Vicente Notario

Cloning of the dihydroxyacid dehydratase-encoding gene (ILV3) from Saccharomyces cerevisiae

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or alpha-ketobutyrate are converted into alpha-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILV genes have been cloned, thus allowing direct molecular analyses of their regulation.

Sequence and expression analysis of bovine pigment epithelium-derived factor

PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival. Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes. Here, we show the cDNA sequence and expression analysis of bovine PEDF. Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE. Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively. It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure. Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF. Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein. Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.

Transplants of fetal substantia nigra regulate glutamic acid decarboxylase gene expression in host striatal neurons

Lesions of the dopaminergic innervation to the striatum result in increased activity of glutamic acid decarboxylase (GAD) and increased GAD mRNA in striatal GABAergic neurons. Here we show that solid transplants of dopamine-containing fetal mesencephalic tissue placed adjacent to the striatum can completely reverse the elevation of GAD mRNA in the striatum of adult rats with complete lesions of the nigrostriatal dopamine projections. The ability of the fetal transplants to re-establish control over gene expression in host target neurons indicates that there is a significant transneuronal influence of the transplanted neurons. Furthermore, striatal GAD mRNA levels appear to be a good marker of the functional impact of dopamine-producing transplants.

Isolation and characterization of SpTRK, a gene from schizosaccharomyces pombe predicted to encode a K+ transporter protein

A novel gene, SpTRK, has been isolated from DNA of the fission yeast Schizosaccharomyces pombe (Sp) by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a sequence fully conserved between the potassium transporter genes TRK1 and TRK2 of Saccharomyces cerevisiae (Sc). SpTRK is a single-copy gene located on Sp chromosome I. Nucleotide sequence analysis of the cloned gene identified an open reading frame (ORF) with coding capacity for a protein of 833 amino acids (aa). The predicted SpTRK aa sequence showed a high level of conservation relative to the potassium transporters of Sc and Saccharomyces uvarum (Su), particularly within their transmembrane (TM) domains and in aa required for their ion transport functions. A single SpTRK transcript of about 2.7 kb is expressed at high levels in exponentially growing Sp cells, but it is downregulated in cells from stationary cultures.

Gradual deregulation and loss of PCPH expression in the progression of human laryngeal neoplasia

PCPH is a gene involved in the regulation of eukaryotic cell proliferation and stress response. Recently, analyses of human and animal solid tumors and cell lines suggested that PCPH protein deregulation may participate in neoplastic progression. To test this possibility, we first examined PCPH expression in several laryngeal carcinoma cell lines by Western analysis. The results showed the presence of altered PCPH polypeptides in these cells, accompanied by the loss of the PCPH form present in normal laryngeal epithelial cells, a deregulated expression pattern similar to that reported previously. We then analyzed PCPH expression in 59 dysplastic lesions of the human larynx, representative of the mild, moderate, and severe stages of the disease. Immunohistochemical data showed that, compared with normal laryngeal mucosa, PCPH expression in the dysplastic samples was associated with areas of epithelial cell maturation rather than with regions of increased proliferation. Furthermore, PCPH expression decreased parallel to the increase in cellular atypia of the dysplastic samples: PCPH either was expressed at very low levels or not expressed in cases of severe dysplasia/carcinoma in situ. This trend toward loss of PCPH expression along malignant progression of the larynx was confirmed by the low to null expression of PCPH in samples of invasive laryngeal carcinoma and by the complete absence of PCPH immunostaining in a laryngeal carcinoma–derived liver metastasis. These results indicated that PCPH protein analysis might allow for the distinction between grades of laryngeal dysplasia. In addition, detection of altered PCPH polypeptides by Western analysis potentially can be applied to the early identification of laryngeal squamous cell carcinoma.

Deregulated expression of the PCPH proto-oncogene in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene

The PCPH proto‐oncogene was identified by its frequent activation in Syrian hamster fetal cells exposed to 3‐methylcholanthrene. We previously isolated human PCPH cDNA and studied its expression in normal human tissues. We report herein the pattern of PCPH expression in normal rat tissues. Each tissue expressed one major PCPH polypeptide that varied in molecular mass in different tissues. Normal mammary gland expressed a single PCPH polypeptide of 27 kDa. This PCPH form also was expressed in lactating mammary glands but at significantly greater levels. These results suggest the existence of tissue‐specific regulatory mechanisms for PCPH expression that may be influenced by the differentiation stage. Our previous studies on the involvement of PCPH in human cancer showed that human breast tumor cell lines have frequent alterations in PCPH, including multiple PCPH polypeptide forms that are not expressed in normal cells. These cell lines also have frequent loss of a 27‐kDa form identified as the only PCPH polypeptide expressed by normal human breast epithelial cells. In this study, we found that these same alterations occurred in vivo during mammary carcinogenesis in Sprague‐Dawley rats treated with 7,12‐dimethylbenz[a]anthracene, in both benign and malignant tumors, indicating that stable changes in PCPH expression took place early in the neoplastic process. Results showed that this experimental system is relevant to human breast carcinogenesis and provides an excellent model to study the molecular basis of the regulation of PCPH expression during normal differentiation and pathologic stages of neoplasia of the mammary gland and to analyze the role of PCPH in the carcinogenic process. Furthermore, the detection of atypical PCPH polypeptides in tumors suggests that PCPH immunodetection may be applied as a diagnostic tool for the early identification of neoplastic breast epithelial cells.

Quercetin as a Modulator of the Cellular Neoplastic Phenotype

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a widely distributed plant-derived flavonoid present in most vegetables and fruits, and it is therefore a common component of the human diet.1 Quercetin has been shown to exert multiple biochemical effects in mammalian cells, including the increase of cAMP levels,2 the inhibition of enzymatic activities such as protein kinase C,3,4 protein tyrosine kinases,5–7 and cAMP and cGMP phosphodiesterases,8,9 as well as the interaction with estrogen type II binding sites.10 These biological actions of quercetin may explain its predominantly inhibitory effect of tumor-derived cell lines,10–14 and its ability to arrest tumor cells in the G1 phase13,15 or, less frequently, in the G2 -M phase16 of the cell cycle. Although some reports17,18 indicate that quercetin could be carcinogenic under certain experimental conditions, the overwhelming evidence demonstrating its ability to inhibit the growth of tumor cells identifies quercetin as an anticarcinogenic phytochemical. Furthermore, the dosage of quercetin could be easily modified in the human diet to attain the appropriate levels for optimum cancer chemopreventive action.

cDNA isolation, expression, and chromosomal localization of the mouse Pcph proto-oncogene

We report here the isolation and characterization of a cDNA from mouse thymus encoding the murine homolog of the protein product of the Syrian hamster Pcph proto‐oncogene. The single open reading frame identified in the cDNA sequence encoded a protein predicted to have 428 amino acids, which shared 93.7% amino acid identity with the Syrian hamster Pcph within the first 412 residues but had a shorter, highly dissimilar C‐terminus. Northern and western analyses revealed that Pcph mRNA and protein were widely distributed in mouse embryo and adult tissues, with the highest expression in adults detected in kidney and liver. The mouse Pcph proto‐oncogene was mapped by linkage analysis to within 3.3±2.3 cM of Pkch‐rs1 on chromosome 12. These data should prove valuable in designing studies to define the cellular function of the Pcph proto‐oncogene. Mol. Carcinog. 26:130–136, 1999.

Increased manganese superoxide dismutase activity, protein, and mRNA levels and concurrent induction of tumor necrosis factor α in radiation‐initiated Syrian hamster cells

The levels of the antioxidant enzyme manganese superoxide dismutase (Mn‐SOD) are frequently decreased in tumor cells and increased in normal cells upon treatment with ionizing radiation. We studied Mn‐SOD at different stages during the neoplastic conversion of radiation‐initiated Syrian hamster embryo HDR‐3 cells. Mn‐SOD activity and the concentration of Mn‐SOD protein and mRNA increased gradually during the malignant transformation of HDR‐3 cells after radiation exposure; fully neoplastic cells showed greater Mn‐SOD levels than preneoplastic and normal 84‐3 cells. Inhibitors of superoxide (SO) anion production (thenoyltrifluoroacetone and rotenone) decreased the concentration of Mn‐SOD mRNA, raising the possibility that the generation of SO radicals participated in the upregulation of Mn‐SOD in cells transformed by exposure to radiation. We observed an increase in the concentration of tumor necrosis factor α (TNFα) in HDR‐3 cells relative to mock‐irradiated cells. Together with the observation that TNFα stimulates the production of SO by mitochondria and increases the level of Mn‐SOD mRNA in other experimental systems, our results suggest that as normal 84‐3 cells undergo malignant transformation induced by ionizing radiation they produce TNFα, to which the cells respond by increasing the concentration of Mn‐SOD mRNA and protein and the activity of the enzyme.

Plumbagin reduces human colon cancer cell survival by inducing cell cycle arrest and mitochondria-mediated apoptosis

Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.