Higher Promoter Activity Research Articles

Genomic organization and structure of the 5′‐flanking region of the TEX101 gene: Alternative promoter usage and splicing generate transcript variants with distinct 5′‐untranslated region

A novel germ cell-specific antigen, TEX101 (TES101-reactive protein), was previously identified using a monoclonal antibody directed against mouse testicular cells. TEX101 is specifically located on the plasma membrane of germ cells, and its expression in gonadal organs is sexually dimorphic. To understand the fundamental mechanism directing gene expression, the genomic organization of TEX101 was studied. The gene consists of five translated exons (exons 2-6) and three 5'-untranslated exons (exon 1a, 1b, and 1c), respectively. TEX101 forms three major transcripts classified by usage of the three 5'-untranslated exons. One form of TEX101 mRNA is transcribed from exon 1c and spliced to the common acceptor site in exon 2. In the second form of the transcript, exon 1a is spliced to exon 1b and exon 2 in a sequential manner. Splicing from exon 1a to exon 2, arises the third form of transcript. Reverse Transcription (RT)-polymerase chain reaction (PCR) analysis demonstrated differential expression pattern of the TEX101 transcripts between testis and ovary. Whereas the expression of transcript-1 is constitutive in male and female gonads, the transcript-2 and -3 are detected only after starting of the spermatogenesis. Luciferase reporter assays using GC-2spd(ts) cells, a cell line from immortalized mouse testicular cells, showed that the 5'-flanking sequence of exon 1c has higher promoter activity than exon 1a. Deletion analysis of the chimeric structures indicated that sequences essential to gene expression are present on the 5'-flanking region between -3186 and +14, where the cluster of five CAAT boxes is located. Taken together, these findings should facilitate an understanding of the regulation of TEX101 expression during gametogenesis.

Involvement of AGO1 and AGO2 in mammalian transcriptional silencing

Duplex RNAs complementary to messenger RNA inhibit translation in mammalian cells by RNA interference (RNAi). Studies have reported that RNAs complementary to promoter DNA also inhibit gene expression. Here we show that the human homologs of Argonaute-1 (AGO1) and Argonaute-2 (AGO2) link the silencing pathways that target mRNA with pathways mediating recognition of DNA. We find that synthetic antigene RNAs (agRNAs) complementary to transcription start sites or more upstream regions of gene promoters inhibit gene transcription. This silencing occurs in the nucleus, requires high promoter activity and does not necessarily require histone modification. AGO1 and AGO2 associate with promoter DNA in cells treated with agRNAs, and inhibiting expression of AGO1 or AGO2 reverses transcriptional and post-transcriptional silencing. Our data indicate key linkages and important mechanistic distinctions between transcriptional and post-transcriptional silencing pathways in mammalian cells.

B29 Gene Silencing in Pituitary Cells Is Regulated by Its 3′ Enhancer

B cell-specific B29 ( Igβ, CD79b) genes in rat, mouse, and human are situated between the 5′ growth hormone (GH) locus control region and the 3′ GH gene cluster. The entire GH genomic region is DNase 1 hypersensitive in GH-expressing pituitary cells, which predicts an “open” chromatin configuration, and yet B29 is not expressed. The B29 promoter and enhancers exhibit histone deacetylation in pituitary cells, but histone deacetylase inhibition failed to activate B29 expression. The B29 promoter and a 3′ enhancer showed local dense DNA methylation in both pituitary and non-lymphoid cells consistent with gene silencing. However, DNA methyltransferase inhibition did not activate B29 expression either. B29 promoter constructs were minimally activated in transfected pituitary cells. Co-transfection of the B cell-specific octamer transcriptional co-activator Bob1 with the B29 promoter construct resulted in high level promoter activity in pituitary cells comparable to B29 promoter activity in transfected B cells. Unexpectedly, inclusion of the B29 3′ enhancer in B29 promoter constructs strongly inhibited B29 transcriptional activity even when pituitary cells were co-transfected with Bob1. Both Oct-1 and Pit-1 bind the B29 3′ enhancer in in vitro electrophoretic mobility shift assay and in in vivo chromatin immunoprecipitation analyses. These data indicate that the GH locus -embedded, tissue-specific B29 gene is silenced in GH-expressing pituitary cells by epigenetic mechanisms, the lack of a B cell-specific transcription factor, and likely by the B29 3′ enhancer acting as a powerful silencer in a context and tissue-specific manner.

Metal dependence and intracellular regulation of the bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803

Investigating the regulation of the cyanobacterial bidirectional hydrogenase on the molecular level is a prerequisite to learn more about the details of its function and to enhance biohydrogen production. Several deletion mutants of histidine kinase genes ( hik) of Synechocystis were analysed concerning their hydrogenase activity. hik7 showed no hydrogenase activity at all. It is supposed that the decreased activity is due to an improper processing of the enzyme. Furthermore, the effect of iron deficiency on the bidirectional hydrogenase was investigated on the level of transcription and enzyme activity. Low iron supply elicited low hydrogenase activity but a high promoter activity. Genetic and phenotypic differences have been repeatedly described for different wild-type (WT) strains of Synechocystis. Here, we report the identification of a substrain that has a severely reduced hydrogenase activity compared to the strain used throughout this study. The hydrogenase activity of the substrain was found to be partly complemented by the addition of nickel. Thus, revealing an inhibited nickel uptake into the cell or insertion into the hydrogenase. It emphasizes the importance of the right choice of WT strain for investigations concerning the hydrogen production capacities.

Hunchback sequence binding protein suppresses mouse TGF-β3 promoter in vitro

Transforming growth factor-β3 (TGF-β3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-β3 5′-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5′-deletion encompassing DNA sequences from −2297 to −1003 bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the −742 to 104 bp regions, suggesting both positive and negative transcriptional regulation in the TGF-β3 promoter. The fragment containing 1.8 kb had the highest luciferase activity. Characterization of this 1.8 kb 5′-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-β3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-β3 gene transcriptional regulator and may be expressed in a cell type-specific manner.

The association of cytokine gene polymorphisms with febrile non‐hemolytic transfusion reaction in multitransfused patients

Cytokines are associated with inflammatory responses including febrile non-hemolytic transfusion reactions (FNHTR). Moreover, there are some polymorphisms of these cytokine genes associated with different levels of gene expression. The aim of the present study was to investigate the association of inflammatory cytokine gene polymorphisms with the occurrence of FNHTR in multitransfused patients. We studied two groups of transfused patients: one presenting FNHTR before 20 transfusions of red blood cells concentrates and the other which never presented FNHTR even after 20 transfusions. The gene polymorphisms studied were IL1B-511C/T and +3953C/T, IL1RN (intron 2, variable number tandem repeat), IL6-174G/C, IL10-1082G/A and -819C/T, TNF-308G/A and LTA+253G/A using polymerase chain reaction and restriction digestion or sequencing methods. An association of IL1RN*2.2 genotype with the occurrence of precocious FNHTR (P < 0.025) was detected. This allele and this genotype have been related with higher serum levels of interleukin (IL)-1beta in vivo and higher promoter activity. No other association was demonstrated. The association of gene polymorphisms related with the increase of inflammatory cytokine gene expression may be a relevant factor in FNHTR and requires confirmation.

1141584386 Cell type specific regulation of human placenta growth factor

Background: Maternal serum levels of PlGF are significantly lower in preeclamptic patients than in normal pregnant women. PlGF mRNA expression is cell type specific with high levels in trophoblast but comparatively low levels in most nontrophoblast. Despite two consensus hypoxia response elements (HREs), PlGF expression is down regulated by hypoxia in trophoblast, but increased in other cell types. These observations suggest unique regulatory mechanisms control PlGF expression. We sought to characterize PlGF promoter regions which function to regulate transcription rates in different cell types and under hypoxia.Methods: Regions of human PlGF gene (−1521/+34), (−828/+34), (−698/+34) were cloned into a reporter vector and (−844/−677) was cloned into a minimal TK promoter containing expression vectors. Trophoblast (JEG3 and JAR), and nontrophoblast (HeLa and hEK‐293) were transiently transfected to determine functional responsiveness of the regions. A PlGF gene region containing two HREs was cloned upstream of a TK containing vector to determine hypoxia (1% O2) responsiveness.Results: PlGF (−1521/+34), (−828/+34) regions showed high promoter activity in trophoblast cells, but little activity in nontrophoblast cells. PlGF (−844/−677) cloned downstream of the TK reporter, augmented promoter activity in trophoblast but not nontrophoblast cells. When cloned upstream to the TK promoter, this region significantly decreased activity in nontrophoblast cells. Surprisingly, the PlGF region containing HREs produced little increase in minimal promoter activity during hypoxia in any of the cells.Conclusions: PlGF transcription is cell type specific and a region of PlGF (−828/−698) may act as an enhancer in trophoblast yet mediates repressor activity in nontrophoblast. The putative HREs in PlGF 5’UTR do not seem to increase PlGF transcription under hypoxia suggesting that they are not essential for governing PlGF expression. Further understanding of transcriptional regulation of PlGF in trophoblast may facilitate approaches to increase expression during preeclampsia.

ACBP – a PPAR and SREBP modulated housekeeping gene

The acyl-CoA binding protein (ACBP) is a 10 kD intracellular lipid binding protein that binds and transports acyl-CoA esters. The protein is expressed in most cell types at low levels; however, expression differs markedly between different cell types with expression being particularly high in e.g. cells with a high turnover of fatty acids. We show here that the relatively high basal promoter activity of the rat ACBP gene in fibroblasts and hepatoma cells relies on sequences between -331 to -182 and on the Sp1 and NF-Y sites at -172 and -143, respectively. The basal transcription is modulated by members of the PPAR and SREBP families. In adipocytes, PPARgamma is in part responsible for the induction during adipocyte differentiation, but other transcription factors appear to play a role as well. In hepatocytes, SREBP-1c is the main regulator of ACBP in response to changes in insulin levels during fasting/refeeding. PPARalpha counteracts this effect by stimulating ACBP expression during fasting. In addition, PPARalpha mediates the induction of ACBP expression in response to peroxisome proliferators. PPARalpha and PPARgamma do not require sequences upstream of -182 for transactivation; however, SREBP-1c requires the synergistic action of sequences in intron 1 for transactivation of the ACBP promoter.

Transcriptional Regulation of Early Transposon Elements, an Active Family of Mouse Long Terminal Repeat Retrotransposons

While early transposon (ETn) endogenous retrovirus (ERV)-like elements are known to be active insertional mutagens in the mouse, little is known about their transcriptional regulation. ETns are transcribed during early mouse embryogenesis in embryonic stem (ES) and embryonic carcinoma (EC) cell lines. Despite their lack of coding potential, some ETns remain transposition competent through their use of reverse transcriptase encoded by a related group of ERVs-MusD elements. In this study, we have confirmed high expression levels of ETn and MusD elements in ES and EC cells and have demonstrated an increase in the copy number of ETnII elements in the EC P19 cell line. Using transient transfections, we have shown that ETnII and MusD LTRs are much more active as promoters in P19 cells than in NIH 3T3 cells, indicating that genomic context and methylation are not the only factors determining endogenous transcriptional activity of ETns. Three sites in the 5' part of the long terminal repeat (LTR) were demonstrated to bind Sp1 and Sp3 transcription factors and were found to be important for high LTR promoter activity in P19 cells, suggesting that as yet unidentified Sp binding partners are involved in the regulation of ETn activity in undifferentiated cells. Finally, we found multiple transcription start sites within the ETn LTR and have shown that the LTR retains significant promoter activity in the absence of its noncanonical TATA box. These findings lend insight into the transcriptional regulation of this family of mobile mouse retrotransposons.

Organization and Promoter Analysis of the Zebrafish (Danio rerio) Interferon Gene

Interferon plays important roles in confronting viral infections as the first line of defense. For the purpose of understanding the molecular mechanism which controls transcription of the interferon gene, we cloned and sequenced the interferon promoter region of the zebrafish interferon gene and characterized its activity using firefly luciferase transient transfection expression assays. Different fragments of the zebrafish interferon 5'-flanking region were transfected into ZFL cells. In these cell lines, maximum promoter activity was located in 2.2 kb of the zebrafish interferon 5' flanking region of the ZFL cell line. In this study, we investigated whether the viral replicative intermediate double-stranded RNA (herein we used synthetic polyinosinic-polycytidylic acid [poly(I):poly(C)] modifies the effects of interferon on gene expression. For this purpose, all zebrafish interferon promoter fragments were treated with either 1, 10, or 100 microg/ml poly(I):poly(C). The results showed that after treatment with 10 microg/ml poly(I):poly(C), high promoter activity was observed in the -2.2-kb interferon promoter fragment. Several putative transcription factors were shown in the promoter region, including IRF-1, C/EBP, NFkappaB, and GATA-1. Further study of the in vivo expression of the interferon promoter during development was carried out in transgenic zebrafish expressing an interferon promoter-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene. Morphological studies of transgenic zebrafish indicated that the interferon promoter-driven GFP transcripts appeared in the yolk, head, and lymphoid organs. These results indicate that the interferon promoter is active in a tissue-specific manner, and suggest that the interferon promoter plays an important role in virus resistance during teleost growth.

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Cloning and Characterization of &amp;beta;-Carotene Ketolase Gene Promoter in &amp;lt;italic&amp;gt;Haematococcus pluvialis&amp;lt;/italic&amp;gt;

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the beta-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

Characterization of the rolB promoter on mikimopine-type pRi1724 T-DNA

The promoter activity of the rolB gene encoded on mikimopine-type pRi1724 ( 1724rolB) was elucidated. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that 1724rolB mRNA was highly accumulated in roots of 1724rolB-transformed tobacco plants. A β-glucuronidase (GUS) fluorometric assay using a 1724rolB promoter-GUS construct also showed high promoter activity in the roots. A GUS histochemical assay revealed that 1724rolB was expressed in the vicinity of root apical meristematic cells of the vascular cylinder in the main roots, in lateral root primordial and meristematic cells, in veins of cotyledons, and faintly in the shoot apex. Since the product of 1724rolB has a N-terminal stretch 17 amino acid longer than the rolB of agropine-type pRi1855 ( 1855rolB), primer extension analysis was used to show that the transcription initiation point of 1724rolB was located beyond the putative TATA box of 1855rolB, suggesting that the locations of the minimal promoters of the two rolB genes differed from each other. 1724rolB promoter analysis using deletion constructs indicated that the minimal region necessary for this promoter activity was, at most, −215 bp upstream of the translation initiation point. A predominant auxin-responsive sequence was located at position −313 to −216 upstream of the transcription initiation point, containing the Nicotiana tabacum rolB domain B factor 1 (NtBBF1) binding motif ACTTTA.

Role of Hepatocyte Nuclear Factors in Transcriptional Regulation of Male-specific CYP2A2

Cytochrome P450 2A2 (CYP2A2) is an adult male-specific rat liver steroid hydroxylase whose sex-dependent expression is regulated at the transcriptional level by sexually dimorphic pituitary growth hormone (GH) secretory patterns. In contrast to CYP2C11 and other male-specific, plasma GH pulse-inducible liver genes, CYP2A2 is highly expressed in hypophysectomized rat liver, despite the absence of GH stimulation. CYP2A2 promoter fragments 0.9-6.2 kb long exhibited unusually high basal promoter activity when transfected into the liver cell line HepG2. A further approximately 2.5-fold increase in activity was obtained by cotransfection of hepatocyte nuclear factor (HNF) 3gamma or HNF4alpha. CYP2A2 promoter activity was inhibited approximately 85% by transfection of HNF3beta or HNF6, both of which are more highly expressed in female than male liver and can strongly trans-activate the female-specific CYP2C12 promoter. The male GH pulse-activated transcription factor STAT5b had no effect on CYP2A2 promoter activity, either alone or in combination with HNF3gamma and HNF4alpha, consistent with the GH pulse-independence of CYP2A2 expression. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4alpha toward two other male-specific liver target genes, Cyp2d9 and CYP8B1. Furthermore, STAT5b in combination with the HNF4alpha coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha strongly enhanced the transcriptional activity of HNF4alpha toward CYP8B1 but not toward CYP2A2. These findings support the hypothesis that sex-dependent HNFs contribute to the sexually dimorphic expression of CYP2A2 and other liver CYPs and highlight the ability of STAT5b to act in concert with HNF4alpha to regulate select male-specific liver CYP genes.

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentally and environmentally. To investigate the regulation of ACC synthase gene expression, the promoters of Arabidopsis ACS genes, AtACS4, AtACS5, and AtACS7, were fused to a GUS reporter gene, and the recombinant transgenes were introduced into Arabidopsis to produce three groups of AtACS::GUS transgenic plants. Histochemic and fluorometric study of these transgenic plants revealed that promoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinated seedlings. AtACS5 has the highest promoter activity in leaves of 2-week-old light-grown seedlings among the three AtACS genes studied. In the mature leaves, AtACS4 and AtACS7 genes are expressed in both veins and areoles, whereas AtACS5 is expressed at a higher level in the areoles and epidermal cells surrounding trichomes. The promoter activities of all these AtACS genes are found in the reproductive organs. AtACS5 and AtACS7 are highly expressed in petals, sepals, carpels, stamens, cauline leaves, inflorescence stems, and siliques, while AtACS4 expression is undetectable in the petals of open flowers. All three AtACS genes are expressed in root tissue. In the 2-week-old light-grown Arabidopsis, the AtACS4 promoter is responsive to the plant hormones IAA, ethylene, and ABA, and to darkness and wounding; the AtACS5 promoter to IAA, ABA, salt, high temperature, and wounding; and the AtACS7 promoter to GA3, ethylene, and ABA, and to darkness and salt. Low-temperature treatment abolishes the darkness-induced AtACS7 gene expression, but not that of AtACS4. Each AtACS gene has a unique expression profile during growth and development. It appears that at any developmental stage or any growth period of Arabidopsis, there is always a member of AtACS multigene family that is actively expressed.

Analysis of a Single Nucleotide Polymorphism in the Promoter of the MxA Gene in Patients with Spontaneous and Treatment Induced Recovery from Hepatitis C Virus Infection

Interferon-α (IFN-α) is an important mediator of the hostrsquor;s innate antiviral defence system. The human recombinant protein is approved for the treatment of persistent viral infections as hepatitis B or C. However, the natural course of infection and treatment response show a great interindividual variation. This study aimed to investigate whether a functional single nucleotide polymorphism in the promoter region of the IFN-α effector MxA is associated with (a) the natural course of hepatitic C virus (HCV) infection and (b) with the outcome of an IFN-α treatment in chronic hepatitis C patients. The rare allele T at position –88 of the MxA promoter has been shown to be associated with a higher promoter activity when compared to the G allele. A total of 190 patients were included. 146 of them developed persistent HCV infection, and 127 were treated with IFN-α. In addition, 44 patients were enrolled who resolved HCV infection spontaneously. We found a genotype distribution of 145: 41: 4 (GG: GT: TT), yielding a rare allele frequency of 0.130. We found no statistically significant difference in genotype distribution or rare allele frequency when patients with a self-limited infection were compared to those with a persistent infection. Moreover, we did not find an association of –88 MxA genotype and response or non-response to an IFN-α therapy. These results apparently contrast with former studies on Japanese chronic hepatitis C patients, which revealed a highly significant association of the T allele and IFN-α therapy response. By comparison, Japanese individuals show a clearly higher rare T allele frequency of 0.290. Of note, the only patient from our group who was homozygous for the T allele and who was treated with IFN-α was of Asian origin and is among those with a sustained response. Another one belongs to the group of patients with self-limiting infection. In conclusion, we assume that associations of the –88 MxA promoter polymorphism and spontaneous or treatment-induced recovery from infection might not become noticeable in the mostly Caucasian individuals enrolled in our study because of the low T allele frequency.

Selective Induction of Apoptosis in Melanoma Cells by Tyrosinase Promoter-Controlled CD95 Ligand Overexpression

Induction of apoptosis has been demonstrated previously by overexpression of CD95 ligand (CD95L) in cultured human melanoma cells. For in vivo approaches based on CD95L, however, targeted expression is a prerequisite and tyrosinase promoters have been considered for selection. Luciferase reporter gene assays performed for a representative panel of melanoma cell lines characterized by strong (SK-Mel-19), moderate (SK-Mel-13, MeWo), weak (A-375), and missing expression (M-5) of endogenous tyrosinase revealed high tyrosinase promoter activities in SK-Mel-19, SK-Mel-13, and MeWo, but only weak activities in A-375 and M-5 as well as in non-melanoma cell lines. After transfection of a CMV promoter CD95L expression construct, melanoma cells were found highly sensitive, as compared with non-melanoma cells. By applying a tyrosinase promoter CD95L construct, apoptosis was selectively induced in SK-Mel-19, SK-Mel-13, MeWo as well as in A-375, which was characterized by high CD95 surface expression and high sensitivity to agonistic CD95 activation. M5 and non-melanoma cell lines remained uninfluenced. Also, resistance to agonistic CD95 activation seen in MeWo characterized by weak CD95 surface expression was overcome by overexpression of CD95L. Our investigations provide evidence that tyrosinase promoter CD95L constructs may be of value for selective induction of apoptosis in therapeutic strategies for melanoma.

The A−336C Insulin-Like Growth Factor Binding Protein-3 Promoter Polymorphism Is Not a Modulator of Breast Cancer Risk in Caucasian Women

The A−336C Insulin-Like Growth Factor Binding Protein-3 Promoter Polymorphism Is Not a Modulator of Breast Cancer Risk in Caucasian Women

Matrix metalloproteinase3 and 9 gene promoter polymorphisms: joint action of two loci as a risk factor for coronary artery complicated plaques

Objective: Matrix metalloproteinases 3 and 9 (MMP3 and MMP9) are present in atherosclerotic plaques and co-operate in the degradation of the fibrous cap of the atheroma, leading to fissuring and ultimately to acute coronary thrombosis. The functional genetic polymorphisms in the promoters of MMP3 and MMP9, which lead to low- and high-transcription activity genotypes, have been shown to be associated with myocardial infarction and angiographically measured atherosclerosis individually, whereas their effects in combination are not yet known. In order to assess the two disease loci simultaneously, we investigated the association of combined low and high promoter activity genotypes with different types of coronary lesions in an autopsy cohort of 300 Caucasian males aged 33–69 years (Helsinki Sudden Death Study). Methods: Genotyping at these loci was performed by PCR, restriction enzyme digestion and minisequencing, and areas of the coronary wall covered with atherosclerotic lesions were measured using computer-assisted morphometry. Results: In analysis of covariance (ANCOVA) with age, body mass index, hypertension, diabetes, smoking and alcohol consumption as covariates, a significant interaction between the MMP3 and MMP9 genotypes was observed on area of complicated lesions ( P = 0.012). Men with high promoter activity genotypes for both loci had, on average, more than two times larger area of complicated lesions (250%) compared with those men who had low promoter activity genotypes ( P = 0.008), but these loci showed no association with myocardial infarction. Conclusions: The joint action of two susceptibility loci, rather than single MMP genes alone, and the particular combination of MMP3 and MMP9 genotypes present at these loci may contribute to heterogeneity in the presentation of atherosclerosis.