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Biomedical subjects

I Bendik

Publications and source records attributed to I Bendik.

15 recordsLinked to original sources
PubMed2005

Bonistein (synthetic genistein), a food component in development for a bone health nutraceutical.

In the discussion of the risk-benefit relation of the hormone replacement therapy (HRT) for elder women phytochemicals with estrogenic activity received a great deal of attention. Phytoestrogens are naturally occurring compounds with structural similarity to 17beta-estradiol. Especially genistein, an isoflavone most abundant in soy, possess a high and selective binding-affinity to the mammalian estrogen receptors. It has been found, that genistein exert in humans both: weak estrogenic and anti-estrogenic effects, similar to the SERMs. Consequently, it was concluded, that genistein might provide an alternative to prevent postmenopausal bone-loss and ameliorate menopausal symptoms without side-effects similar to HRT. Pre-clinical experiments and results from clinical pilot studies with pure genistein confirmed its efficacy in these indications. Nevertheless, currently some open issues still exist to recommend its intake thoughtlessly. Bonistein, pure synthetic genistein developed by DSM Nutritional Products, was tested extensively in appropriate models for bone health. A battery of toxicological studies was conducted to determine safe intake levels. In the early clinical development pharmacokinetic studies were performed in healthy volunteers and in postmenopausal women. Now large-scale studies are in preparation to investigate Bonistein's efficacy in postmenopausal bone-loss and climacteric syndrome.

Animals↗
PubMed2003

The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.

In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.

Adipocytes↗
PubMed2001

A novel polymorphism in the promoter of the RAGE gene is associated with non-small cell lung cancer.

The receptor for advanced glycosylation endproducts (RAGE) is abundant at both the transcriptional and translational level in normal lung but is not expressed in non-small cell lung cancer (NSCLC). In order to determine whether sequence variations might be responsible for the inactivation of RAGE in NSCLC, we investigated the RAGE gene in primary NSCLCs and in the corresponding normal tissues of nine patients. Although sequence analysis revealed no somatic, tumor-associated mutations, six novel sequence variants were identified: T-->A in the promoter region 388 bp upstream of the start codon: T-->A in exon 1 (Ala2Ala), C-->G in exon 3 (Val89Val), C-->T in intron 6, G-->C and C-->G in exon 10 (Arg365Ser and Arg369Gly). In addition, we detected a 63 bp deletion in the promoter region (358-421 bp upstream of the start codon) in one NSCLC patient. The T-->A transversion in the promoter region was detected in three of nine patients. Further analysis of this polymorphic locus in 54 NSCLC patients and 59 non-cancer controls revealed a significant difference in the genotype distribution between NSCLC patients and controls. Interestingly, the AA genotype was more common in NSCLC patients (20.8%) than in controls (3.5%). The cumulative occurrence of the AA variant in NSCLC suggests that this genotype is a putative risk factor for NSCLC development.

Aged↗
PubMed2001

Genomic organization and chromosomal mapping of SPARC-like 1, a gene down regulated in cancers.

Human SPARC-like 1 (SPARCL1), also known as MAST9 or hevin, is a member of the SPARC protein family. Originally we identified SPARCL1 as one of the genes down regulated in human non-small cell lung cancer (NSCLC). Recent reports indicate that the down regulation of SPARCL1 also occurs in prostate and colon carcinomas, suggesting that SPARCL1 inactivation is a common event not only in NSCLCs but also in other tumors of epithelial origin. In the present work we report the cloning and mapping of the genomic locus of human SPARCL1. Using fluorescence in situ hybridization analysis, SPARCL1 was localized to chromosome 4q22-25, a region often deleted in human cancers. Furthermore, we show that the intron/exon organization of the human SPARCL1 gene is similar to its murine homologue SC1. SPARCL1 contains 11 exons and 10 introns which span approximately 47 kb of the genome. We also sequenced the 5'-flanking region of the human SPARCL1 gene containing 2.4 kb of the putative promoter region. The data presented herein are a prerequisite for deletion/mutation analysis of the SPARCL1 gene in tumors. In addition, knowledge of the SPARCL1 promoter sequence allows to investigate the regulation of SPARCL1 expression on the transcriptional level. Taken together our results will help to clarify the function of SPARCL1 in tumor formation.

Base Sequence↗
PubMed2001

Expression of putative anticancer targets in familial adenomatous polyposis and its association with the APC mutation status.

Several substances interfering with colorectal carcinogenesis may reduce or prevent adenoma formation in familial adenomatous polyposis (FAP), an inherited predisposition to colorectal cancer. This study determined the expression of genes coding for putative anticancer targets (COX-2, iNOS, MMP-7, ODC, PKCbeta, PPARgamma, RXRalpha, RXRbeta, RXRgamma) in FAP patients to provide one of the rationales for the design of chemotherapy and -prevention strategies. Gene expression was assessed by TaqMan analysis in colonic tissue of 9 FAP patients with mutations in the APC gene (APCpos), 5 FAP patients without identified genetic defect (APCneg), and 3 healthy individuals. Among the examined genes, PKCbeta and MMP-7 were most consistently altered in adenoma tissue relative to matched mucosa. Intriguingly, ODC was clearly overexpressed in polyps from APCpos but not APCneg patients. Furthermore, PKCbeta, MMP-7, ODC, and COX-2 as well as all RXRs displayed altered expression in apparently healthy FAP mucosa as opposed to that of healthy individuals. Our data suggests PKCbeta and MMP-7 to be the most suited as anticancer targets among the genes studied.

Adenomatous Polyposis Coli↗
PubMed2000

Germline alterations in the cyclooxygenase-2 gene are not associated with the development of extracolonic manifestations in a large swiss familial adenomatous polyposis kindred.

Familial adenomatous polyposis (FAP) is an autosomal dominant condition leading to the development of multiple colorectal polyps and other features. Intrafamilial variation in phenotype is known to occur in FAP; despite carrying the same causing mutation in the APC gene, disease expression may considerably differ in affected individuals, likely due to the existence of modifier genes. Several lines of evidence suggest the cyclooxygenase-2 (COX-2) gene to be a candidate modifier in FAP. Since COX-2 appears to be expressed in tissues prone to be affected in FAP, it might influence the occurrence of extracolonic manifestations in this disorder. Herein, we investigated whether alterations in the COX-2 gene are involved in the development of extracolonic polyps and extragastrointestinal features. Mutational analysis using single-strand conformation polymorphism (SSCP) in 130 members of a FAP family displaying strong phenotype variation revealed 3 polymorphic sites within the coding region of the COX-2 gene. None of these allelic variants, however, segregated with a particular disease phenotype. In addition, expression analysis was performed in 31 family members with representative phenotypes. Neither of the two polymorphisms detected within the COX-2 promoter was associated with a given phenotype nor was there a significant difference in quality or quantity of COX-2 mRNA in lymphocytes as measured by reverse transcription- and real time quantitative reverse transcription PCR (RT-PCR and TaqMan). In conclusion, germline alterations in the COX-2 gene are unlikely to account for the development of extracolonic disease in FAP patients.

Adenomatous Polyposis Coli↗
PubMed1998

Characterization of MAST9/Hevin, a SPARC-like protein, that is down-regulated in non-small cell lung cancer.

In the search for genes down-regulated in non-small cell lung cancer (NSCLC), we have identified a cDNA fragment, termed MAST9. Cloning, sequencing, and characterization of the full-length MAST9 cDNA revealed that the entire 2808 nucleotide sequence had an open reading frame of 1992 nucleotides encoding a Mr 75,000 protein. Sequence analysis disclosed a striking homology to SPARC, known to be involved in tumorigenesis. The recently identified "Hevin" cDNA isolated from high endothelial venules is identical to MAST9. Using Northern and Western blot analysis, we showed that MAST9 was down-regulated in the tumor samples of nine non-small cell lung carcinoma patients. Furthermore, we demonstrated that both the bacterially expressed and the endogenous MAST9 proteins form homodimers. The lack of expression in non-small cell lung carcinomas and its homology to SPARC suggest a putative role of MAST9 in lung tumor formation.

Amino Acid Sequence↗
PubMed1997

Differential messenger RNA and protein expression of the receptor for advanced glycosylated end products in normal lung and non-small cell lung carcinoma.

The receptor for advanced glycosylated end products (RAGE), a member of the immunoglobulin superfamily, was one of the cDNA subtraction clones that was found to be differentially expressed in human lung and the corresponding tumor tissue. In nine additional matched normal/tumor pairs, a strongly reduced or missing expression, not only on a transcriptional level but also on a protein level, was demonstrated in the non-small cell lung carcinoma tissue. Because amphoterin, a physiological ligand of RAGE that is highly expressed in the lung, mediates cell differentiation via RAGE, a down-regulation of the receptor may be considered as a critical step in lung tumor formation. Furthermore, we determined the complete reading frame of RAGE.

Base Sequence↗
PubMed1995

Similar ligand-induced conformational changes of thyroid hormone receptors regulate homo- and heterodimeric functions.

Thyroid hormone receptors (TRs) bind specific thyroid hormone response elements (TREs) as heterodimers with retinoid X receptors (RXRs) and act as transcriptional activators. As homodimers, TRs can bind a distinct set of sequences and function as ligand sensitive repressors. In our study, we compared the natural malic enzyme TRE (ME-TRE) as a model system for the TR/RXR heterodimer pathway to the chicken lysozyme silencer element F2-TRE which is strongly bound and regulated by TR/TR homodimers. Using electrophoretic mobility shift assays, transient transfections with a variety of natural and synthetic triiodothyronine and thyroxine derivatives as well as limited proteolytic analysis, we show that the natural homo- and heterodimeric pathways show similar ligand requirements. Furthermore, we observe that the ligand-induced conformational changes in the receptor proteins that either result in a loss of TR/TR homodimer binding and release of transcriptional repression or in transcriptional activation of TR/RXR heterodimers are indistinguishable. Therefore, we propose that in TR/TR homodimers and TR/RXR heterodimers very similar moieties of the receptors are involved in ligand binding and subsequent conformational changes that lead to loss of gene repression (TR/TR homodimer) and gain of gene activation (TR/RXR heterodimer).

Animals↗
PubMed1995

Thyroid hormone receptor homodimers can function as ligand-sensitive repressors.

Unlike the steroid hormone receptors that bind their response elements as homodimers, thyroid hormone receptor (TRs) as well as retinoic acid receptors and several other receptors have been shown to require heterodimerization with retinoid X receptors (RXR) for efficient binding to most response elements. In this article we have compared in detail TR DNA binding and its gene-regulatory characteristics in the presence and absence of RXR. We observe that in the absence of RXR, TRs are able to bind with high affinity as homodimers to a subset of thyroid hormone response elements consisting of two AGGTCA motifs arranged as inverted palindromes. This binding is inhibited by T3, which prevents TR homodimers from functioning as ligand-dependent transcriptional activators. We demonstrate that TR homodimers can act as potent ligand-responsive repressors, in particular when binding to sites 3' of the TATA box. Thus, TRs appear to have important regulatory functions in the absence of RXRs. This is strongly supported by our observations that some naturally occurring TR beta mutants that have been associated with generalized thyroid hormone resistance as well as the v-erbA oncogene are defective in this activity. Thus ligand-sensitive repression by TRs is an important regulatory mechanism.

Animals↗
PubMed1993

Interaction between retinoic acid and vitamin D signaling pathways.

The nuclear signaling pathways for retinoids and vitamin D differ in the specificity of the respective receptors for response elements. Two pathways for the action of both retinoic acid receptors (RARs) and vitamin D receptors (VDRs) have been identified, one being retinoid X receptor (RXR)-dependent and the other being RXR-independent. Moreover, RXRs were found to function as homodimers. In several steps we converted the retinoid specific response element of the human retinoic acid receptor beta promoter into the vitamin D/retinoic acid response element of the human osteocalcin promoter. We found that VDR homodimers only bind to the motif RGGTGA. The extended osteocalcin element also contains an imperfect direct repeat based on the motif RGGTGA spaced by three nucleotides, which is bound by RXR homodimers and activated by 9-cis-retinoic acid. The responsiveness of the osteocalcin element to all-trans-retinoic acid is mediated neither by RAR homodimers nor by RAR-RXR heterodimers. However, a VDR-RAR heterodimer binds to the osteocalcin response element and mediates activation by all-trans-retinoic acid. This heterodimer also binds to pure retinoid response elements, but it does not mediate activation by vitamin D alone. In combination with all-trans-retinoic acid, however, vitamin D enhances VDR-RAR heterodimer-mediated gene expression. This finding suggests a direct interaction between nuclear signaling by retinoic acid and vitamin D increasing the combinatorial possibilities for gene regulation by the nuclear receptors involved.

Base Sequence↗
PubMed1993

Two nuclear signalling pathways for vitamin D.

The dihydroxylated form of vitamin D3 (1,25-dihydroxy-D3)mediates a biological response by binding to intracellular receptors which belong to the steroid receptor superfamily. These receptors act as ligand-dependent transcription factors that bind to specific DNA sequences (reviewed in refs 6-9). We have identified two classes of vitamin D response elements that are activated either by the vitamin D receptor (VDR) alone or by heterodimers of VDR and the retinoid-X receptor-alpha (RXR-alpha). The motif GGGTGA arranged as a direct repeat with a spacing of six nucleotides or as a palindrome without spacing, or as an inverted palindrome with a 12-nucleotide spacing, confers vitamin D inducibility mediated by VDR alone. A second class of response elements, composed of directly repeated pairs of motifs (GGTCCA, AGGTCA, or GGGTGA) spaced by three nucleotides, is synergistically activated by RXR and VDR, but only in the presence of both ligands. Thus, the RXR ligand and the nature of the response element determine whether a nuclear receptor is co-regulated by RXR.

Base Sequence↗
PubMedDate not supplied

A spin-column procedure for estrogen receptor equilibrium and competition binding analysis.

Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.

Alphavirus Infections↗
PubMedDate not supplied

Simple and fast method to test the receptor for advanced glycosylated endproducts (RAGE) for its tumor suppressive potential using the Tet-On system.

The Tet gene expression system, that allows tightly controlled gene expression in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 293 cells in semi-solid medium. Establishing a Tet-On gene expression system involves two consecutive stable transfections. Here, we describe an alternative procedure to obtain a Tet-On gene expression system in a single transfection step for the use in tumor biology. The plasmids necessary for the regulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 clones were analyzed using PCR revealing 8 colonies to be double stably transformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression system could be completed in half of the time required for the original two step method. The generated 293 double stable cells were used in the clonogenic assay for the testing of the tumor suppressive potential of RAGE.

Base Sequence↗