YB-1 protein levels are elevated in most human breast cancers, and high YB-1 levels have been correlated with drug resistance and poor clinical outcome. YB-1 is a stress-responsive, cell cycle-regulated transcription factor with additional functions in RNA metabolism and translation. In this study, we show in a novel transgenic mouse model that human hemagglutinin-tagged YB-1 provokes remarkably diverse breast carcinomas through the induction of genetic instability that emerges from mitotic failure and centrosome amplification. The increase of centrosome numbers proceeds during breast cancer development and explanted tumor cell cultures show the phenotype of ongoing numerical chromosomal instability. These data illustrate a mechanism that might contribute to human breast cancer development.

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.

The study of signal transduction processes using antisense oligonucleotides is often complicated by low intracellular stability of the antisense reagents or by nonspecific effects that cause toxicity. Here, we introduce a new class of antisense molecules, so-called GeneBlocs, which are characterized by improved stability, high target RNA specificity, and low toxicity. GeneBlocs allow for efficient downregulation of mRNA expression at nanomolar concentrations, and they do not interfere with cell proliferation. We demonstrate these beneficial properties using a positive readout system. GeneBloc-mediated inhibition of tumor suppressor PTEN (phosphatase and tension homologue detected on chromosome 10) expression leads to hyperactivation of the phosphatidylinositol (PI) 3-kinase pathway, thereby mimicking the loss of PTEN function and its early consequences observed in mammalian cancer cells. Specifically, cells treated with PTEN GeneBlocs show functional activation of Akt, a downstream effector of PI 3-kinase signaling, and exhibit enhanced proliferation when seeded on a basement membrane matrix. In addition, GeneBlocs targeting the catalytic subunit of PI 3-kinase, p110, specifically inhibit signal transduction of endogenous or recombinant PI 3-kinase. This demonstrates that GeneBlocs are powerful tools to analyze and to modulate signal transduction processes and, therefore, represent alternative reagents for the validation of gene function.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17 beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+. (C) 1999 Elsevier Science Ltd. All rights reserved.

Five types of 17 beta-hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The porcine peroxisomal 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The 17 beta-HSD 4 reveals only 25% amino acid similarity with 17 beta-HSD 1, 2, 3 and 5 enzymes. The highest levels of 17 beta-HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. In porcine gonads the immunofluorescence assigned the 17 beta-HSD 4 to granulosa cells, Leydig and Sertoli cells. A 2.9 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17 beta HSDs. Although five Asn-Xaa-Ser/Thr (Xaa - unspecified amino acid) sites are found in the 80 kDa protein the enzyme is not glycosylated. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are the first enzymes that are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The unique multidomain structure of the 80 kDa protein permits the catalysis of several reactions so far thought to be performed by complexes of different enzymes.

. The 17β-hydroxysteroid dehydrogenase type IV (17β-HSD IV) is a multifunctional enzyme that is localized in the peroxisomes. The N-terminal part has dehydrogenase activity, the central part has hydratase activity, and the carboxy-terminal part is responsible for sterol transport. Recent observations of mutations in the human 17β-HSD IV cDNA leading to a severe peroxisomal disorder motivated us to define the genomic organization of this gene mapped to Chromosome (Chr) 5q2. We show here that this gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. By mapping the regulatory region of this gene, we have shown that the first 400 bp upstream of the transcription start site are sufficient to activate transcription. The data presented here will permit sequence analysis of patients with peroxisomal disorders.

The 2.9-kb mRNA of 17β-hydroxysteroid dehydrogenase IV codes for an 80-kDa (737 amino acids) protein featuring domains that are not present in the other human 17β-hydroxysteroid dehydrogenases. The N-terminal part reveals conserved motifs of the short-chain alcohol dehydrogenase family. The central- and C-terminal domains are similar to peroxisomal enzymes for β-oxidation of fatty acids and to sterol carrier protein 2. The 80-kDa protein is N-terminally cleaved to a 32-kDa fragment (amino acids 1–323). Both the 80-kDa and the N-terminal 32-kDa peptides are able to catalyze the dehydrogenation with steroids at the C17 position and with 3-hydroxyacyl-CoA. The central part of the 80-kDa protein (amino acids 324–596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80-kDa protein (amino acids 5977ndash;737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The unique multidomain structure of the 80-kDa protein permits the catalysis of several reactions previously thought to be performed by complexes of different enzymes.

The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.

Annals of the New York Academy of SciencesVolume 784, Issue 1 p. 124-136 New 17β-Hydroxysteroid Dehydrogenases Molecular and Cell Biology of the Type IV Porcine and Human Enzymes JERZY ADAMSKI, JERZY ADAMSKI Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorJOSHUA CARSTENSEN, JOSHUA CARSTENSEN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorBETTINA HUSEN, BETTINA HUSEN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorMEYKE KAUFMANN, MEYKE KAUFMANN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorYVAN De LAUNOIT, YVAN De LAUNOIT Unite d'Oncologie Moleculaire Institut Pasteur de Lille Lille, FranceSearch for more papers by this authorFRAUKE LEENDERS, FRAUKE LEENDERS Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorMONIKA MARKUS, MONIKA MARKUS Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorPETER W. JUNGBLUT, PETER W. JUNGBLUT Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this author JERZY ADAMSKI, JERZY ADAMSKI Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorJOSHUA CARSTENSEN, JOSHUA CARSTENSEN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorBETTINA HUSEN, BETTINA HUSEN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorMEYKE KAUFMANN, MEYKE KAUFMANN Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorYVAN De LAUNOIT, YVAN De LAUNOIT Unite d'Oncologie Moleculaire Institut Pasteur de Lille Lille, FranceSearch for more papers by this authorFRAUKE LEENDERS, FRAUKE LEENDERS Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorMONIKA MARKUS, MONIKA MARKUS Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this authorPETER W. JUNGBLUT, PETER W. JUNGBLUT Max-Planck-Institut für Experimentelle Endokrinologie 30603 Hannover, GermanySearch for more papers by this author First published: April 1996 https://doi.org/10.1111/j.1749-6632.1996.tb16232.xCitations: 10AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Engel, L. L. & E. V. Gromann. 1974. Recent Prog. Horm. Res. 30: 139–169. 2 Tseng, L. & E. Gurpide. 1974. Endocrinology 94: 419–425. 3 Tseng, L. & J. Mazella. 1981. J. Steroid Biochem. 14: 437–442. 4 Peltoketo, H., V. Isomaa, O. 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Four types of 17 beta-hydroxysteroid dehydrogenases have been identified so far. The porcine peroxisomal 17 beta-hydroxysteroid dehydrogenase type IV catalyzes the oxidation of estradiol with high preference over the reduction of estrone, A 2.9-kilobase mRNA codes for an 80-kDa (737 amino acids) protein featuring domains which are not present in the other 17 beta-hydroxysteroid dehydrogenases. The 80-kDa protein is N terminally cleaved to a 32-kDa fragment with 17 beta-hydroxysteroid dehydrogenase activity, Here we show for the first time that both the 80-kDa and the N-terminal 32 kDa (amino acids 1-323) peptides are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part of the 80-kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency, The C-terminal part of the 80-kDa protein (amino acids 597-737) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions so far thought to be performed by complexes of different enzymes.

Multifunctional protein-2 (MFP2), also known as D-bifunctional protein, is a central enzyme of the peroxisomal β-oxidation pathway. Defects in this enzyme are associated with a spectrum of neurological disorders encompassing developmental and degenerative pathologies. In order to investigate the cellular and molecular mechanisms of these neuropathologies, mouse models with general and cell type selective loss of MFP2 were generated. In this review the distinct anomalies in the CNS of adult Mfp2 knockout mice are discussed, in particular the cerebellar degeneration and neuroinflammation. The potential underlying mechanisms are considered with regard to the cellular origin and biochemical causes. Finally, the similarities and differences between the CNS phenotypes of mice lacking MFP2 and mice with peroxisome biogenesis disorders are assessed.

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) IV is coded by 2.9 kb mRNA translated to an 80 kDa protein which is N-terminally cleaved to a 32 kDa enzyme. The 17 beta-HSD IV is dedicated to steroid inactivation and reveals only 25% amino acid similarity with 17 beta-HSD I-III enzymes. Despite five Asn-Xaa-Ser/Thr (Xaa = unspecified amino acid) sites in the 80 kDa protein the enzyme is not glycosylated. The porcine 32 kDa 17 beta-HSD IV forms dimers of 75 kDa. The highest 17 beta-HSD IV mRNA expression and specific activities are found in liver and kidney followed by ovary and testes. In porcine gonads the immunofluorescence assigned the 17 beta-HSD IV to granulosa cells and to Leydig and Sertoli cells. As shown by the treatment with phorbol-myristate-acetate in vitamin D-differentiated monocytic leukemia THP1 cells, steroid synthesis and inactivation are regulated differentially by the protein kinase C pathway: an increase in aromatase is accompanied by a decrease in 17 beta-HSD IV mRNA levels.

Porcine 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD4) was recently purified and cloned.(1,2) It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. The 32-kDa fragment (estradiol dehydrogenase, EDH) is cut from an 80-kDa (737 amino acid) primary translation product with a multidomain structure. The N-terminal 32-kDa sequence reveals similarities to the conserved motives of the short chain alcohol dehydrogenase family. The similarity to human hydroxysteroid dehydrogenases 1, 2, and 3 is below 25%. The central domain of HSD4 is 40% identical with the central domains of the fatty acid hydratase/dehydrogenase of Saccharomyces cerevisiae. The C-terminal HSD4 segment is 39% similar to the sterol carrier protein 2 (SCP2) featuring the peroxisome-targeting signal AKI. We have studied the multifunctionality of the protein and its subcellular localization.