CArG Elements Research Articles

Construction of artificial promoters sensitively responsive to sonication in vitro.

To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro. The binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay. Of 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm(2) for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication. A promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

MRTF-A transactivates COMT gene and decreases the anti-tumor effects of tamoxifen

Myocardin-related transcription factors A (MRTF-A) is a myocardin-related transcription factor that have been found strongly activated CarG box–containing genes through its direct binding to serum response factor (SRF). In the present study, the MRTF-A ex-pression vector was constructed. The MTT assay showed that transfection of MRTF-A could significantly decrease the anti-tumor effect of tamoxifen on MCF-7 breast cancer cells. The bioinformatics analysis found that the CarG element existed in the pro-moter region of COMT gene of many familiar verte-brates, including of human, rhesus macaque, chimpanzee, etc. The results of RT-PCR assay further showed that MRTF-A could enhance the transcrip-tion level of COMT. These results are the first to indicate that COMT might be a target gene which could be regulated by MRTF-A/SRF, and such transactivation event might be involved in the process of tamoxifen resistance.

Myocardin Is Sufficient for a Smooth Muscle-Like Contractile Phenotype

Myocardin (Myocd) is a strong coactivator that binds the serum response factor (SRF) transcription factor over CArG elements embedded within smooth muscle cell (SMC) and cardiac muscle cyto-contractile genes. Here, we sought to ascertain whether Myocd-mediated gene expression confers a structural and physiological cardiac or SMC phenotype. Adenoviral-mediated expression of Myocd in the BC(3)H1 cell line induces cardiac and SMC genes while suppressing both skeletal muscle markers and cell growth. Immunofluorescence microscopy shows that SRF and a SMC-like cyto-contractile apparatus are elevated with Myocd overexpression. A short hairpin RNA to Srf impairs BC(3)H1 cyto-architecture; however, cotransduction with Myocd results in complete restoration of the cyto-architecture. Electron microscopic studies demonstrate a SMC ultrastructural phenotype with no evidence for cardiac sarcomerogenesis. Biochemical and time-lapsed videomicroscopy assays reveal clear evidence for Myocd-induced SMC-like contraction. Myocd is sufficient for the establishment of a SMC-like contractile phenotype.

Serum response factor neutralizes Purα- and Purβ-mediated repression of the fetal vascular smooth muscle α-actin gene in stressed adult cardiomyocytes

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

A Cysteine-rich LIM-only Protein Mediates Regulation of Smooth Muscle-specific Gene Expression by cGMP-dependent Protein Kinase

Vascular smooth muscle cells (VSMCs) undergo phenotypic modulation, changing from a differentiated, contractile to a de-differentiated, synthetic phenotype; the change is associated with decreased expression of smooth muscle (SM)-specific genes and loss of cGMP-dependent protein kinase (PKG), but transfection of PKG into de-differentiated VSMCs restores SM-specific gene expression. We show that small interference RNA-mediated down-regulation or pharmacologic inhibition of PKG reduced SM-specific gene expression in differentiated VSMCs and provide a mechanism for cGMP/PKG regulation of SM-specific genes involving the cysteine-rich LIM-only protein CRP4. PKG associated with CRP4 and phosphorylated the protein in intact cells. CRP4 had no intrinsic transcriptional activity, but exhibited adaptor function, because it acted synergistically with serum response factor (SRF) and GATA6 to activate the SM-alpha-actin promoter. cGMP stimulation of the promoter required PKG and CRP4 co-expression with SRF and GATA6. A phosphorylation-deficient mutant CRP4 and a CRP4 deletion mutant deficient in PKG binding did not support cGMP/PKG stimulation of the SM-alpha-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-alpha-actin promoter CArG (CC(AT)(6)GG) element. CRP4 and SRF associated with CArG elements of endogenous SM-specific genes in intact chromatin. Small interference RNA-mediated down-regulation of CRP4 prevented the positive effects of cGMP/PKG on SM-specific gene expression. In the presence of CRP4, cGMP/PKG increased SRF- and GATA6-dependent expression of endogenous SM-specific genes in pluripotent 10T1/2 cells. Thus, CRP4 mediates cGMP/PKG stimulation of SM-specific gene expression, and PKG plays an important role in regulating the phenotype of VSMCs.

Abstract 985: BAC Transgenic Analysis of the Smooth Muscle Calponin Gene

Regulatory elements controlling gene expression are often found in separate, sometimes remote, regions around gene loci. Artificial chromosomes offer a means to capture all regulatory elements for study of gene regulation in a near-correct genomic context. The smooth muscle calponin gene (CNN1) encodes for a multifunctional protein involved in signaling, contractile force generation, and growth regulation. While CNN1’s physiology has been studied extensively, its transcriptional regulation has proven to be intractable to conventional in vivo assays. Four evolutionarily-conserved serum response factor (SRF) binding sites (called CArG boxes) are present in the first intron of CNN1 and appear to be required for full transcriptional competence of CNN1 in cultured smooth muscle cells (SMC). To assess the functionality of CNN1 CArG elements in vivo , we exploited a 103-kb bacterial artificial chromosome (BAC) containing the human CNN1 locus shown previously to completely recapitulate endogenous mouse CNN1 expression in vivo . We now show similar SMC-restricted expression of human CNN1 from only 38-kb of BAC sequence. BAC recombineering studies in which the first intron (containing four CArG elements) of CNN1 is replaced with a bacterial selection gene ( galK ) reveal complete loss of human CNN1 expression in vascular SMC, with residual expression in some visceral SMC-containing tissues such as bladder. Transgenic mice carrying the 38-kb BAC with point mutations in all four intronic CArG elements show similar findings suggesting that SRF directly mediates in vivo expression of CNN1 primarily through intronic CArG elements. Bioinformatic, luciferase, and ChIP assays reveal the presence of distal, conserved CArG elements, including a previously unrecognized one located more than 3-kb upstream of the CNN1 transcription start site. The latter CArG-like element is responsive to the SRF coactivator myocardin and is essential for full CNN1 promoter activity in vitro . Moreover, this distal CArG-like element is embedded in a cis regulatory module with conserved binding sites to other distinct transcription factors. We propose that full in vivo CNN1 gene expression requires multiple elements, including five CArG boxes, spread over 14-kb of genomic sequence.

2007 Russell Ross Memorial Lectureship in Vascular Biology—Epigenetic Control of Vascular Smooth Muscle Differentiation in Development and Disease

There is clear evidence that alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the pathogenesis of a number of major human diseases, including atherosclerosis and postan-gioplasty restenosis. This process is referred to as “phenotypic switching” and likely evolved to promote repair of vascular injury. However, the mechanisms controlling phenotypic switching as well as normal differentiation of SMCs in vivo are poorly understood. This talk will provide an overview of molecular mechanisms that control differentiation of SMCs during vascular development. A particular focus will be to consider the role of CArG elements found within the promoters of many SMC differentiation marker genes, as well as regulation of their activity by serum response factor and the potent SMC-selective serum response factor coactivator myocardin. In addition, I will summarize recent work in our laboratory showing that SMC- and gene-locus–selective changes in chromatin structure play a critical role both in normal control of SMC differentiation and in phenotypic switching in response to vascular injury. Finally, I will present evidence based on conditional knockout experiments in mice showing that krupple-like factor 4 is induced in SMCs after vascular injury and regulates SMC phenotypic switching and growth through: binding to G/C repressor elements located in close proximity of CArG elements within the promoters of many SMC marker genes, suppressing expression of myocardin, and inducing epigenetic modifications of SMC marker gene loci associated with chromatin condensation and transcriptional silencing. Supported by NIH grants P01 HL19242, R37 HL57353, and R01 HL 38854.

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis.

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.

Regulation of Ncx1 Gene Expression in the Normal and Hypertrophic Heart

The Na+/Ca2+ exchanger (NCX1) is crucial in the regulation of [Ca2+]i in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy, ischemia, and failure. This upregulation can have an effect on Ca2+ transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that regulate the exchanger during development, growth, and hypertrophy. The Ncx1 gene is upregulated in response to alpha-adrenergic stimulation. We have shown that this is via p38alpha activation of transcription factors binding to the Ncx1 promotor at the -80 CArG element. Interestingly, most of the elements, including the CArG element, which we have demonstrated to be important for regulation of Ncx1 expression are in the proximal 184 bp of the promotor. Using a transgenic mouse, we have shown that the proximal 184 bp is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for upregulation in response to pressure overload.

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22alpha promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22alpha promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22alpha transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22alpha transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells.

Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells

Smooth muscle cell (SMC) differentiation is an essential component of vascular development and these cells perform biosynthetic, proliferative, and contractile roles in the vessel wall. SMCs are not terminally differentiated and possess the ability to modulate their phenotype in response to changing local environmental cues. The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms involved in controlling phenotypic switching of SMC with particular focus on examination of processes that contribute to the repression of SMC marker genes. We discuss the environmental cues which actively regulate SMC phenotypic switching, such as platelet-derived growth factor-BB, as well as several important regulatory mechanisms required for suppressing expression of SMC-specific/selective marker genes in vivo, including those dependent on conserved G/C-repressive elements, and/or highly conserved degenerate CArG elements found in the promoters of many of these marker genes. Finally, we present evidence indicating that SMC phenotypic switching involves multiple active repressor pathways, including Krüppel-like zinc finger type 4, HERP, and ERK-dependent phosphorylation of Elk-1 that act in a complementary fashion.

New Insights to Vascular Smooth Muscle Cell and Pericyte Differentiation of Mouse Embryonic Stem Cells In Vitro

The molecular mechanisms that regulate pericyte differentiation are not well understood, partly because of the lack of well-characterized in vitro systems that model this process. In this article, we develop a mouse embryonic stem (ES) cell-based angiogenesis/vasculogenesis assay and characterize the system for vascular smooth muscle cell (VSMC) and pericyte differentiation. ES cells that were cultured for 5 days on OP9 stroma cells upregulated their transcription of VSMC and pericyte selective genes. Other SMC marker genes were induced at a later time point, which suggests that vascular SMC/pericyte genes are regulated by a separate mechanism. Moreover, sequence analysis failed to identify any conserved CArG elements in the vascular SMC and pericyte gene promoters, which indicates that serum response factor is not involved in their regulation. Gleevec, a tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF) spell-receptor signaling, and a neutralizing antibody against transforming growth factor (TGF) beta1, beta2, and beta3 failed to inhibit the induction of vascular SMC/pericyte genes. Finally, ES-derived vascular sprouts recruited cocultured MEF cells to pericyte-typical locations. The recruited cells activated expression of a VSMC- and pericyte-specific reporter gene. We conclude that OP9 stroma cells induce pericyte differentiation of cocultured mouse ES cells. The induction of pericyte marker genes is temporally separated from the induction of SMC genes and does not require platelet-derived growth factor B or TGFbeta1 signaling.

Angiotensin II induced reactive oxygen species modulate smooth muscle α‐actin gene expression via increased SRF binding to CArG cis elements in SHR renal microvascular smooth muscle cells

We tested the hypothesis that Superoxide (O2·−) mediates Ang II-dependent increase in expression of smooth muscle α-actin (SMα-actin) gene in cultured renal preglomerular microvascular smooth muscle cells (PGSMCs) from SHR. O2·− quantified by lucigenin method was significantly increased by 85% in intact PGSMCs and by 47% in cell membranes at 12 hr after incubation with Ang II (1uM). Expression of SMα-actin mRNA determined by real time RT-PCR was enhanced by 81% by Ang II and inhibited by 50% in Ang II-treated cells by the thiol antioxidant, N-acetyl-L-cysteine (NAC, 20mM) which also reduced Ang II-induced O2·− generation to 20%. Serum response factor (SRF) and its co-activator, myocardin (Myo) mediate CArG-dependent transcription of SMα-actin gene. Although Ang II treatment did not modify mRNA for SRF, it increased Myo expression by 22%. Promoter repoter assays indicated that Ang II increased SMα-actin promoter activity by 2.3 fold and this was inhibited by 43% by NAC. Site-directed mutations demonstrated that CArG elements are required for SMα-actin promoter activity. Quantitative chromatin immunoprecipitation assay revealed that Ang II increased SRF enrichment of CArG regions in SMα-actin promoter in intact chromatin by 57%, whereas NAC reduced it by 43% without affecting histone 3 (H3) acetylation or histone 3 lysine-9 (H3K9) dimethylation. In conclution; Ang II induces O2·− in SHR PGSMCs that increases SMα-actin gene expression via up-regulation of Myo and enhanced SRF bound to CArG elements without modifications of H3 acetylation and H3K9 methylation in the SMα-actin promoter regions.

Increase in the Expression of an Alpha-Amylase Gene and Sugar Accumulation Induced during Cold Period Reflects Shoot Elongation in Hyacinth Bulbs

The aim of this study was to investigate physiological and biochemical mechanisms of shoot elongation after cold period in hyacinth (Hyacinthus orientalis L. cv. Delft Blue). Hyacinth shoot rapidly elongated during hydro-culture period in cooled bulbs, but not in non-cooled bulbs. Alpha-amylase (EC 3.2.1.1.) is a key enzyme involved in starch hydrolysis. Alpha-amylase activity increased during the cold storage period and was low during rapid shoot elongation in hyacinth. In the non-cooled bulbs, its activity remained at the similar level. Sucrose content increased during the cold storage period in the shoot, but not in the scales. We, for the first time, isolated cDNA for cold-responsive alpha-amylase gene (HoAmy1A, accession No. AB198975) from hyacinth, and presented that HoAmy1A expression increased in the scale during the cold storage period, but the level was very low during shoot elongation. We also found that promoter region of HoAmy1A contained CArG element, which is related to the response to low temperature. In tulip (Tulipa genesriana L.), the most studied bulbous plant, dramatic increase in alpha-amylase activity and translocation of sugars from the scales to shoot occurred during the growth stage, following cold treatment (Komiyama et al., 1997; Lambrechts et al., 1994). Our results suggest that there are two types (tulip and hyacinth types) of sprouting mechanisms in bulbous plants.

Molecular pathways for pericyte and mural cell differentiation

Pericytes and mural cells express a vascular smooth muscle cell (SMC) phenotype in response to endothelial cell-derived factors. Most SMC target genes depend upon serum response factor (SRF) binding to CArG box elements. Recent studies emphasize the importance of transcriptional coactivators and corepressors in coupling SRF-dependent transcription to environmental cues. Thus myocardin and myocardin-related transcription factors (MRTFs) interact with SRF and strongly stimulate SMC gene expression, whereas cysteine-rich LIM only proteins (CRPs) enhance the binding affinity of SRF for SMC CArG elements. MRTFs and CRPs shuttle between the cytoplasm and nucleus thereby transmiting intracellular signals to SRF. Docking sites on SRF also recruit Ets domain-containing factors, like Elk1, that inhibit SMC gene expression via displacement of myocardin. Furthermore, notch-dependent corepressors can inhibit SMC gene expression by pathways yet to be fully defined. Our studies have focused on mural cell differentiation in coronary vessel formation. We found that sonic hedgehog activates a positive pathway for SMC differentiation, while notch signaling prevents rhoA-GTPase-mediated stimulation of SRF coactivators. These studies suggest that pericyte and mural cell differentiation is controlled by both positive and negative coregulators of SRF-dependent transcription. Supported by HL19242.

Contribution of serum response factor and myocardin to transcriptional regulation of smoothelins

Smoothelin-A and -B isoforms are highly restricted to contractile smooth muscle cells (SMCs). Serum response factor (SRF) and myocardin are essential for contractile SMC differentiation. We evaluated the contribution of SRF/myocardin to transcriptional regulation of smoothelins. Rat vascular SMCs were transfected with smoothelin-A and smoothelin-B promoter reporter constructs and promoter activity was analyzed. The effects of mutations in the smoothelin-A promoter CArG-boxes and co-transfections with a myocardin expression plasmid were assessed. Electrophoretic mobility shift assays and chromatin immunoprecipitations were performed to investigate SRF-binding to the smoothelin-A CArG-boxes. Smoothelin promoter activity was detected in vascular SMCs. Comparative sequence analysis revealed two conserved CArG elements in the smoothelin-A promoter that bind SRF as shown by chromatin immunoprecipitation. The proximal CArG-near bound SRF stronger than CArG-far in gel shift assays. Mutagenesis studies also indicated that CArG-near is more important than CArG-far in regulating smoothelin-A promoter activity. Myocardin augmented smoothelin-A promoter activity 2.5-fold in a CArG-near-dependent manner. In contrast, myocardin had little effect on the smoothelin-B promoter. Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF. Our data show a role for SRF/myocardin in regulating smoothelin-A whereas the higher smoothelin-B expression appears to be SRF/myocardin-independent.

Hypoxia- and radiation-activated Cre/loxP ‘molecular switch’ vectors for gene therapy of cancer

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or 'molecular switch', based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated 'molecular switch' vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.

Cisplatin‐controlled p53 gene therapy for human non‐small cell lung cancer xenografts in athymic nude mice via the CArG elements

Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 (Egr-1) promoter through specific cis-acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53, and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr-p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr-p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr-p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr-p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr-p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC.

Radiation, Hypoxia and Chemotherapy-mediated Gene Therapy and the Potential for Tumour Delivery via Macrophages

Purpose/Objective: Hypoxia is an important prognostic indicator of poor tumour treatment outcome after radio- and chemo-therapy. We have developed suicide gene therapy vectors containing radiation-responsive CArG elements and hypoxia-responsive elements (HREs), which are activated by clinical levels of ionizing radiation and hypoxia. We have successfully shown vector responses to both fractionated X-ray doses of 1–5 Gy and 0–1% O2. Furthermore, we are testing the use of human macrophages for delivery of gene therapy to tumours.

Nuclear Factor of Activated T Cells and Serum Response Factor Cooperatively Regulate the Activity of an α-Actin Intronic Enhancer

Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.