Inhibition Of Gene Transcription Research Articles

Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)

BackgroundGene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3).ResultsThe deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison of the genome sequences of the two strains showed that BL21 possesses two mutations in the promoter region of iclR and rare codons are present in arcA implying a lower tRNA acceptance. Both phenomena presumably result in a reduced ArcA and IclR synthesis in BL21, which contributes to the similar physiology as observed in E. coli K12 ΔarcAΔiclR.ConclusionsThe deletion of arcA results in a decrease of repression on transcription of TCA cycle genes under glucose abundant conditions, without significantly affecting the glyoxylate pathway activity. IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under glucose limitation, Crp is responsible for the high glyoxylate flux, but IclR still represses transcription. Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

Tacrolimus: 10 years of successful clinical application

Tacrolimus (FK506) is the first drug from the new class of drugs - calcineurin inhibitors. It has a great potential and can change the current approaches to treatment of different dermatoses. Topical forms of Tacrolimus are activated due to the effect on T-lymphocyte signal transduction, and they also inhibit gene transcription. As a result, the T-cell response to antigens is alleviated. Percutaneous absorption of Tacrolimus in affection foci is higher than in healthy skin. Due to this a smaller amount of the drug is absorbed by the skin as long as the skin is healed. Systemic absorption of Tacrolimus is low in the course of treatment. The maximum clinical experience has been gained in the field of applying Tacrolimus for treatment of atopic dermatitis. In a number of clinical studies, 0.03-0.3% Tacrolimus ointment efficiently reduced the severity of symptoms and course of atopic dermatitis in adults and children. Moreover, topical treatment with Tacrolimus did not involve any substantial toxic effects. The patients most often complained of local irritations in the area where the ointment was applied, which were transient by nature and did not demand the drug withdrawal. Today Tacrolimus is a valuable and safer alternative to glucocorticosteroids for treatment of atopic dermatitis. It can also be used for treating other dermatoses, which demands additional all-round examination.

Absence of NPM1 promoter hypermethylation in human myelodysplastic syndrome

Npm1+/− heterozygous mice develop a haematological disorder with features resembling human myelodysplastic syndrome (MDS). Promoter hypermethylation of the NPM1 gene may lead to suppressed gene transcription and hence functional haploinsufficiency,...

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

BackgroundAlpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. α-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of α-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken.Methodology and FindingsWe analyzed α-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-α-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic α-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC.ConclusionsMBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer.

Doxorubicin In Vivo Rapidly Alters Expression and Translation of Myocardial Electron Transport Chain Genes, Leads to ATP Loss and Caspase 3 Activation

BackgroundDoxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Methodology/Principal FindingsMice were treated with an acute dose of either doxorubicin (DOX) (15 mg/kg) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (25 mg/kg). DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO). Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.Conclusions/SignificanceThese data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these events probably account for the heart damage.

PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy.

Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs

Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.

Abstract 208: Low-dose actinomycin D decreases Mcl-1 expression and acts synergistically with ABT-737 to induce apoptosis of SCLC cell lines

Abstract SCLC is initially a highly chemotherapy-responsive disease but relapse and progressive development of chemotherapy resistance is the rule. Overexpression of pro-survival members of the Bcl-2 family, including Bcl-2, Bcl-XL and Mcl-1 are thought to play a role in the pathogenesis and chemotherapy resistance of SCLC. ABT-737, a BH3-mimetic drug which blocks the function of Bcl-2 and Bcl-XL, has shown single agent activity in preclinical models of SCLC and enhances the activity of a variety of cytotoxic agents. Low levels of Mcl-1 expression and high levels of Noxa expression have been characterized as markers of sensitivity to ABT-737 in SCLC and other malignancies. We have also noted that Noxa-mediated downregulation of Mcl-1 expression is absent in 4/4 SCLC cell lines examined, suggesting roles for this defect in the pathogenesis of SCLC and in resistance to ABT-737. Because of the short half-life of Mcl-1 mRNA and protein, we studied the effects of combining ABT-737 and actinomycin D, a general transcriptional inhibitor used in the treatment of Wilms tumor, sarcomas, germ cell and trophoblastic tumors. Actinomycin D decreased Mcl-1 expression in a dose-dependent fashion in the low ng/ml range, 3 logs lower than that required for general transcriptional inhibition. While exposure to actinomycin resulted in a cell line-dependent increase in Noxa expression, Noxa was not required for the decrease in Mcl-1 expression, as it occurred in the H209 cell line, which does not express Noxa, as well as the H69, WBA and H526 cell lines which do. Concentrations of actinomycin D from 0.4-4 ng/ml showed single agent activity across a panel of SCLC cell lines in 72 hr MTT viability assays and specifically induced a high grade G2-M cell cycle blockade. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with combination indices in the 0.5 range, indicating synergy by Chou-Talalay analysis, without a significant effect on MRC-5 pulmonary fibroblasts. In addition to loss of Mcl-1 expression and enhanced Noxa expression, correlates of apoptosis induced by the combination included marked Bid cleavage. Exposure to 4 ng/ml actinomycin was only required for the first 24 hrs of the combined incubation but the presence of ABT-737 was required for the full 72 hrs to see maximum loss of viability and clonogenic potential. This actinomycin exposure mimics a clinically achievable AUC of 100 ng.hr/ml after bolus administration, suggesting the feasibility of combination studies utilizing bolus actinomycin D administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 208.

Abstract 4877: Polyamine analogues modulate gene expression by inhibiting KDM1/LSD1 and altering chromatin structure in human breast cancer cells

Abstract Gene expression can be modulated through histone methylation, which is tightly control by the balance of histone methytransferase and demethylase. Methylation (mono-, di- and tri-methylation) of lysine 4 of histone 3 is associated with active gene transcription. Lysine-specific demethylase 1 (KDM1/LSD1) can enzymatically demethylate mono- and di-methyl H3K4 that leads to suppression of gene transcription. Our previous studies demonstrated that a novel class of polyamine analogues can effectively inhibit KDM1/LSD1 and induce re-expression of candidate epigenetically silenced genes. The purpose of this study was to examine the molecular effects of polyamine analogues, 2d and PG11144, on global gene expression profile by inhibition of KDM/LSD1 in estrogen receptor-negative MDA-MB-231 breast cancer cells. The possible mechanisms of polyamine analogues mediating modulation of gene expression were also investigated. Treatment with polyamine analogous, 2d or PG11144, significantly increases global histone 3 dimethylation at lysine 4 (H3K4me2), as well as altering gene expression. There are 274 and 625 genes changed at least 2-fold after treatment with 2d and PG11144 respectively. The genes’ expression levels of twelve 2d up-regulated and fourteen PG11144 up-regulated genes were confirmed by real-time RT-PCR. Chromatin immunoprecipitation analysis demonstrated that up-regulated gene expression by polyamine analogous is associated with up-regulated active histone marks (H3K4me, H3K4me2 and ActH3K9) and down-regulated repressive histone marks (H3K9me2 and H3K27me3) in the promoter regions of the relevant target genes. Supported by NIH CA 88843, CA51085, Breast Cancer Research Foundation, and Komen for the Cure KG080923. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4877.

Abstract 4869: Transcriptional upregulation of histone deacetylase 2 promotes Myc-induced oncogenic effects

Abstract Myc oncoproteins induce cancer cell proliferation by transcriptional modulation. Histone deacetylases (HDACs) enhance cancer cell proliferation and survival, in part, by effects on tumour suppressor gene transcription, and thus HDAC inhibitors are among the most promising new classes of anticancer drugs. Here we show that N-Myc and c-Myc up-regulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Affymetrix microarray revealed that a subset of genes, including cyclin G2 (CCNG2), was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells. PCR analysis confirmed that CCNG2 gene transcription was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. BrdU incorporation assays demonstrated that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc and HDAC2-induced cell proliferation. Dual cross-linking chromatin immunoprecipitation and protein co-immunoprecipitation assays showed that N-Myc acted as a transrepressor, by recruiting the HDAC2 protein to an Sp1-binding site at the CCNG2 core promoter, in a manner distinct from its action as a transactivator. Moreover, HDAC2 was up-regulated, and CCNG2 down-regulated, in pre-cancerous and neuroblastoma tissues from MYCN (N-Myc) transgenic mice, and c-Myc over-expression correlated with up-regulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of up-regulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4869.

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Opposing Effects of Two Tissue Transglutaminase Protein Isoforms in Neuroblastoma Cell Differentiation

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.

Histone acetylation and its role in embryonic stem cell differentiation

The understanding of mechanisms leading to cellular differentiation is the main aim of numerous studies. Accessibility of DNA to transcription factors depends on local chromatin structure and chromatin compaction inhibits gene transcription. Histone acetylation correlates with an open chromatin structure and increased gene expression. Gene transcription levels are changed in early embryonic stem cells differentiation in a tissue-specific manner and epigenetic marks are modified, including increased global acetylation levels. Manipulation of histone deacetylases activity might be an interesting tool to generate populations of specific cell types for transplantation purposes. Thus, this review aims to show recent findings on histone acetylation, a post translational modification and its manipulation in embryonic stem cells differentiation.

Sequential steps underlying neuronal plasticity induced by a transient exposure to gabazine

Periods of intense electrical activity can initiate neuronal plasticity leading to long lasting changes of network properties. By combining multielectrode extracellular recordings with DNA microarrays, we have investigated in rat hippocampal cultures the temporal sequence of events of neuronal plasticity triggered by a transient exposure to the GABA(A) receptor antagonist gabazine (GabT). GabT induced a synchronous bursting pattern of activity. The analysis of electrical activity identified three main phases during neuronal plasticity induced by GabT: (i) immediately after termination of GabT, an early synchronization (E-Sync) of the spontaneous electrical activity appears that progressively decay after 3-6 h. E-Sync is abolished by inhibitors of the ERK1/2 pathway but not by inhibitors of gene transcription; (ii) the evoked response (induced by a single pulse of extracellular electrical stimulation) was maximally potentiated 3-10 h after GabT (M-LTP); and (iii) at 24 h the spontaneous electrical activity became more synchronous (L-Sync). The genome-wide analysis identified three clusters of genes: (i) an early rise of transcription factors (Cluster 1), primarily composed by members of the EGR and Nr4a families, maximally up-regulated 1.5 h after GabT; (ii) a successive up-regulation of some hundred genes, many of which known to be involved in LTP (Cluster 2), 3 h after GabT likely underlying M-LTP. Moreover, in Cluster 2 several genes coding for K(+) channels are down-regulated at 24 h. (iii) Genes in Cluster 3 are up-regulated at 24 h and are involved in cellular homeostasis. This approach allows relating different steps of neuronal plasticity to specific transcriptional profiles.

4-Hydroxytamoxifen inhibits K + currents in mouse ventricular myocytes

Tamoxifen is a widely used chemotherapeutic agent, which has been associated with prolongation of the QT interval. Other studies have reported that acute exposure to tamoxifen can reduce cardiac K + currents. However, in vivo tamoxifen is largely metabolized and most of its activity is attributable to its major metabolite, 4-hydroxytamoxifen (4OH-tamoxifen). Accordingly, in the present study, we performed voltage-clamp experiments to directly investigate the effects of 4OH-tamoxifen on the repolarizing K + currents in adult mouse ventricular myocytes in order to determine whether the effects of tamoxifen on repolarization could be ascribed to 4OH-tamoxifen. K + currents were recorded before and after acute exposure to 4OH-tamoxifen (0.5, 1 and 10 μM). 4OH-tamoxifen reduced the density of the Ca 2+-independent transient outward ( I to), the ultrarapid delayed rectifier ( I Kur) and the inward rectifier ( I K1) K + currents (by up to 43%, 41% and 26%, respectively) but had no significant effect on the steady-state outward K + current ( I ss). Voltage dependence of steady-state inactivation and reactivation time of I to and I Kur were not affected by 4OH-tamoxifen. Experiments using the pure estrogen receptor antagonist, ICI 182,780 and the inhibitor of gene transcription, actinomycin D, were undertaken to assess the involvement of estrogen receptor. Administered alone these compounds did not affect the density of K + currents. Moreover, pretreatment of the cells with ICI 182,780 or actinomycin D did not prevent the inhibitory response to 4OH-tamoxifen. Overall, 4OH-tamoxifen reduced K + currents in mouse ventricle and this effect is unrelated to gene transcription and does not involve interaction of the drug with estrogen receptor.

Emerging treatment combinations: Integrating therapy into clinical practice

To review data supporting the effectiveness of emerging treatment options for metastatic breast cancer. Recent research has focused on several signal-transduction pathways important in the pathogenesis of breast cancer. Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that is involved in cell growth and survival. Everolimus, an orally active inhibitor of mTOR, has demonstrated promising efficacy results and a favorable safety profile in initial studies. Epidermal growth factor receptor (EGFR), a cell-surface molecule that has been implicated in the pathogenesis of breast cancer, may also be important in the emergence of resistance to endocrine therapy. Initial clinical studies have suggested that EGFR inhibitors such as gefitinib may delay the development of resistance to endocrine therapy in patients with breast cancer when given concurrently with tamoxifen or an aromatase inhibitor. Finally, considerable recent research has examined the role of epigenetic gene silencing, in which acetylation or deacetylation of DNA modifies the expression of tumor-suppressing genes. The enzyme histone deacetylase (HDAC) suppresses gene transcription by modifying chromatin into a more compact form. HDAC inhibitors have emerged as a potential new treatment option for several cancer types, including breast cancer. The HDAC inhibitor vorinostat has recently been examined in combination with other treatments, including cytotoxic agents and bevacizumab, for the treatment of breast cancer. In one small Phase I and II study, first-line treatment with the combination of vorinostat, paclitaxel, and bevacizumab produced objective responses (partial or complete) in more than 50% of patients with recurrent or metastatic breast cancer. Although the results of the described studies are promising, randomized controlled clinical trials are needed to better understand the efficacy and safety of emerging treatment options for patients with metastatic breast cancer.

Targeted Knockdown of EGR-1 Inhibits IL-8 Production and IL-8-mediated Invasion of Prostate Cancer Cells through Suppressing EGR-1/NF-κB Synergy

IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.

Cellular stress created by intermediary metabolite imbalances

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway--the D-galactose amphibolic pathway--changes the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.

Synergistic Interplay between Promoter Recognition and CBP/p300 Coactivator Recruitment by FOXO3a

FOXO3a is a transcription factor belonging to the forkhead box O-Class (FOXO) subfamily, and it regulates metabolism, cell-cycle arrest, cell differentiation, and apoptosis through activating or suppressing gene transcription. FOXO3a contains a well-folded DNA-binding forkhead (FH) domain, but a large portion of the remaining protein sequence (75% of the total) is predicted to comprise intrinsically disordered regions (IDRs). Within the IDRs, there are three conserved regions (CR1-CR3), and it has been shown that CR3 (residues D610-N650) is a transactivation domain that recruits the coactivator histone acetyltransferase (HAT) CBP/p300, through binding to its KIX domain. In a previous study, we determined the solution structure of the FH domain and identified an intramolecular interaction between FH and CR3 domains of FOXO3a. Here we illustrate that the KIX domain of CBP interacts with the central core region (L620-A635) of CR3, which also internally interacts with the FH domain. In this heterotypic interplay, FH prevents CR3 from binding to KIX; however, upon binding to the Forkhead response element (FRE) DNA, the FH domain releases the CR3 domain, allowing it to interact with KIX. While previous studies have shown that the transactivation domains of c-Myb and MLL bind to distinct sites on KIX, our results indicate that FOXO3a CR3 has an ability to bind to both of these sites. These results suggest a model of FOXO3a-dependent coactivator recruitment in which the dynamic interplay between KIX and FH domains for binding to CR3 plays a key regulatory role in gene transcription activation.

Glucocortıcoıds: As A Clınıcıan We Use Much, We Know Less

Glucocorticoids (GC) are frequently used important drugs in clinical practice of pediatrics. As well as, they have important physiologic functions; they have immune suppressive, anti-inflammatory and anti-allergic effects that they exert on primary and secondary immune cells, tissues and organs. Their therapeutic effects are considered to be mediated by four different mechanisms of action: the classical genomic mechanism of action caused by the cytosolic glucocorticoid receptor (cGR); secondary non-genomic effects which are also initiated by the cGR; membrane-bound glucocorticoid receptor (mGR)-mediated non-genomic effects and finally non-specific, non-genomic effects caused by interactions with cellular membranes. Mechanisms of GR action are transactivation (GR binds to a related element in the promoter region of GC sensitive genes, inducing gene transcription) and transrepression (GR binds to a negative related element in a promoter region of GC-regulated genes that inhibit gene transcription by interfering with the binding of activating transcription factors). The underlying molecular mechanisms for their side effects are complex and frequently only partly understood. Recent data suggest that certain side effects are predominantly mediated via transactivation (e.g., diabetes, glaucoma), whereas others are predominantly mediated via transrepression (e.g., suppression of the hypothalamic-pituitary-adrenal axis). In this review, we aimed to look at new molecular mechanisms of effects and side effects in relationship with apoptosis and caveolin, intracellular transport and endoplasmic reticulum stress, resistance mechanisms and new drugs developed based on these topics of GCs.