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At least 19 recordsLinked to original sources
PubMed1990

Induction of the expression of differentiation-related antigens on human colon carcinoma cells by stimulating protein kinase C.

This study was undertaken to determine whether the phorbol diester, phorbol 12-myristate 13-acetate (PMA), causes differentiation of the human colon carcinoma cell line, SW 48. Under routine growth conditions, the cells are round, have a high nuclear-to-cytoplasmic ratio, and lack cytoplasmic vacuoles. After treatment for 1 hour with 100 nmol/L of PMA at 37 degrees C, the cells assumed a spread-out, flasklike shape, displayed a low nuclear-to-cytoplasmic ratio, and exhibited cytoplasmic vacuoles. An inert but lipophilic phorbol diester, 4 phorbol 12,13-didecanoate, failed to induce these morphological changes. Cell kinetic studies showed that whereas SW 48 cells have a doubling time of 35 hours, those incubated with 100 nmol/L of PMA have a doubling time of 90 hours. Although the flow cytometry histograms were similar until 8 hours into the cell cycle, the PMA-treated cells ultimately spent proportionately less time in S and more in G2/M. Finally, under routine growth conditions, SW 48 cells express neither carcinoembryonic antigen nor G7 antigen. These antigens, which are present on the surface of well-differentiated cells, were expressed after treatment of SW 48 with PMA. The data suggest that PMA causes profound changes in structure, cell growth kinetics, and antigen expression, consistent with induction of differentiation of the cell line SW 48.

Antibodies, Monoclonal↗
PubMed1992

Post-transcriptional regulation of urokinase plasminogen activator gene expression occurs in the nucleus of BC1 rat mammary tumor cells.

The regulation of urokinase plasminogen activator (uPA) expression was investigated in 2 highly metastatic rat mammary adenocarcinoma cell lines, BC1 and MAT 13762. BC1 cells were observed to synthesize, on average, 10 times less uPA enzyme and mRNA than MAT 13762 cells; however this difference was not accounted for by differences in uPA gene copy number/structure or in the rate of uPA gene transcription in the cell lines studied. Moreover, Northern blot analysis of invasive sub-populations derived in vitro from the BC1 cell line revealed levels of uPA expression similar to those of the parent, but a 3-fold elevation in expression of the metalloprotease gene, transin. Further investigation showed that treatment of BC1 cells with either of the protein synthesis inhibitors, cycloheximide or anisomycin, increased the level of both nuclear and cytoplasmic uPA RNA 6- to 18-fold in 4 hr, whilst inducing a maximum 2.6-fold increase in the rate of uPA gene transcription. This increase in uPA gene expression may therefore reflect, in part, an increase in the stability and/or processing of nuclear uPA transcripts. These results suggest that the degree of uPA gene expression does not correlate directly with BC1 tumor-cell invasion in vitro, and that the uPA gene is down-regulated, at least in part, post-transcriptionally in the nucleus of BC1 mammary tumor cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Clonal variation of expression of the genes coding for plasminogen activators, their inhibitors and the urokinase receptor in HT1080 sarcoma cells.

The human sarcoma cell line HT1080 was found, by in situ hybridization, to consist of cells expressing various levels of urokinase (uPA) and tissue type (tPA) plasminogen activator (PA) suggesting clonal variation of expression of these genes. Colonies originating from single HT1080 cells were, therefore, established and screened for PA activity using a fibrin agarose overlay. Colonies inducing lysis (clone C+ and H+) or no lysis (clones B- and M-) were isolated and tested for mRNA levels of uPA, tPA, uPA receptor (uPAR) and the 3 PA inhibitors (PAI), PAI-1, PAI-2 and protease-nexin I. The different clones revealed considerable variation of expression of the different PA and PAI genes, with lysis-inducing clones expressing mainly the PA genes, whereas non-lysing clones demonstrated higher expression of the PAI genes. Amplification or loss of specific genes was excluded by Southern blotting. The protein levels of cellular and secreted PA and PAI determined by ELISA and Western blots demonstrated a pattern similar to that observed for PA and PAI mRNA concentrations, suggesting clonal differences either on the level of transcription or in RNA processing and/or stability. Due to complex interactions between PA and PAI, neither mRNA nor protein levels of the different genes were predictive for the amount of functional PA activity present in the supernatant or on the cell surface of the different clones. Receptor-bound uPA activity was found to be considerably higher in lysis-inducing than in non-lysing clones and the activity was dependent on neutralization by PAI-1 rather than on the level of uPAR mRNA.

Fibrinolysin↗
PubMed1989

Cell-type-specific expression of mouse DNA polymerase beta-gene is regulated by silencer elements.

RNA blot hybridization analysis revealed that the steady-state level of DNA polymerase beta-mRNA in mouse neuroblastoma N18TG2 cells was approximately five-fold higher than that in NIH/3T3 cells. In order to examine the function of DNA polymerase beta-gene silencers in these two cell lines, we employed a chloramphenicol acetyltransferase (CAT)-transient expression assay using the CAT plasmids containing the silencers linked to various promoter-enhancers. In NIH/3T3 cells, DNA polymerase beta-gene silencers effectively repressed the function of its own promoter and those of several other heterologous promoter-enhancers. In contrast, the silencers only marginally affected the CAT expression directed by DNA polymerase beta-gene promoter and heterologous promoter-enhancers in N18TG2 cells. The extent of the increase of CAT expression by removing silencer elements in NIH/3T3 cells was very similar to the ratio of DNA polymerase beta-mRNA content in N18TG2 cells to that in NIH/3T3 cells. These results indicate that cell-type-specific expression of DNA polymerase beta-gene is primarily controlled by the function of its silencer elements.

Animals↗
PubMed1991

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.

Blotting, Northern↗
PubMed1992

Transcripts encoding protein kinase C-alpha, -delta, -epsilon, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells.

The protein kinase C (PKC) family of phospholipid-dependent serine-threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca(2+)-mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca2+. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta, but not PKC-beta or -gamma. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca2+. Interestingly, the PKC-zeta transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme. An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca2+ or the PKC activator 12-O-tetradecanoylphorbol-13-acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP-1, which contain an activating mutation of the c-Ha-ras gene. Like normal keratinocytes. 308 and SP-1 cells expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta. However, the abundance of PKC-zeta transcripts in both cell lines was reduced by 74-89% when compared with normal keratinocytes at similar Ca2+ levels. In addition, SP-1 but not 308 cells exhibited a sevenfold increase in PKC-eta mRNA when cultured in medium with 1.4 mM Ca2+. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v-Ha-ras oncogene. As with normal, 308, and SP-1 cells, v-Ha-ras keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta and -eta. The level of PKC-zeta transcripts was similar in normal and v-Ha-ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP-1 cells was not a direct result of ras activation. As in SP-1 cells, PKC-eta in v-Ha-ras keratinocytes was responsive to extracellular Ca2+, with a four-fold increase in transcript abundance in 0.12 mM Ca2+ medium relative to 0.05 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Characterization of the cytochrome c gene from the starch-fermenting yeast Schwanniomyces occidentalis and its expression in Baker's yeast.

A cytochrome c protein gene, CYC10, of the dextran- and starch-fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein-coding region shares close homologies to the cytochrome c genes. A S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein-encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiae. The CYC10 gene was oxygen-induced but not subject to catabolite repression. The expression of the CYC10 gene was studied in the heterologous yeast S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indicating that these two genes share similar or closely related cis- and trans-acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. occidentalis cells. Search in the 5' untranslated region of the CYC10 gene revealed some homologies at -425 to -405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae. Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.

Amino Acid Sequence↗
PubMedDate not supplied

Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis.

Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.

Alcohol Dehydrogenase↗
PubMed1992

Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene.

The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.

Base Sequence↗
PubMed1990

Stromelysin in tumor progression and metastasis.

There are several characteristics of stromelysin that suggest that expression of this enzyme may play an important role in tumor invasion and metastasis; the stromelysin gene is expressed in response to stimulation by oncogenes and tumor promoters, and the protein product of this gene is a metalloproteinase capable of degrading multiple components of the extracellular matrix. Experimental evidence to support this hypothesis has been derived from several animal model systems, in which a positive correlation has been observed between stromelysin expression and tumor progression and metastasis. In addition, in vivo experiments in which the levels of TIMP, the tissue inhibitor of metalloproteinases, were altered also strongly suggest a causal role for metalloproteinases in tumor metastases. The expression of active stromelysin in tumor cells requires the fulfillment of several criteria, and this multistep process is reminiscent of the molecular events that are currently understood to contribute to tumor progression and carcinogenesis. Expression of stromelysin mRNA requires both a stimulus, a step which may correspond to the activation of an oncogene in multistep carcinogenesis, as well as the lifting of transcriptional repression, which may correspond to the loss of tumor suppressor function. Both positive and negative modulation of stromelysin transcription appear to utilize pathways that involve the protooncogenes c-fos and/or c-jun. The expression of active stromelysin enzyme also requires conversion of the proenzyme to an active form, and a proper balance between the expression of inhibitors and the levels of active enzyme. The multiple levels of stromelysin regulation support the concept of multistep carcinogenesis and may provide a tool for further understanding of the molecular nature of the events that lead to tumor progression, invasion, and metastasis.

Animals↗
PubMed1990

Cathepsin D: a protease involved in breast cancer metastasis.

Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Ad12 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.

Breast Neoplasms↗
PubMed1990

Regulation of chorismate mutase in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ARO7 gene was cloned by screening a wild-type gene bank for complementation of an aro7 auxotrophic mutant. In vitro mutagenesis of the isolated plasmid (pJFB1) gave several transformants resistant to levels of the phenylalanine analogue 2-thienylalanine inhibitory to the wild-type transformant. Chorismate mutase assays indicated that two of the mutants (J14-26IV6 and J14-26IV9) were resistant to feedback inhibition by tyrosine displayed by wild-type strains. Analysis of the effect of other aromatic amino acids on chorismate mutase activity showed that tryptophan counteracted this inhibition. Analysis of the effect of tyrosine in the growth medium on enzyme activity indicated that the wild-type ARO7 gene was repressed by tyrosine, a phenomenon not previously reported. Two of the 2-thienylalanine resistant mutants (J14-26IV3 and J14-26IV9) appeared to be resistant to this repression. Transcriptional analysis confirmed that the level of ARO7 transcript decreased with increasing tyrosine concentration. In stain J14-26IV9 the ARO7 transcript level was not affected. J14-26IV9, therefore, appears to be a double mutant, resistant to both feedback inhibition and repression by tyrosine.

Chorismate Mutase↗
PubMed1991

Positive and negative elements upstream of the meiosis-specific glucoamylase gene in Saccharomyces cerevisiae.

The SGA1 gene encoding glucoamylase is specifically expressed late in meiotic development of the yeast Saccharomyces cerevisiae. We found that accumulation of both enzyme activity and transcripts was regulated negatively by both nutritional signals and a haploid-specific negative regulator gene of meiosis. RME1, and positively by the inducer genes for meiosis, IME1 and IME2. To study the role of sequences upstream of the SGA1 gene in its expression and regulation, we generated internal deletions in the 5' non-coding region of the gene and chimeric genes with portions of the upstream sequence inserted into a reporter gene. By analyzing the expression of these genes, we have identified both a 19 bp upstream activation sequence (UAS) and a 49 bp negatively regulating element (NRE). The UAS activated transcription with no requirement for heterozygosity at the mating-type locus, but this activation was still under negative control by nutrients. The NRE showed no UAS-like activity but conferred IME2-dependent (or meiosis-specific) expression on a heterologous promoter. These results suggest that meiosis-specific expression of the SGA1 gene is established by a regulatory hierarchy including positive and negative factors, the actions of which are mediated through the two separate upstream regulatory elements, UAS and NRE, respectively. Also, that two independently acting cascades exist for the regulation of SGA1 expression: one transduces both the mating-type and nutritional signals and includes the IME2 product, which acts to relieve the repression through NRE; and another transduces only the nutritional signal independently of the above pathway and inhibits positive factors acting on UAS.

Autoradiography↗
PubMed1991

The CCR1 (SNF1) and SCH9 protein kinases act independently of cAMP-dependent protein kinase and the transcriptional activator ADR1 in controlling yeast ADH2 expression.

cAMP-dependent protein kinase (cAPK) is implicated in the inactivation of the yeast transcriptional activator ADR1, which regulates glucose-repressible ADH2 gene expression. The interdependence of cAPK, SCH9 (a protein kinase that when overexpressed can functionally substitute for cAPK), and the CCR1 (SNF1) protein kinase that is required for ADH2 expression was studied. SCH9 was found to be required for ADH2 expression in contrast to the inhibitory role played by cAPK. CCR1 and SCH9 were observed to affect ADH2 expression independently of both ADR1 and cAPK. In contrast, cAPK was shown to exert its effects on ADH2 solely through ADR1. These results indicate that the SCH9 and CCR1 protein kinases are components of regulatory pathways separate from that utilized by cAPK to control ADR1 activity and ADH2 expression.

Alcohol Dehydrogenase↗
PubMed1992

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.

The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Amino Acid Sequence↗
PubMed1992

Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans.

The amdR gene of Aspergillus nidulans regulates a number of structural genes in response to omega amino acid inducers. The site of action of the amdR product on expression of the amdS gene was investigated by studying the effects of changes in the 5' region of amdS, generated in vitro, on the induction, and on responses of an amdS-lacZ fusion gene to an amdRc allele. A sequence was identified that is sufficient for amdR regulation and that shows identity with sequences involved in amdR regulation of the gatA and lam genes. This sequence includes a CCAAT sequence and it was shown that this sequence is an important element in setting the basal level of amdS expression.

Amidohydrolases↗
PubMed1992

Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation.

The regulation of the expression of the A. nidulans niiA and niaD genes (encoding nitrite reductase and nitrate reductase activities, respectively) was investigated by Northern blotting. It was demonstrated that expression of the niiA and niaD genes is controlled at the level of mRNA accumulation and that mutations within the nirA and areA regulatory genes, as well as certain mutations within niaD itself or cnxE (for its molybdenum cofactor), markedly affect niiA and niaD transcript levels.

Aspergillus nidulans↗