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Biomedical subjects

G A Puca

Publications and source records attributed to G A Puca.

At least 19 recordsLinked to original sources
PubMed2001

Loss of estrogen receptor beta expression in malignant human prostate cells in primary cultures and in prostate cancer tissues.

The aim of this study was to investigate the expression of estrogen receptor (ER) beta and alpha genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERbeta and ERalpha messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ERalpha messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERbeta (C-terminal) antibody, demonstrated ERbeta protein in all NPEC lysates and in one of the six CPECS: ERalpha protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ERalpha N-terminal domain was used. In contrast, ERalpha protein was not detected in two of the six CPEC lysates when ERalpha C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6-8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERbeta gene is expressed together with ERalpha in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECS: Moreover, in some CPECs, the ERalpha gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways.

Blotting, Western↗
PubMed2001

Differentiation of myeloid cell lines correlates with a selective expression of RIZ protein.

BACKGROUND: The retinoblastoma-interacting zinc-finger gene RIZ is expressed in two forms (RIZ1 and RIZ2) that differ for the presence near the N-terminus of RIZ1 of a conserved domain, defined PR (PRDI-BF1-RIZ homology), homologous to a similar domain present in other proteins recognized as tumor suppressor gene products. The RIZ1 form is usually absent or expressed at low levels in tumor cells, whereas RIZ2 is frequently expressed. We investigated a possible involvement of RIZ1 in differentiation control using a myeloid cell maturation model that is easily modulated by retinoids and other agents. MATERIALS AND METHODS: HL60 or NB4 cell lines or patients' leukemic promyelocytes were treated with all- trans -retinoic acid or other agents to induce differentiation. RIZ gene expression was determined with reverse transcriptase polymerase chain reaction (RT-PCR) and RNase protection assay. Immunocytochemistry was performed to assess variation of the intracellular distribution of RIZ protein on all- trans-retinoic acid treatment. Forced expression of RIZ1 protein was obtained with a recombinant adenovirus containing RIZ1 cDNA. RESULTS: Treatment with retinoic acid induced a selective expression of RIZ1 in HL60 cell line. Retinoic acid effect was maximal at 7 days and correlated to the granulocytic differentiation of cells. A similar effect was obtained in retinoic acid-sensitive NB4 cell line or in patients' leukemic promyelocytes, but not in the retinoic acid-resistant cell line NB4.007/6 or in the U937 cell line. Selective expression of RIZ1 was also induced by 12-O-tetradecanoyl-phorbol-13-acetate in the U937 and HL60 cell lines and by 1,25-dihydroxyvitamin D(3) only in HL60 cells. In HL60 cells, RIZ1 was also induced by activation of a retinoid alpha receptor-independent maturation pathway based on retinoid X receptor agonist and protein kinase A synergism. In addition, retinoic acid produced a redistribution of the antigen within the nucleus in these cells. Forced expression of RIZ1 protein induced growth arrest and death of HL60 cells. CONCLUSIONS: The correlation between the selective expression of RIZ1 induced by retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate, or 1,25-dihydroxyvitamin D(3) and differentiation suggested that RIZ protein was involved in myeloid cell differentiation induced by these agents.

Adenosine Monophosphate↗
PubMed2000

Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells.

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

Amino Acid Sequence↗
PubMed2000

Estradiol induces functional inactivation of p53 by intracellular redistribution.

Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm.

Blotting, Western↗
PubMed2000

The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action.

Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control.

Base Sequence↗
PubMed1999

Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein).

Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins.

Base Sequence↗
PubMed1998

Interaction of vault particles with estrogen receptor in the MCF-7 breast cancer cell.

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.

Animals↗
PubMed1997

Identification of the Syrian hamster cardiomyopathy gene.

The BIO14.6 hamster is a widely used model for autosomal recessive cardiomyopathy. These animals die prematurely from progressive myocardial necrosis and heart failure. The primary genetic defect leading to the cardiomyopathy is still unknown. Recently, a genetic linkage map localized the cardiomyopathy locus on hamster chromosome 9qa2.1-b1, excluding several candidate genes. We now demonstrate that the cardiomyopathy results from a mutation in the delta-sarcoglycan gene that maps to the disease locus. This mutation was completely coincident with the disease in backcross and F2 pedigrees. This constitutes the first animal model identified for human sarcoglycan disorders.

Amino Acid Sequence↗
PubMed1996

Prostaglandin E2 induction of binding activity to CRE and AP-2 elements in human T lymphocytes.

Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses. Since it is known that PGE2 is able to increase cAMP levels, we investigated whether it can affect gene expression through the activation of the transcription factors which bind enhancer elements in the promoter regions of cAMP-regulated genes. Using electrophoretic mobility shift assay, we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2, but not AP-1, DNA elements. Since the okadaic acid, a potent protein phosphatase inhibitor, prolongs the induction of the binding activity, phosphorylation events are likely to occur. This activity seems to be due to increased cAMP levels because forskolin and IBMX mimic the effects of PGE2. More interestingly, transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter. These data support the positive role for PGE2 on some immune functions.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Identification of a novel sarcoglycan gene at 5q33 encoding a sarcolemmal 35 kDa glycoprotein.

Mutations in any of the genes encoding the alpha, beta or gamma-sarcoglycan components of dystrophin-associated glycoproteins result in both sporadic and familial cases of either limb-girdle muscular dystrophy or severe childhood autosomal recessive muscular dystrophy. The collective name 'sarcoglycanopathies' has been proposed for these forms. We report the identification of a fourth member of the human sarcoglycan family. We named this novel cDNA delta-sarcoglycan. Its mRNA expression is abundant in striated and smooth muscles, with a main 8 kb transcript, encoding a predicted basic transmembrane glycoprotein of 290 amino acids. Antibodies specifically raised against this protein recognized a single band at 35 kDa on western blots of human and mouse muscle. Immunohistochemical staining revealed a unique sarcolemmal localization. FISH, radiation hybrid and YAC mapping concordantly linked the delta-sarcoglycan gene to 5q33, close to D5S487 and D5S1439. The gene spans at least 100 kb and is composed of eight exons. The identification of a novel sarcoglycan component modifies the current model of the dystrophin-glycoprotein complex.

Amino Acid Sequence↗
PubMed1994

A novel p53 mutant in human breast cancer revealed by multiple SSCP analysis.

DNA from tumor tissue and peripheral blood lymphocytes of primary breast cancer patients was screened for the presence of p53 mutations. In DNA from one tumor we found that the histidine codon 193 (CAT) was somatically converted to arginine (CGT). This amino acid residue is highly conserved in many species, thus suggesting that such mutation plays an important role in the loss of wt-p53 function.

Amino Acid Sequence↗
PubMed1993

Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor.

A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor.

Animals↗
PubMed1992

Purified estrogen receptor enhances in vitro transcription.

An in vitro transcription system was developed to investigate the mechanisms of gene regulation by the estrogen receptor (ER). ER purified from calf uterus was highly active in enhancing RNA transcription from a template DNA containing estrogen response elements (EREs) upstream from a minimal promoter. Under the conditions employed, no addition of tissue specific factors was required and both estrogen or antiestrogens were ineffective. The stimulation of transcription correlated with the copy number of EREs in the template. The addition of competitor ERE oligonucleotides specifically inhibited the ER-induced transcription. We suggest that the ER may be involved in the formation of the stable initiation complex.

Animals↗
PubMed1992

Detection of a nonsense mutation in the dystrophin gene by multiple SSCP.

A combination of multiplex PCR with the single strand conformation polymorphism (SSCP) technique was employed to screen for point mutations in the human dystrophin gene. Co-amplification of 11 exons from genomic DNA of Duchenne and Becker muscular dystrophy (DMD/BMD) patients with no deletion or duplication was performed and the samples subjected to multiple SSCP analysis. We report the case of a nonsense mutation in a Duchenne patient identified by this approach. The mutation introduces a termination codon within exon 8 of the dystrophin gene. It is predicted to cause a very premature translational termination accounting for the severe phenotype observed. The patient inherited this mutation from his mother. In addition the analysis revealed 5 polymorphisms useful for internal control.

Codon↗