RESUMEN
Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.
Asunto(s)
Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Mucinas/genética , Neoplasias Gástricas/genética , Empalme Alternativo , Secuencia de Bases , Biopsia , Western Blotting , Células Epiteliales/metabolismo , Mucosa Gástrica/patología , Humanos , Inmunohistoquímica , Mucinas/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
Helicobacter pylori shows a rather high variability of several biochemical markers including lipopolysaccharide structures. This study aimed to determine whether Helicobacter pylori has a potential for phenotypic variability and to describe its effects on bacterial pathogenesis. From colonies of three clinical strains of Helicobacter pylori with rough (R) colony morphology, spontaneous phenotypic variants with smooth (S) colony morphology were isolated that occurred with a frequency of 10(-2) to 10(-3), irrespective of growth conditions. R-variant bacteria produced exclusively low-molecular-mass lipopolysaccharide. They exhibited increased lysis in the presence of plain air. In contrast, the S variants produced low- and high-molecular-mass lipopolysaccharide and did not exhibit increased lysis in the presence of plain air. Cocultivation of bacterial cells with AGS stomach cancer cells revealed that R-variant bacteria but not S-variant bacteria effected an inhibition of high molecular-weight glycoprotein biosynthesis and secretion by the host cells. Skirrow supplement added as selective agent to liquid and/or solid media was tolerated to a similar extent among R- and S-variant bacteria, while all variants proved sensitive to metronidazole, amoxicillin and clarithromycin except for the R and S isolates of strain Hp57, which showed resistance to the latter compound. It was concluded that R- and S-variants of Helicobacter pylori may have distinct roles in pathogenesis; nevertheless, these bacteria may be isolated by traditional methods and eradicated by conventional anti-infective therapy.
Asunto(s)
Antibacterianos/farmacología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Fenotipo , Células Tumorales CultivadasRESUMEN
A clone from a lambda gt11 cDNA expression library of HeLa cells was isolated, sequenced, and shown to encode a new human zinc finger protein. The cDNA of the gene termed ZFP161 has an open reading frame of 1347 bp. The predicted protein comprises 449 amino acid residues and contains five zinc finger motifs of the Krüppel type near the C-terminus and a BTB/POZ domain in the N-terminal region. The protein is 98% homologous to a murine zinc finger protein, ZF5 (M. Numoto et al., 1993, Nucleic Acids Res. 21: 3767-3775), which is a putative transcriptional repressor of c-myc and exhibits growth-suppressive activity in mouse cell lines. Through the use of a panel of somatic cell hybrids for chromosomal assignment and DNAs of somatic cell hybrids containing a deleted chromosome 18 for fine mapping, the human gene ZFP161 was localized to 18p11.21-pter. Therefore, ZFP161 is a candidate gene by position for the holoprosencephaly type 4 gene, HPE4, which is involved in congenital malformations. With DNAs from an interspecific backcross, two homologous mouse genes, Zfp161 and Zfp161-rs1, were mapped to chromosome 17 and the X chromosome, respectively. Mapping of Zfp161 confirms and extends a region of homology between distal mouse chromosome 17 and human 18p.
Asunto(s)
Cromosomas Humanos Par 18/genética , Genes myc/genética , Proteínas Represoras/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Holoprosencefalia/genética , Humanos , Factores de Transcripción de Tipo Kruppel , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares , Proteínas Represoras/química , Proteínas Represoras/fisiología , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Homología de Secuencia de Aminoácido , Cromosoma X/genéticaRESUMEN
The distribution of gelsolin, a calcium-dependent actin-modulating protein, and the expression of the corresponding gene, have been characterized with respect to morphogenetic processes in mouse ovary. Substantial amounts of gelsolin have been detected in the ovary and uterus of the mouse by immunoblot analysis. The similar relative ratio of mRNA of alpha-smooth muscle actin (alpha-SM actin) and gelsolin in the two organs suggests that expression of these two genes is coordinated at the transcriptional level. Immunofluorescence has demonstrated gelsolin predominantly in three types of cells in the ovary: (1) cells of the theca externa and stroma, (2) endothelial cells lining blood vessels, and (3) cells of the superficial epithelium of ovary. In the smooth-muscle-like cells of the theca externa, gelsolin appears tightly associated with the microfilamentous cytoskeleton, which is also rich in alpha-SM actin. The presence of gelsolin in myoid cells suggests that this protein, possibly by modulation of the activity of the actomyosin ATPase, plays a critical role in contractile and morphogenetic processes, e.g., during growth and maturation of the follicle or during ovulation. In cells of the endothelium, intracellular gelsolin is associated with the F-actin cytoskeleton around the nucleus. The circumferential belt lining the lateral cell membranes in cells of the superficial epithelium at the ovarian surface is also rich in gelsolin. Our observations indicate that the function of gelsolin as a calcium- and phospholipid-dependent modulator of actin assemblies is pivotal for the regulation of the dynamic alterations of the actin cytoskeleton in the superficial epithelium when cells become attenuated and retract their microvilli during growth of the follicle.