Gene Transcription Assay Research Articles

Interleukin-13, but Not Indomethacin, Increases Cysteinyl-Leukotriene Synthesis in Human Lung Macrophages

Aspirin-exacerbated respiratory disease (AERD) is associated with constitutively elevated synthesis of bronchoconstrictor cysteinyl-leukotrienes, associated with increased expression of leukotriene (LT)C4 synthase and Th2 cytokines and airway eosinophilia. We examined whether interleukin-13 can increase LTC4 synthase gene transcription and cysteinyl-leukotriene synthesis in macrophages isolated from resected human lung tissue and whether an NSAID (indomethacin) can trigger further cysteinyl-leukotriene synthesis in these cells. Overnight culture of human lung macrophages with IL-13 (10 ng/mL) increased spontaneous and ionophore-stimulated production of cysteinyl-leukotrienes by 42% (P = 0.02) and 52% (P = 0.005), respectively, as quantified by enzyme immunoassays, but PCR gene transcription assays did not demonstrate an effect on LTC4S mRNA. The addition of indomethacin (100 μM) did not modulate cysteinyl-leukotriene production in either IL-13-treated or untreated macrophages. We conclude that while IL-13 enhances cysteinyl-leukotriene synthesis in human lung macrophages, it does not replicate the enhanced LTC4 synthase expression observed in the AERD lung nor confer sensitivity to NSAIDs.

Signal transduction pathways mediating the effect of adrenomedullin on osteoblast survival

Adrenomedullin (ADM) plays an important role in the regulation of osteoblastic cells through both a proliferative and an anti-apoptotic effects. The present study investigated mechanisms involved in the effect of ADM on survival. We report that ADM can act in osteoblasts both through a non-transcriptional action, by phosphorylation of different kinases and components, and through a transcriptional effect by activation of CREB. So, we observed by Western blot analysis, modifications in the downstream targets of ERK, the pro-apoptotic protein Bad, which is inactivated by increase in Ser155 phosphorylation, and the transcription factor CREB, which is activated by phosphorylation at Ser133. CREB activation was confirmed by a CRE-dependent gene transcription assay and an immunocytochemical study. This increase in CREB phosphorylation could lead to its enhanced transcriptional activity, as indicated by the induced expression of the proliferation marker, PCNA. Moreover, ADM could also activate the tyrosine kinase Src and the PI3-Kinase, both of which are implicated in survival. The use of specific pharmacological inhibitors allowed to establish that ADM could activate a signaling cascade involving Src, MEK, ERK, p90RSK, and that the effect of ADM, in particular on the CREB protein, greatly depends on the regulatory control of interfering signaling pathways. Moreover, as Wnt signaling plays an important role in the control of osteoblast apoptosis, we explored a major component of this pathway, protein GSK3β. ADM-induced inactivation of GSK3β by phosphorylation at Ser9, highly suggests that ADM could also exert its survival effect in osteoblast via components of the Wnt pathway.

The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor.

The X-ray crystal structure of the turkey β-adrenoceptor has recently been determined. However, mutations were introduced into the native receptor that was essential for structure determination. These may cause alterations to the receptor pharmacology. It is therefore essential to understand the effects of these mutations on the pharmacological characteristics of the receptor. This study examined the pharmacological effects of both the m23 mutations and the β36 deletions, both alone and then in combination in the β36–m23 mutant used in the crystallisation and structure determination of the turkey β-adrenoceptor. Stable CHO-K1 cell lines were made of each of the receptor mutants and the affinity and efficacy of ligands assessed by 3H-CGP 12177 whole cell ligand binding, 3H-cAMP accumulation, and CRE-SPAP gene transcription assays. The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the β36 deletions alone had no effect on ligand affinity. Both sets of changes appeared to reduce the agonist activation of the receptor. Both the m23 and the β36 receptors retained two active agonist-induced receptor conformations similar to that of the original tβtrunc receptor. The combined β36–m23 receptor bound ligands with similar affinity to the m23 receptor; however, agonist activation was only observed with a few agonists including the catecholamines. Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (β36–m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

Molecular determinants of transient and reversible induced up-regulation of CaCDR1 in azole susceptible clinical isolates of Candida albicans

The present study examines the molecular mechanism underlying in vitro-induced resistance to FLC (fluconazole), KTC (ketaonazole), MCZ (miconazole) and CHX (cycloheximide) in AS (azole-susceptible) strains of Candida albicans when exposed to CaCDR1/CaCDR2 inducers like FPZ (fluphenazine) and steroids [PRG (progesterone) and β-EST (β-oestradiol)]. By employing spot and checkerboard titre assays, we provide evidence of an in vitro-induced antagonism between tested drugs and inducers, which was accompanied with a concomitant increase in CaCDR1 and CaCDR2 transcript levels. Notably, unlike AS isolates, parental WT (wild-type) and Δcdr2 null strains, Δcdr1 as well as Δcdr1/Δcdr2 nulls, when challenged with the inducers could not display antagonism. Our results validated by Northern blotting, reporter gene transcription and TRO (transcription run on) assays show that in vitro-induced antagonism between tested drugs and inducer in AS isolates was mainly due to a transient and reversible transcriptional activation of CaCDR1. Notwithstanding our earlier observation that consistent high transcript levels of CaCDR1 in clinical AR (azole-resistant) isolates were maintained due to the combination of its transcriptional activation and enhanced mRNA stability via elongated poly(A) tails, this study shows that transient and reversible transcriptional activation of CaCDR1 was the major determinant of induced antagonism in AS isolates. The distinct strategies between sustained (in AR isolates) and transiently induced resistance mechanisms (in AS isolates) adopted by Candida should become useful in improving therapeutic approaches.

Physiologic and Pharmacologic Modulation of Glucose-Dependent Insulinotropic Polypeptide (GIP) Receptor Expression in β-Cells by Peroxisome Proliferator–Activated Receptor (PPAR)-γ Signaling

OBJECTIVEWe previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARγ in GIP-R transcription.RESEARCH DESIGN AND METHODSChromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARγ knockout mice (PANC PPARγ−/−), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone.RESULTSIn vitro studies of INS-1 cells confirmed that PPAR-γ binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARγ−/− mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress β-cell PPARγ. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARγ and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px.CONCLUSIONSOur studies have shown physiologic and pharmacologic regulation of GIP-R expression in β-cells by PPARγ signaling. Also disruption of this signaling pathway may account for the lowered β-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Bibenzyl- and stilbene-core compounds with non-polar linker atom substituents as selective ligands for estrogen receptor beta

A series of structurally simple bibenzyl-diol and stilbene-diol core molecules, structural analogs of the well-known hexestrol and diethylstilbestrol non-steroidal estrogens, were prepared and evaluated as estrogen receptor (ER) subtype-selective ligands. Analysis of their ERα and ERβ binding showed that certain substitution patterns engendered binding affinities that were >100-fold selective for ERβ. When further investigated in cell-based gene transcription assays, some molecules showed similarly high relative transcriptional potency selectivity in favor of ERβ. Interestingly, the most ERβ-selective molecules were those bearing non-polar substituents on one of the internal carbon atoms. These compounds should be useful probes for determining the physiological roles of ERβ, and they might lead to the development of more selective and thus safer pharmaceuticals.

A study of antagonist affinities for the human histamine H2 receptor

Ligand affinity has been a fundamental concept in the field of pharmacology and has traditionally been considered to be constant for a given receptor-ligand interaction. Recent studies have demonstrated that this is not true for all three members of the G(s)-coupled beta-adrenoceptor family. This study evaluated antagonist affinity measurements at a different G(s)-coupled receptor, the histamine H(2) receptor, to determine whether antagonist affinity measurements made at a different family of GPCRs were constant. CHO cells stably expressing the human histamine H(2) receptor and a CRE-SPAP reporter were used and antagonist affinity was assessed in short-term cAMP assays and longer term CRE gene transcription assays. Nine agonists and seven antagonists, of sufficient potency at the H(2) receptor to examine in detail, were identified. Measurements of antagonist affinity were the same regardless of the efficacy of the competing agonist, time of agonist incubation, cellular response measured or presence of a PDE inhibitor. Antagonist affinity at the G(s)-coupled histamine H(2) receptor obeys the accepted dogma for antagonism at GPCRs. This study further confirms that something unusual is indeed happening with the beta-adrenoceptors and is not an artefact related to the transfected cell system used. As the human histamine H(2) receptor does not behave in a similar manner to any of the human beta-adrenoceptors, it is clear that information gathered from one GPCR cannot be simply extrapolated to predict the behaviour of another GPCR. Each GPCR therefore requires careful and detailed evaluation on its own.

Three Novel Missense Mutations within the LHX4 Gene Are Associated with Variable Pituitary Hormone Deficiencies

The LHX4 LIM-homeodomain transcription factor has essential roles in pituitary gland and nervous system development. Heterozygous mutations in LHX4 are associated with combined pituitary hormone deficiency. Our objectives were to determine the nature and frequency of LHX4 mutations in patients with pituitary hormone deficiency and to examine the functional outcomes of observed mutations. The LHX4 gene sequence was determined from patient DNA. The biochemical and gene regulatory properties of aberrant LHX4 proteins were characterized using structural predictions, pituitary gene transcription assays, and DNA binding experiments. A total of 253 patients from 245 pedigrees with GH deficiency and deficiency of at least one additional pituitary hormone was included in the study. In five patients, three types of heterozygous missense mutations in LHX4 that result in substitution of conserved amino acids were identified. One substitution is between the LIM domains (R84C); the others are in the homeodomain (L190R; A210P). The patients have GH deficiency; some also display reductions in TSH, LH, FSH, or ACTH, and aberrant pituitary morphology. Structural models predict that the aberrant L190R and A210P LHX4 proteins would have impaired DNA binding and gene activation properties. Consistent with these models, EMSAs and transfection experiments using pituitary gene promoters demonstrate that whereas the R84C form has reduced activity, the L190R and A210P proteins are inactive. LHX4 mutations are a relatively rare cause of combined pituitary hormone deficiency. This report extends the range of phenotypes associated with LHX4 gene mutations and describes three novel exonic mutations in the gene.

Small-Molecule Inhibitors of Histone Acetyltransferase Activity: Identification and Biological Properties

Starting from a yeast phenotypic screening performed on 21 compounds, we described the identification of two small molecules (9 and 18) able to significantly reduce the S. cerevisiae cell growth, thus miming the effect of GCN5 deletion mutant. Tested on a GCN5-dependent gene transcription assay, compounds 9 and 18 gave a high reduction of the reporter activity. In S. cerevisiae histone H3 terminal tails assay, the H3 acetylation levels were highly reduced by treatment with 0.6-1 mM 9, while 18 was effective only at 1.5 mM. In human leukemia U937 cell line, at 1 mM 9 and 18 showed effects on cell cycle (arrest in G1 phase, 9), apoptosis (9), and granulocytic differentiation (18). When tested on U937 cell nuclear extracts to evaluate their histone acetyltransferase (HAT) inhibitory action, both compounds were able to reduce the enzyme activity when used at 500 microM. Another quinoline, compound 22, was synthesized with the aim to improve the activity observed with 9 and 18. Tested in the HAT assay, 22 was able to reduce the HAT catalytic action at 50 and 25 microM, thereby being comparable to anacardic acid, curcumin, and MB-3 used as references. Finally, in U937 cells, compounds 9 and 18 used at 2.5 mM were able to reduce the extent of the acetylation levels of histone H3 (9) and alpha-tubulin (9 and 18). In the same assay, 22 at lower concentration (100 microM) showed the same hypoacetylating effects with both histone and non-histone substrates.

Gene expression profiling changes induced by a novel Gemini Vitamin D derivative during the progression of breast cancer

We investigated gene expression changes induced by a novel Gemini Vitamin D 3 analog, RO-438-3582 (1α,25-dihydroxy-20 S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 ( Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D 3 analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-β systems, differently in the two cell lines. The Gemini Vitamin D 3 analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D 3 analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

Estrogenic activity of dicofol with the human estrogen receptor: Isomer- and enantiomer-specific implications

Dicofol is a non-systemic acaricide/miticide currently registered in the US and Canada for use on a wide variety of crops. This agrochemical has been identified as a potential candidate substance for the United Nations Economic Commission for Europe (UN-ECE) Persistent Organic Pollutant (POP) Protocol and implicated as a potential “endocrine disrupting compound”. The technical product is usually synthesized from technical DDT and consists of approximately 80% and 20% of p, p′- and o, p′-dicofol isomers. The o, p′-substituted isomer of dicofol is chiral and may have enantiomer-specific activity; however, the stereospecific activity of o, p′-dicofol has not been reported. In this study, we examined the isomer- and enantiomer-specific endocrine disruption potential of dicofol using yeast-based steroid hormone receptor gene transcription assay designed with the human estrogen receptor (hER). Estrogenic activity of (+)-17-β estradiol (positive control), p, p′-dicofol, racemic o, p′-dicofol [(±)- o, p′-dicofol] and the individual o, p′-dicofol enantiomers was measured via quantification of β-galactosidase. The (±)- o, p′- and p, p′-dicofol were weak estrogen mimics (EC 50: 4.2 × 10 −6 and 1.6 × 10 −6 M, respectively) relative to estradiol (3.7 × 10 −10 M). For o, p′-dicofol, the β-galactosidase induction by (−)- o, p′-dicofol (EC 50: 5.1 × 10 −7 M) was greater than the racemic mixture. However, the (+)- o, p′-dicofol enantiomer was found to have negligible estrogenic activity. These data indicate that dicofol is a weak hER agonist due to activity of the achiral p, p′-isomer and (−)- o, p′-substituted enantiomer and emphasizes the influence of chemical structure and configuration on biological responses to exposure from chiral compounds.

Enhanced Coactivator Binding and Transcriptional Activation of Mutant Vitamin D Receptors From Patients With Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets by Phosphorylation and Vitamin D Analogs

In this study, we report that the function of certain mutant VDRs from patients with hereditary HVDRR can at least be partially restored by phosphorylation and hexafluoro 1,25(OH)2D3 analogs. Our study provides new insights into mechanisms involved in enhancement of mutant VDR function. 1,25-Dihydroxyvitamin D-resistant rickets (HVDRR) is a rare genetic disorder caused by inactivating mutations in the vitamin D receptor (VDR). In this study, we examined VDR from patients with HVDRR having mutations in the ligand-binding domain (F251C, I268T, H305Q, E420K). We examined methods of restoring transcriptional activity of these mutants and the mechanisms involved. Reporter gene transcriptional assays were used to examine the activation of mutant VDRs. Western-blot analysis, glutathione S-transferase (GST) pull-down assays, and chromatin immunoprecipitation (ChIP) assays were also used in this study. Using mutant VDRs, H305Q, F251C, I268T, and 10(-8) M 1,25(OH)2D3, only 10-30% of the activity of wildtype (WT) VDR in activating 24(OH)ase transcription was observed. The transcriptional response of mutant VDR mutants was significantly enhanced 2- to 3-fold by co-treatment of VDR mutant transfected COS-7 cells with 1,25(OH)2D3 and okadaic acid (OA; inhibitor of phosphatase; 50 nM). The H305Q mutant was the most responsive (90% of the response exhibited by WT VDR was restored). The E420K mutant was unresponsive to 1,25(OH)2D3 in the presence or absence of OA. The increased transcriptional response correlated with an increase in the interaction between DRIP205 and the mutant VDR. We further provide evidence that OA induces the phosphorylation of CREB-binding protein (CBP), indicating for the first time a correlation between phosphorylation of CBP and enhanced VDR function. Hexafluoro 1,25(OH)2D3 analogs (RO-26-2198 and RO-4383561) also resulted in at least a partial restoration of the transcriptional responsiveness of mutant VDRs I268T, F251C, and H305Q. Our data indicate that the enhanced potency of the hexafluoro analogs may be caused by increased DRIP205 and glucocorticoid receptor interacting protein 1 (GRIP-1) binding to VDRs and enhanced association of VDRs with DNA, as suggested by results of ChIP assays. Our study provides new insights into the mechanisms involved in the enhancement of VDR function by both phosphorylation and hexafluoro analogs and forms a basis for future study of vitamin D analogs or specifically designed kinase activity mediators as potential therapy for the treatment of selected patients with HVDRR.

Isocoumarins as estrogen receptor beta selective ligands: Isomers of isoflavone phytoestrogens and their metabolites

In a search for new ligands selective for the estrogen receptor beta (ERβ), we prepared a series of non-steroidal compounds having an isocoumarin core structure. An interesting feature of these derivatives is that they bear the same functionalities as the well-known ERβ -selective, isoflavone phytoestrogens daidzein and genistein, but in an isomeric arrangement. These compounds could be prepared efficiently by electrophilic cyclization of acetylenic ester precursors, followed by simple manipulations to introduce additional substituents. Through a reduction of some of the isocoumarins, we also obtained isomeric analogs of the isoflavone metabolites equol and dehydroequol. The compounds we prepared were evaluated in ER binding assays, and selected compounds were studied further in cell-based gene transcription assays. Several of the isocoumarins and their analogs are high-affinity ligands that show considerable selectivity for ERβ in terms of binding affinity, and strikingly high ERβ selectivity in terms of potency in gene transcription assays. Two of the best compounds, which combine high transcriptional potency with an ERβ selectivity greater than 1000, should prove to be excellent probes of ERβ function in vivo.

Growth Suppression of Ovarian Cancer Xenografts in Nude Mice by Vitamin D Analogue EB1089

The poor response of advanced epithelial ovarian cancer to current treatments necessitates the development of alternative therapeutic strategies. Inhibition of cancer growth by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compounds represents an innovative approach for cancer therapy. The current study evaluated the therapeutic potential of a synthetic 1,25(OH)2D3 analogue EB1089 in the treatment of ovarian cancer. The response of human ovarian cancer cells to 1,25(OH)2D3 and EB1089 were first compared in cell growth, gene transcription, and apoptotic assays. Then, nude mice bearing OVCAR3 tumor xenografts were treated with EB1089 at different dosages, and tumor volumes were monitored. The effect of EB1089 and 1,25(OH)2D3 on the level of serum calcium was also examined. After the treatment, tumors were excised and processed for histologic examination, Ki-67 staining, and tissue terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays to evaluate the morphologic, proliferative, and apoptotic changes induced by EB1089, respectively. The study shows that EB1089 suppresses the in vitro growth of ovarian cancer cells and transcriptionally activates the GADD45 reporter gene more effectively than 1,25(OH)2D3. Clinically more importantly, EB1089 suppresses the growth of OVCAR3 tumor xenografts in nude mice without inducing hypercalcemia. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis contribute to the EB1089-induced tumor suppression in vivo. This study is the first demonstration that ovarian cancer responds positively in vivo to treatment with a 1,25(OH)2D3 compound and thus supports continued development of 1,25(OH)2D3 analogues for possible use as an alternative or complementary therapy for human ovarian cancer.

HOXB13 homeodomain protein suppresses the growth of prostate cancer cells by the negative regulation of T-cell factor 4. Jung C, Kim RS, Lee SJ, Wang C, Jeng MH, Department of Urology, Walther Oncology Center, Indiana University, Indianapolis, IN.: Cancer Res 2004;64:3046–51

In prostate gland, HOXB13 is highly expressed from the embryonic stages to adulthood. However, the function of HOXB13 in normal cell growth and tumorigenesis is not yet known. We investigated the role of HOXB13 and mechanism by which it functions in HOXB13-negative cells. Expression of HOXB13 was forced in HOXB13-negative PC3 prostate cancer cells using a liposome-mediated gene transfer approach. Compared with the control clones, HOXB13-expressing PC3 cells exhibited significant inhibition of in vitro and in vivo cell growth with G1 cell cycle arrest mediated by the suppression of cyclin D1 expression. Because cyclin D1 is mainly regulated by beta-catenin/T-cell factor (TCF), TCF-4 response element was used in a reporter gene transcription assay, demonstrating that HOXB13 significantly inhibits TCF-4-mediated transcriptional activity in both prostate and nonprostate cells. This inhibition occurred in a dose-responsive manner and was specific to TCF-4 response element. Western blot analysis demonstrated that HOXB13 down-regulates the expression of TCF-4 and its responsive genes, c-myc and cyclin D1. HOXB13 also suppressed the activity of natural c-myc promoter. This study suggests that HOXB13, a transcription factor, functions as a cell growth suppressor by negatively regulating the expression of TCF-4, which eventually provides negative signals for cell proliferation. This observation will provide valuable insight into the molecular basis of prostate tumorigenesis.

HOXB13 homeodomain protein suppresses the growth of prostate cancer cells by the negative regulation of T-cell factor 4. Jung C, Kim RS, Lee SJ, Wang C, Jeng MH, Department of Urology, Walther Oncology Center, Indiana University, Indianapolis, IN.Cancer Res 2004;64:3046–51

In prostate gland, HOXB13 is highly expressed from the embryonic stages to adulthood. However, the function of HOXB13 in normal cell growth and tumorigenesis is not yet known. We investigated the role of HOXB13 and mechanism by which it functions in HOXB13-negative cells. Expression of HOXB13 was forced in HOXB13-negative PC3 prostate cancer cells using a liposome-mediated gene transfer approach. Compared with the control clones, HOXB13-expressing PC3 cells exhibited significant inhibition of in vitro and in vivo cell growth with G1 cell cycle arrest mediated by the suppression of cyclin D1 expression. Because cyclin D1 is mainly regulated by beta-catenin/T-cell factor (TCF), TCF-4 response element was used in a reporter gene transcription assay, demonstrating that HOXB13 significantly inhibits TCF-4-mediated transcriptional activity in both prostate and nonprostate cells. This inhibition occurred in a dose-responsive manner and was specific to TCF-4 response element. Western blot analysis demonstrated that HOXB13 down-regulates the expression of TCF-4 and its responsive genes, c-myc and cyclin D1. HOXB13 also suppressed the activity of natural c-myc promoter. This study suggests that HOXB13, a transcription factor, functions as a cell growth suppressor by negatively regulating the expression of TCF-4, which eventually provides negative signals for cell proliferation. This observation will provide valuable insight into the molecular basis of prostate tumorigenesis.

HOXB13 homeodomain protein suppresses the growth of prostate cancer cells by the negative regulation of T-cell factor 4.

In prostate gland, HOXB13 is highly expressed from the embryonic stages to adulthood. However, the function of HOXB13 in normal cell growth and tumorigenesis is not yet known. We investigated the role of HOXB13 and mechanism by which it functions in HOXB13-negative cells. Expression of HOXB13 was forced in HOXB13-negative PC3 prostate cancer cells using a liposome-mediated gene transfer approach. Compared with the control clones, HOXB13-expressing PC3 cells exhibited significant inhibition of in vitro and in vivo cell growth with G1 cell cycle arrest mediated by the suppression of cyclin D1 expression. Because cyclin D1 is mainly regulated by beta-catenin/T-cell factor (TCF), TCF-4 response element was used in a reporter gene transcription assay, demonstrating that HOXB13 significantly inhibits TCF-4-mediated transcriptional activity in both prostate and nonprostate cells. This inhibition occurred in a dose-responsive manner and was specific to TCF-4 response element. Western blot analysis demonstrated that HOXB13 down-regulates the expression of TCF-4 and its responsive genes, c-myc and cyclin D1. HOXB13 also suppressed the activity of natural c-myc promoter. This study suggests that HOXB13, a transcription factor, functions as a cell growth suppressor by negatively regulating the expression of TCF-4, which eventually provides negative signals for cell proliferation. This observation will provide valuable insight into the molecular basis of prostate tumorigenesis.

NF-kappaB induction by bisperoxovanadium compounds requires CD45, p36(LAT), PKC, and IKK activity and exhibits kinetics of activation comparable to those of TCR/CD28 coengagement.

We have previously shown that bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors can potently activate NF-kappaB. We have already determined that p56(lck), ZAP-70, SLP-76, capacitative entry of calcium, and calcium-regulated effectors are important in bpV-induced NF-kappaB activation. In this study, we evaluated whether other signal transducers previously reported in NF-kappaB induction by T cell activating stimuli are also activated by bpV compounds. Nuclear translocation of NF-kappaB was evaluated in cell lines deficient for either CD45 or p36(LAT) to assess the role of these signal transducers in bpV-mediated NF-kappaB activation. A deficiency of either protein greatly reduced the extent of NF-kappaB nuclear translocation following bpV treatment. Isoform-specific PKC inhibitors were then used to show that bpV compounds activate NF-kappaB through both calcium-sensitive and -insensitive PKC isoforms. The implication of the IkappaB-kinase complex was then investigated through the use of an IkappaBalpha-specific kinase assay and plasmids expressing catalytically inactive forms of IKKalpha and IKKbeta. Upstream kinases involved in IKK complex activation such as TPL-2/COT, NIK, and IKKepsilon were also shown to play an important role in bpV-mediated NF-kappaB activation. Finally, reporter gene transcriptional assays and gel shift assays were performed to compare the kinetics of activation of NF-kappaB by bpV with those of antigenic and TNFalpha stimulation. We demonstrate, both in Jurkat cells and in primary T cells, that bpV-mediated NF-kappaB activation kinetics are comparable to those of an antigenic stimulation but occur much slower than the kinetics seen upon TNFalpha treatment.

Antiestrogenic and antiprogesteronic activity of tire extracts with yeast-based steroid hormone receptor gene transcription assay.

Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater & Biotechnol, Wuhan 430072, Peoples R China; Chinese Acad Sci, Res Ctr Ecoenvironm Sci, Lab Environm Chem & Ecotoxicol, Beijing 100085, Peoples R China; GSF, Natl Res Ctr Environm & Hlth, Inst Ecol Chem, D-85764 Neuherberg, Germany

Comparison of antiandrogenic activities of vinclozolin and d,l-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo

A chemical substance used as a photoinitiator for light-cure resin compositions, d,l-camphorquinone (CQN) was found to be weakly antiandrogenic in vitro. It competitively antagonized dihydrotestosterone (DHT)-induced transcriptional activity on the yeast-based androgen receptor gene transcription assay (YAA). Antiandrogenic activity of CQN was shown at higher concentration than 10 −4 M while the well-known antiandrogen, vinclozolin (VCZ) showed the activity at concentrations of 10 −6 M and above in YAA. The antiandrogenic activity of CQN was reconfirmed in the human cell line-based androgen receptor gene transcription assay (HCAA). To determine whether CQN affect male reproductive development, CQN or VCZ was administered to pregnant mice daily from gestational days 10 to 18 by gavage. In utero exposure to VCZ at 100 mg/kg/day caused a significant decrease in anogenital distance (AGD) of F1 neonates and reduced spermatogenesis in F1 males at 42 days of age. In contrast, maternal doses (100 and 300 mg/kg/day) of CQN had no affect on these endpoints in F1 offspring. Further, VCZ or CQN had no adverse affect on F1 male fertility. From these observations, CQN is potentially antagonistic to androgen receptor (AR) in vitro, but is estimated to be less antiandrogenic in vivo when it is administered to pregnant mice by gavage. Furthermore, these findings are the first to demonstrate that VCZ exerts significant antiandrogenic effects on reproductive tract development during gestation in mice.