by (3)H-thymidine incorporation. T3 modulation of BTG2 expression was investigated using immunoblots, Northern blots, and transient gene expression assays. The putative T3 response element was determined by electrophoretic mobility shift assay. T3 (0.1-1,000 nM) enhanced threefold the proliferation of prostate carcinoma cells and human androgen-dependent prostate carcinoma cells (LNCaP), but not PC-3 cells. T3 also inhibited BTG2 gene expression in LNCaP cells. Reporter assays showed that T3 downregulates by 50% promoter activity of the BTG2 gene in LNCaP cells but not PC-3 cells or thyroid-hormone receptor (TRbeta1)-overexpression PC-3 cells. Deleting the putative thyroid hormone response element (TRE; AGCGATGACCTCAGCG) blocked the inhibitory effect of T3 on BTG2 promoter activity. Electrophoretic mobility shift assays with purified TRbeta1 from in vitro translation, or with nuclear extracts from LNCaP cells and PC-3 cells, demonstrated the presence of T3 receptor binding sites in the TRE region. These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.

Vitamin D receptor is essential for normal keratinocyte stem cell function

The major physiological role of the vitamin D receptor (VDR) is the maintenance of mineral ion homeostasis. Mutation of the VDR, in humans and mice, results in alopecia. Unlike the effects of the VDR on mineral ion homeostasis, the actions of the VDR that prevent alopecia are ligand-independent. Although absence of the VDR does not prevent the development of a keratinocyte stem cell niche in the bulge region of the hair follicle, it results in an inability of these stem cells to regenerate the lower portion of the hair follicle in vivo and impairs keratinocyte stem cell colony formation in vitro. VDR ablation is associated with a gradual decrease in keratinocyte stem cells, accompanied by an increase in sebaceous activity, a phenotype analogous to that seen with impaired canonical Wnt signaling. Transient gene expression assays demonstrate that the cooperative transcriptional effects of beta-catenin and Lef1 are abolished in keratinocytes isolated from VDR-null mice, revealing a role for the unliganded VDR in canonical Wnt signaling. Thus, absence of the VDR impairs canonical Wnt signaling in keratinocytes and leads to the development of alopecia due to a defect in keratinocyte stem cells.

Methods for transient assay of gene function in floral tissues

BackgroundThere is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.ResultsTwo constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum) chalcone synthase gene (CHS) and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression.ConclusionWe demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to insert the gene sequence of interest. These techniques should allow analysis of gene function in a much broader range of flower species.

Manganese antagonizes iron blocking mitochondrial aconitase expression in human prostate carcinoma cells

To investigate the possible role of manganese in the regulation of mitochondrial aconitase (mACON) activity human prostate carcinoma cell line PC-3 cells. The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays. In vitro study revealed that manganese chloride (MnCl2) treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC-3 cells. Although results from transient gene expression assays showed that MnCl2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by co-treatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl2 on the gene expression of mACON. These findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.

Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana.

Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.

Cloning of crucian carp ( Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region

The genomic DNA of crucian carp ( Carassius cuvieri) metallothionein-II (ccMT-II), with its upstream region, was obtained. The sequence analysis of its upstream region revealed several putative cis-acting elements including seven metal regulatory elements (MREs), three activator protein 1 (AP1), two glucocorticoid response elements (GREs), etc. The seven MREs locate into two clusters, a distal cluster with four MREs within −800/−600 bp from the translation start site and a proximal cluster with three MREs close to TATA box. In transient luciferase gene expression assays, both of the distal and proximal cluster MREs have significantly shown synergistic effects in the transcription of ccMT-II gene; the proximal cluster of MREs serves as the major elements in metal inducing activity; Zn 2+ and Cd 2+ served as much stronger inducers than Cu 2+ shown in ccMT-II expression. The two GRE homologous sequences in ccMT-II promoter showed not to be inductive in either HepG2 or HEK293.

Role of V protein RNA binding in inhibition of measles virus minigenome replication

Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein–RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3′ terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein–RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238–266 were necessary for efficient RNA binding and overlapped with residues (238–278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238–247) that blocked RNA binding and repression indicating that these two activities were related.

Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (Ac MNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for Ac MNPV infection. It has been well established that 19 Ac MNPV late expression factors ( lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the Ac MNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an Ac MNPV late promoter–reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding Ac MNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding Ac MNPV homolog in the context of the remaining Ac MNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the Ac MNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.

Zinc blocks gene expression of mitochondrial aconitase in human prostatic carcinoma cells

Mitochondrial aconitase (mACON) contains a [4Fe-4S] cluster as the key enzyme for citrate oxidation in the human prostatic epithelial cell. Although there is accumulating evidence indicating that accumulation of high levels of zinc in prostate epithelial cells causes reduced efficiency of citrate oxidation, zinc regulation on the mACON is still not well understood. From in vitro studies, zinc chloride treatment has been developed using humic acid as the carrier (Zn-HA) in human prostatic carcinoma cells, PC-3. Zn-HA treatment (0.1-10 microM) restricts mACON enzymatic activity, which attenuates citrate utility and decreases intracellular ATP levels in PC-3 cells, whereas the effect is blocked by adding the zinc chelator, diethylenetriaminepentaacetic acid (DTPA). Immunoblot, ribonuclease-protection and transient gene-expression assays indicate that Zn-HA treatments inhibit mACON gene expression. Mutation of the putative metal response element (MRE) from CTCGCCTTCA to TGATCCTTCA abolishes Zn-HA inhibition of mACON promoter activity. Our results have demonstrated that zinc possesses a specific regulatory mechanism on the mACON gene, and a biologic function of the putative metal regulatory system in mACON gene transcription has been identified.

Ethyl caffeate suppresses NF‐κB activation and its downstream inflammatory mediators, iNOS, COX‐2, and PGE2in vitro or in mouse skin

Ethyl caffeate, a natural phenolic compound, was isolated from Bidens pilosa, a medicinal plant popularly used for treating certain inflammatory syndromes. The purpose of this study was to investigate the structural activity, and the anti-inflammatory functions and mechanism(s) of ethyl caffeate. Ethyl caffeate was found to markedly suppress the lipopolysaccharide (LPS)-induced nitric oxide (NO) production (IC(50) = 5.5 microg ml(-1)), mRNA and protein expressions of inducible nitric oxide synthase (iNOS), and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Transient gene expression assays using human cox-2 promoter construct revealed that ethyl caffeate exerted an inhibitory effect on cox-2 transcriptional activity in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells. Immunohistochemical studies of mouse skin demonstrated that TPA-induced COX-2 expression was significantly inhibited by ethyl caffeate with a superior effect to that of celecoxib, a nonsteroidal anti-inflammatory drug. The phosphorylation and degradation of inhibitor kappaB (IkappaB) and the translocation of nuclear transcription factor-kappaB (NF-kappaB) into the nucleus, as well as the activation of mitogen-activated protein kinases (MAPKs) induced by LPS in macrophages, were not affected by ethyl caffeate. Ethyl caffeate, however, could inhibit NF-kappaB activation by impairing the binding of NF-kappaB to its cis-acting element. These results suggest that ethyl caffeate suppresses iNOS and COX-2 expressions partly through the inhibition of the NF-kappaB.DNA complex formation. Structure-activity relationship analyses suggested that the catechol moiety and alpha,beta-unsaturated ester group in ethyl caffeate are important and essential structural features for preventing NF-kappaB.DNA complex formation. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory and therapeutic properties of ethyl caffeate.

A human‐specific TNF‐responsive promoter for Goodpasture antigen‐binding protein

The Goodpasture antigen-binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head-to-head with POLK, the gene encoding for DNA polymerase kappa (pol kappa), and shares with it a 140-bp promoter containing a Sp1 site, a TATA-like element, and a nuclear factor kappa B (NFkappaB)-like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFkappaB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFkappaB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFkappaB to a DNA element encompassing a NFkappaB-like site that is pivotal for the 140-bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA-like element but is devoid of the NFkappaB-like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human-specific TNF-responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

Effects of baculovirus transactivators IE-1 and IE-2 on the Drosophila heat shock 70 promoter in two insect cell lines

Baculovirus infection of permissive cells proceeds in a cascade fashion with the transcription of early, late and very late genes. The structure of a number of baculovirus early gene promoters has been dissected in detail and the viral factors necessary to stimulate their expression have been identified. Early baculovirus gene promoters in general have a resemblance to host promoters while late and very late gene promoters are different from early baculovirus promoters and are more defined. In this study we investigated whether two key Autographa californica M nucleopolyhedrovirus (AcMNPV) transactivators have the ability to regulate the commonly used cellular promoter from the Drosophila heat shock 70 protein gene, during transient gene expression assays in two insect cell lines permissive for AcMNPV infection, SF-21 and TN-368, or during viral infection. The AcMNPV ie-1 transactivator gene stimulated gene expression of this cellular promoter in both cell lines when the promoter was cis-linked to an enhancer element, but stimulation in the absence of enhancer elements was either undetected or lower than in the presence of enhancer elements in SF-21 and TN-368 cells, respectively. The transactivator ie-2 stimulated gene expression in the presence of cis-linked enhancer elements and ie-1 in SF-21 cells. During viral infection, the heat shock 70 promoter was maximally activated at 12 hours post infection. We discuss how these results affect the interpretation of transient gene expression assays performed in the presence of viral transcription factors.

Molecular characterization of mlo mutants in North American two‐ and six‐rowed malting barley cultivars

SUMMARY Barley lines PRU1, URS1 and URS2 represent three candidate mlo mutants induced in either the two-rowed cultivar Prudentia or the six-rowed cultivar Ursula. Both Prudentia and Ursula are North American malting barley varieties with specific malting properties. Here, we analysed the three candidate mutants at the molecular level. We identified lesions in the Mlo gene of all three lines, causing either a premature stop codon (PRU1), a shift in the reading frame (URS1) or a single amino acid replacement (URS2). In a transient gene expression assay, the URS2 mlo allele fails to complement a barley null mutant genotype, indicating that URS2 is a genuine mlo mutant (here designated as mlo-33). The MLO-33 mutant variant accumulates to similar levels as the wild-type MLO protein in Arabidopsis protoplasts, suggesting that MLO-33 is stable in planta. We show that the mlo-33 allele can be readily detected in barley genomic DNA by a cleaved amplified polymorphic sequence marker, rendering this allele particularly suited for marker-assisted breeding.

DOWN-REGULATION OF THE PROSTATE SPECIFIC ANTIGEN PROMOTER BY p53 IN HUMAN PROSTATE CANCER CELLS

The WT p53 gene appears to have a broad role in suppressing prostatic tumorigenesis. We identified the mechanisms responsible for the effect of p53 on prostate specific antigen (PSA) expression by prostate cancer cell lines in vitro and investigated the role of a putative p53 response element in the PSA promoter region in prostate cancer cells. LNCaP cells were used to determine the effect of doxorubicin on p53 and PSA expression. The putative p53 response element in the human PSA promoter was identified by transient gene expression with site direct mutagenesis assays using a PSA reporter vector. Quantitative PSA secretions were assessed using enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent and immunoblot assays indicated that doxorubicin treatment increased p53 expression but inhibited PSA levels in LNCaP cells. Transient gene expression assays showed that human PSA promoter activity was blocked by doxorubicin treatment. Mutation of the p53 response element GGGCATGTCT to GGGAGGATCT abolished the blocking effects of doxorubicin on PSA gene promoter activity. Results demonstrate that p53 regulates PSA gene expression through a putative p53 response element in the PSA promoter within human prostate cancer cells.

Modulation of iron on mitochondrial aconitase expression in human prostatic carcinoma cells

The mitochondrial aconitase (mACON) containing a [4Fe-4S] cluster is regarded as the key enzyme for citrate oxidation in the epithelial cells of human prostate. In vitro studies using the human prostatic carcinoma cells, PC-3 cells, found that both hemin and ferric ammonium citrate (FAC) significantly increased mACON enzymatic activity and gene expression. The effect of FAC on mACON was enhanced 2-fold by co-treating with ascorbic acid but blocked by co-treating with iron chelator, deferoxamine mesylate. Hemin treatments blocked 30% of citrate secretion from PC-3 cells but upregualted 2-fold of intracellular ATP biosynthesis. Results from reporter assay by using a cytomegalovirus enhance/promoter driven luciferase mRNA ligated to the iron response element (IRE) of mACON as a reporter construct demonstrated that modulation of FAC on gene translation of mACON gene is dependent on the IRE. Transient gene expression assays indicated that upregulation of mACON gene transcription by FAC may through the putative antioxidant response element (ARE) signal pathway. This study provides the first evidence of the biologic mechanism of human mACON gene translation/transcription and suggests a regulatory link between the energy utilization and the iron metabolism in human prostatic carcinoma cells.

Testosterone modulates mitochondrial aconitase in the full-length human androgen receptor-transfected PC-3 prostatic carcinoma cells.

In vitro studies indicated that dihydrotestosterone (DHT) stimulates the enzymatic activity of the mitochondrial aconitase (mACON) in androgen-sensitive prostatic carcinoma cells, LNCaP. Cell proliferation assay determined that DHT doubles the optimal proliferation response of LNCaP cells. The androgen-insensitive human prostatic carcinoma cells, PC-3, were overexpressed in the human androgen receptor to assess the involvement of the native androgen receptor in the regulation by DHT of mACON gene expression. A stable-transfected clone that expresses the full-length androgen receptor was selected and termed PCAR9. The results revealed that DHT-treated PCAR9 cells paradoxically not only reduced the enzymatic activity of mACON but also blocked the biosynthesis of intracellular ATP attenuating cell proliferation. Transient gene expression assay indicated that DHT divergently regulates the promoter activity of the mACON gene in LNCaP and PCAR9 cells. This study suggested that DHT regulates mACON gene expression and the proliferation of cells in a receptor-dependent model through modulation by unidentified non-receptor factors.

Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line

The coding region of cDNA and genomic DNA, with its promoter region, of zebrafish metallothionein ( zMT) gene homologous to the piscine MT-II was obtained. The A/T-rich promoter region contains four metal regulatory elements (MREs), three activator protein 1 (AP1) and one specific protein 1 (Sp1) binding sites. The four MREs are organized into two clusters, a distal cluster with one MRE lying around 740 bp upstream of the transcription start point and a proximal cluster with three MREs located close to the TATA box. The metal induction ability of the promoter was assessed by transient luciferase gene expression assays in HepG2 cells. The zMT promoter was inducible by Zn 2+, Cd 2+, Cu 2+ and Hg 2+ ions in decreasing inducibility, while inert to Ni 2+, Pb 2+ and Co 2+ ions, and H 2O 2 treatment in vitro . Deletion of the putative cis-acting elements in the promoter region revealed that the distal MRE (MREd) was important in mediating metal inducibility. Despite the binding of HepG2 cell nuclear protein factors to all MREs as confirmed by electrophoretic mobility shift assay (EMSA), the proximal MREs did not provide significant contribution to metal induction of zMT gene in HepG2 cells. The metal inducibility of zMT promoter required the cooperative effect of at least three MRE sites.