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R Urrutia

Publications and source records attributed to R Urrutia.

At least 19 recordsLinked to original sources
PubMed1998

KRAB-independent suppression of neoplastic cell growth by the novel zinc finger transcription factor KS1.

The study of zinc finger proteins has revealed their potential to act as oncogenes or tumor suppressors. Here we report the molecular, biochemical, and functional characterization of KS1 (KRAB/zinc finger suppressor protein 1), a novel, ubiquitously expressed zinc finger gene initially isolated from a rat pancreas library. KS1 contains 10 C2H2 zinc fingers, a KRAB-A/B motif, and an ID sequence that has been shown previously to participate in growth factor-regulated gene expression. Northern blot analysis using pancreatic cell lines demonstrates that KS1 mRNA is inducible by serum and epidermal growth factor, suggesting a role for this gene in cell growth regulation. Biochemical analysis reveals that KS1 is a nuclear protein containing two transcriptional repressor domains, R1 and R2. R1 corresponds to the KRAB-A motif, whereas R2 represents a novel sequence. Transformation assays using NIH3T3 cells demonstrate that KS1 suppresses transformation by the potent oncogenes Ha-ras, Galpha12, and Galpha13. Deletion of the R1/ KRAB-A domain does not modify the transformation suppressive activity of KS1, whereas deletion of R2 abolishes this function. Thus, KS1 is a novel growth factor-inducible zinc finger transcriptional repressor protein with the potential to protect against neoplastic transformation induced by several oncogenes.

3T3 Cells↗
PubMed1998

Identification and chromosomal localization of CTNNAL1, a novel protein homologous to alpha-catenin.

Sodium butyrate (NaB) was shown to induce growth inhibition and apoptosis in a human pancreatic cancer cell line (AsPC-1). To identify the genes that are differentially regulated by NaB, we generated subtracted EST libraries highly enriched for up- or down-regulated transcripts using the suppression subtraction hybridization technique. One of the ESTs identified in the down-regulated library showed significant homology to human alpha-catenin. A cDNA of 2.45 kb that codes for a putative protein of 734 aa was cloned. The cloned cDNA was used as a template in an in vitro transcription-translation reaction. This produced a protein product of about 82 kDa in size. The gene designated as CTNNAL1 (catenin (cadherin-associated protein), alpha-like 1) was found to be ubiquitously expressed in many tissues including pancreas, heart, and skeletal muscle. A human BAC clone containing the gene was isolated and used as a probe for fluorescence in situ hybridization (FISH). Both radiation hybrid and FISH analysis mapped the gene to chromosome band 9q31.2, a region where frequent abnormalities have been observed in bladder carcinoma, esophageal cancers, and several other tumors.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor-beta-inducible Sp1-like zinc finger-encoding genes involved in the regulation of cell growth.

Sp1-like zinc finger transcription factors are involved in the regulation of cell growth and differentiation. Recent evidence demonstrating that mammalian cells express novel, yet uncharacterized, Sp1-like proteins has stimulated a search for new members of this family. We and others have recently reported that the transforming growth factor (TGF)-beta-regulated gene TIEG encodes a new Sp1-like protein that inhibits cell growth in cultured cells. Here we report the identification, nuclear localization, DNA binding activity, transcriptional repression activity, and growth inhibitory effects of TIEG2, a novel TGF-beta-inducible gene related to TIEG. TIEG2 is ubiquitously expressed in human tissues, with an enrichment in pancreas and muscle. TIEG2 shares 91% homology with TIEG1 within the zinc finger region and 44% homology within the N terminus. Biochemical characterization reveals that TIEG2 is a nuclear protein, which, as predicted from the primary structure, specifically binds to an Sp1-like DNA sequence in vitro and can repress a promoter containing Sp1-like binding sites in transfected Chinese hamster ovary epithelial cells. Furthermore, functional studies using [3H]thymidine uptake and MTS (3-(4, 3-dimethyltiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-su lfophenyl)-2 H-tetrazolium) assays demonstrate that the overexpression of TIEG2 in Chinese hamster ovary cells inhibits cell proliferation. Thus, TIEG2, together with TIEG1, defines a new subfamily of TGF-beta-inducible Sp1-like proteins involved in the regulation of cell growth.

Amino Acid Sequence↗
PubMed1998

1996 Comfort Symposium on Pancreatic Carcinogenesis.

This article summarizes the proceedings of the 1996 Comfort Symposium on Pancreatic Carcinogenesis that took place at the Mayo Clinic in Rochester, Minnesota, September 11-13, 1996. The annual series of Comfort lectures are aimed at discussing leading theories and advanced technological developments in the area of pancreatic research. The goals of this year's symposium were to summarize epidemiologic and experimental findings in the field of pancreatic cancer research, to foster communications among scientists studying this disease, and to identify areas of research that are likely to bridge the gaps between basic science and patient care. The topics discussed included (i) current algorithms for the diagnosis of early pancreatic cancer, (ii) animal and cellular models of pancreatic carcinogenesis, (iii) mechanisms of pain in pancreatic cancer, (iv) the role of signaling cascades and transcription factors in the regulation of pancreatic cell growth and differentiation, (v) methods to study genetic alterations associated with neoplastic diseases, and (vi) recent developments in gene-targeting techniques. The lectures and discussions during the symposium successfully achieved the goals outlined above and resulted in the identification of novel areas of research that may increase our understanding of the etiology and pathogenesis, and lead to early diagnosis and treatment of pancreatic cancer.

Animals↗
PubMed1998

Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p.

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

Animals↗
PubMed1997

Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells.

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Adult↗
PubMed1997

The dynamins: redundant or distinct functions for an expanding family of related GTPases?

In the 7 years since dynamin was first isolated from bovine brain in search of novel microtubule-based motors, our understanding of this enzyme has expanded significantly. We now know that brain dynamin belongs to a family of large GTPases, which mediate vesicle trafficking. Furthermore, this enzymatic activity is markedly increased through association with microtubules, acidic phospholipids, and certain regulatory proteins that contain Src homology 3 (SH3) domains. From functional, genetic, and cellular manipulations, it is now generally accepted that dynamin participates in the endocytic uptake of receptors, associated ligands, and plasma membrane following an exocytic event. These observations have confirmed at least one function of dynamin that was predicted from seminal studies on a pleiotropic mutant, shibire(ts) (shi(ts)) in Drosophila melanogaster. Of equal interest is the finding that there are multiple dynamin gene products, including two that are expressed in a tissue-specific manner, and they share marked homology with a larger family of distinct but related proteins. Therefore, it is attractive to speculate that the different dynamins may participate in related cellular functions, such as distinct endocytic processes and even secretion. In turn, dynamin could play an important role in cell growth, cell spreading, and neurite outgrowth. The purpose of this review is to enumerate on the expansive dynamin literature and to discuss the nomenclature, expression, and putative functions of this growing and interesting family of proteins.

Alternative Splicing↗
PubMed1997

Exploring the role of homeobox and zinc finger proteins in pancreatic cell proliferation, differentiation, and apoptosis.

Transcription factors are DNA binding proteins that regulate gene expression in response to a large variety of extracellular stimuli, and thereby act as key molecular switches for controlling cell differentiation, proliferation, and apoptosis. During the last decade, a myriad of these proteins have been identified and classified into different structural families, including homeobox, zinc finger, leucine zipper, and helix-loop-helix transcription factors. Members of the homeobox and zinc finger superfamilies are among the best-characterized transcription factors known to act as potent regulators of normal development in organisms ranging from insects to humans. In addition, mutations or aberrant expression in genes encoding these proteins can result in neoplastic transformation in several different cell types, further supporting their role as "guardians" of normal cell growth and differentiation. Therefore, the purpose of this article is to review this field of research with a particular emphasis on the role of homeobox- and zinc finger-containing transcription factors in pancreatic cell growth, cell differentiation, and apoptosis. The potential participation of these proteins in neoplastic transformation is also discussed.

Animals↗
PubMed1996

A novel profile of expressed sequence tags for zinc finger encoding genes from the poorly differentiated exocrine pancreatic cell line AR4IP.

Genes encoding for C2H2 zinc finger proteins are known to regulate normal cell proliferation and differentiation and have often been found to be mutated in different forms of cancer. We are interested in understanding the role of these genes as regulators of cell proliferation and differentiation in the exocrine pancreas. Therefore, we have generated expressed sequence tags (ESTs) encoding pancreas-enriched zinc finger peptides using the polymerase chain reaction and hybridization techniques [Adams, M.D. et al. (1991) Science, 252, 1651-1656]. Here we report the primary structure and expression pattern of 18 different zinc finger-encoding cDNAs (DZF-1-18) from the azaserine-derived tumoral cell line AR4IP which displays a poorly differentiated phenotype. Sequence analysis shows that all of these clones encode peptides which share the consensus DNA-binding motif with the Drosophila zinc finger transcription factor krüppel. High stringency Northern blot analysis shows that eight different zinc finger transcripts are expressed at high levels in normal adult rat pancreas and therefore constitute good candidates to play a role as transcription factors in exocrine pancreatic cells.

Animals↗
PubMed1996

Identification and characterization of zinc finger encoding genes from the tumoral exocrine pancreatic cell line AR42J.

Zinc finger transcription factors are DNA-binding proteins that are known to determine the identity of cells by regulating cell-specific gene expression. In addition, mutations in some members of this family have been found to be associated with several neoplasias, including Wilms' tumor and leukemias. Because the mechanisms that regulate normal, as well as neoplastic, pancreatic cell differentiation are poorly understood, we are searching for pancreas-enriched transcription factors that may determine the identity of pancreatic cells. Towards this end, we have used the polymerase chain reaction and degenerate primers against the highly conserved (Cys2-His2) zinc finger domain to amplify novel transcription factor encoding cDNAs from the well-characterized pancreatic acinar cell line AR42J. Using this approach, we have identified 17 different zinc finger encoding cDNAs (AZF-1 to -17). Sequence analysis shows that all of these clones encode for different zinc finger peptides which share the consensus DNA binding motif with the Drosophila transcription factor krüppel. As a first step in the characterization of these genes, the purified PCR products were used to determine their spatial pattern of expression by northern blot analysis. Using these techniques, we find that numerous zinc finger encoding genes are expressed in AR42J acinar cells as well as in normal adult rat pancreas and suggest that they may play a role as transcription factors in exocrine pancreatic cells.

Age Factors↗
PubMed1996

Expression of dynamin immunoreactivity in experimental pancreatic tumors induced in rat by mancozeb-nitrosomethylurea.

Dynamins are GTPases which support receptor-mediated endocytosis and bind to several tyrosine kinase receptor-associated proteins known to mediate cell proliferation and differentiation. We have recently established that dynamin expression correlates with normal neuronal (Torre et al., J. Biol. Chem., 269 (1994) 32411-32417) and acinar pancreatic cell differentiation (Cook et al., Mol. Biol. Cell, 6 (1995) 405a). To begin to understand the role of dynamin in neoplastic pancreatic cell differentiation, we have followed the expression of this protein by immunohistochemistry during the development of pancreatic tumors in a mancozeb-nitrosomethylurea (NMU)-based carcinogenesis model recently developed in our laboratory (Monis and Valentich, Carcinogenesis, 14 (1993) 929-933). After a single intraperitoneal injection (50 mg/g body wt) of this carcinogen, rats fed with mancozeb develop pancreatic focal acinar hyperplasia (FACH), dysplastic foci (DYF) displaying acinar-like and ductular-like structures, and ductular-like carcinoma in situ (CIS). After histochemical staining using a monoclonal anti-dynamin antibody, high levels of this protein are consistently observed in well-differentiated acinar tumors (FACH). In contrast, dynamin immunoreactivity is almost undetectable in more advanced lesions showing a ductular-like phenotype (ductular-like DYF and CIS). This change in the expression pattern of dynamin during the progression of acinar into ductular-like DYF and CIS lesions correlates with recent findings from our laboratory showing a differential expression pattern for dynamin in pancreatic cells during embryonic development, with ductular-like precursor cells expressing low levels of this protein. Based upon these results, we conclude that more advanced ductular-like neoplastic cells induced by the carcinogen NMU in rat pancreas behave phenotypically like pancreatic precursor cells in their pattern of expression for dynamin.

Animals↗
PubMed1996

Upregulation of molecular motor-encoding genes during hepatocyte growth factor- and epidermal growth factor-induced cell motility.

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are known to stimulate the locomotion of epithelial cells in culture. However, the molecular mechanisms which mediate these important changes are poorly understood. Here we have determined the effects of HGF and EGF on hepatocyte morphology, cytoskeletal organization, and the expression of molecular motor-encoding genes. Primary cultures of hepatocytes were treated with 10 ng/ml of HGF or EGF and observed with phase and fluorescence microscopy at 10, 24, and 48 h after treatment. We found that, over time, treated cells spread and became elongated after 24 h of treatment while forming long processes with dramatic alterations in the microtubule and actin cytoskeletons by 48 h. Quantitative Northern blot analysis was performed to measure expression of cytoskeletal-(beta-actin, alpha-tubulin) and molecular motor-(dynein, kinesin, and myosin I alpha and II) encoding genes which may contribute to this change in form. We observed the highest increase in levels of expression for myosin II (3.3-fold), kinesin (2.7-fold), myosin I alpha (2.2-fold), and alpha-tubulin (1.9-fold) after only 2 h of treatment with HGF. In contrast, EGF upregulated the expression of myosin I alpha (2.4-fold), kinesin (1.5-fold), and dynein (1.5-fold) at 10 h. The expression of the beta-actin gene remained constant in HGF-treated cells, while EGF induced a slight upregulation after 10 h of treatment. These results show for the first time that a selective upregulation of molecular motor-encoding genes correlates with alterations in cell shape and motility induced by HGF and EGF.

Actins↗
PubMed1996

Three dynamin-encoding genes are differentially expressed in developing rat brain.

Dynamin proteins are members of a recently described family of GTPases involved in receptor-mediated processes. To date, three different dynamin-encoding genes have been identified in mammalian tissues. Dynamin I is expressed only in neurons, whereas dynamin II is ubiquitously expressed. A third isoform, dynamin III, was originally isolated from a rat testis cDNA library and shown to be testis-specific. However, here we report the cloning and characterization of dynamin III from brain and lung, demonstrating a more extended pattern of expression for this isoform. In addition, we have investigated the temporal pattern of expression of these three genes during brain development. We find that both dynamin I and dynamin III mRNA levels are up-regulated during embryogenesis, whereas dynamin II mRNA levels remain unchanged. From these results, we conclude that dynamin III is not a testis-specific isoform and, furthermore, that rat brain expresses three different dynamin-encoding genes that are differentially regulated during development. Therefore, this large isoform diversity of dynamin proteins in brain predicts a significant complexity in the understanding of dynamin-based processes in this tissue.

Alternative Splicing↗
PubMed1995

A soluble motor from the alga Nitella supports fast movement of actin filaments in vitro.

In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.

Actins↗
PubMed1994

Dynamin 1 antisense oligonucleotide treatment prevents neurite formation in cultured hippocampal neurons.

Dynamin 1 (D100) is a microtubule-activated GTPase that is believed to play a role in the early steps of receptor-mediated endocytosis. Previous studies on the characterization of the Drosophila dynamin gene homolog, known as shibire, suggest that this protein may also participate in the formation of neuronal processes. To understand the role of rat brain dynamin 1 in neuronal morphogenesis, we correlated the intracellular levels of dynamin with the formation of neurites in rat hippocampal neurons and neuroblastoma N1E-115 cells in vitro. Quantitative Western and Northern blot analyses show that dynamin levels increase during neurite formation in both the hippocampal neurons and N1E-115 cells and decrease in N1E-115 cells during serum-induced neurite retraction. Furthermore, using antisense technology, we investigated the effect of decreased intracellular levels of dynamin on neurite formation in cultured embryonic hippocampal neurons. Antisense oligonucleotides against sequences surrounding the initiation codon of the rat brain dynamin gene (D100) reduce the intracellular levels of dynamin by approximately 90% and impair the formation of the minor and axon-like processes in a dose-dependent manner. Taken together, these results suggest that dynamin-mediated processes are necessary for normal neuronal morphogenesis.

Amino Acid Sequence↗
PubMed1994

Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues.

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.

Amino Acid Sequence↗
PubMed1993

Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

Base Sequence↗