Quercetin mediates the down-regulation of mutant p53 in the human breast cancer cell line MDA-MB468.

The effects of the bioflavonoid quercetin (3,3',4',5,7-pentahydroxyflavone) on the growth and cell cycle progression of the human breast cancer cell line MDA-MB468 have been studied. Quercetin inhibited cell proliferation with an IC50 (a drug concentration which inhibited growth by 50% following a 3-day exposure) value of 7 micrograms/ml. In actively growing cultures, the addition of quercetin resulted in the accumulation of cells at the G2-M phase. We have correlated these effects on cell proliferation with the observation that quercetin strongly inhibited, in a time- and dose-dependent fashion, the expression of the mutated p53 protein, which is the only form present at high levels in this cell line. This inhibition takes place at the translational level. Quercetin did not affect the steady-state mRNA levels of p53, but prevented the accumulation of newly synthesized p53 protein. This quercetin action appeared to be somewhat specific for p53 because the drug did not alter the amount of other proteins present in MDA-MB468 cells such as P-glycoprotein and did not prevent the induction of the synthesis of epidermal growth factor receptor in response to epidermal growth factor.

Activation of phospholipase D participates in signal transduction pathways responsive to gamma-radiation.

Early responses of mammalian cells to ionizing radiation include the activation of a protein kinase C implicated in the regulation of gene expression, the stimulation of tyrosine kinase activities, and the enhancement of phosphatidylinositol turnover. In the present report we show that clinically relevant doses of gamma-radiation (2 Gy) stimulate phosphatidylcholine hydrolysis in human squamous carcinoma cells. Radiation induced the accumulation of intracellular [3H]choline and the simultaneous increase in [3H]myristoyl-phosphatidic acid, followed by a small increase in the levels of [3H]myristoyl-diacylglycerol. Furthermore, in the presence of ethanol, gamma-radiation stimulated the appearance of [32P]phosphatidylethanol, an indicator of phospholipase D transphosphatidylation activity. These data demonstrate for the first time that phospholipase D activation participates in signaling pathways in response to gamma-radiation.

cph, a novel oncogene which cooperates with H-ras in the transformation of NIH3T3 fibroblasts.

We have performed the molecular cloning of the non-ras transforming sequences previously detected in neoplastic Syrian hamster embryo fibroblasts initiated in vitro with 3-methylcholanthrene (MCA) (Notario et al., 1990). These sequences were isolated using cosmid-rescue techniques from a third-cycle NIH3T3 transformant obtained by sequential transfections of genomic DNA from MCA-initiated hamster fetal cells. Rescued (C-5) clones encompassed about 42.5 kbp of Syrian hamster genomic DNA containing hamster-specific repetitive elements (HRS). An internal 19 kbp BamHI fragment (B-1) was the only C-5 fragment which recognized specific transcripts in poly(A)+ RNA from hamster embryo cells. The same mRNA species were present in both normal and MCA-initiated neoplastic cells: a major transcript of about 2.5 kb, and other less abundant ones, ranging from approximately 2.0 kb to 5.0 kb. These mRNA species were detected consistently by each of several B-1 DNA subfragments located at positions spanning almost the entire B-1 length. The nucleotide sequence of some transcript-positive (S5P2 and S6) genomic B-1 fragments was determined. No significant homology exists between the nucleotide sequences of these B-1 subfragments and established DNA databases. Therefore, the C-5 cosmid clone contains novel genomic sequences. Transfection of C-5 DNA into mouse NIH3T3 cells resulted in the appearance of transformed foci (about five foci per microgram of DNA) within 25 days post-transfection, thus demonstrating the transforming activity of the C-5 clone, which was consequently renamed as the cph oncogene. Co-transfection of the cph oncogene with the human H-ras oncogene (T24), demonstrated a synergistic action between the two oncogenes in the transformation of murine fibroblasts.

Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X.

Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe.

Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity. Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated. The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway. Cells disrupted for wis] or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished. In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells. Taken together, the data suggest that the elevation of trehalase activity in Schiz. pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress. The wisl-phhl MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress.

Trehalose-6P synthase is essential for trehalase activation triggered by glucose, nitrogen source or heat shock, but not by osmostress, in Schizosaccharomyces pombe.

Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source. Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock. Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock. In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar. Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells. Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells. These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock. However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz. pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress.

Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe.

We have cloned and sequenced the ntp1+ gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe. The ntp1+ gene product (Ntp1p) showed a 45-55% identity with neutral trehalases from other yeasts at the amino acid sequence level. However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p. Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1+ expression during osmostress.

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.

Activation of neutral trehalase by glucose and nitrogen source in Schizosaccharomyces pombe strains deficient in cAMP-dependent protein kinase activity.

Schizosaccharomyces pombe cells carrying a disruption in the PKA1 gene, that encodes the catalytic subunit of cAMP-dependent protein kinase (PKA), lacked the glucose- and nitrogen-source-induced activation of trehalase at stationary-phase but rised trehalase activity in response to these compounds during the exponential phase of growth. Treatment by phosphatase of either glucose- or nitrogen-source-activated trehalase resulted in trehalase deactivation suggesting that phosphorylation of the enzyme protein occurs during activation. These data indicate that in growing cells of this yeast the mechanism responsible for the activation of trehalase can be independent of interactions with free catalytic subunits of PKA and related to a signaling pathway involving a type of protein kinase different from PKA.

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.

Curing of the killer character of Saccharomyces cerevisiae with acridine orange.

Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains). The results showed a high curing percentage of the killer character. This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation. The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger.

Resting cells of the fission yeast Schizosaccharomyces pombe, suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen tannophilus, the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.

Characterization of killer-resistant strains of Saccharomyces cerevisiae isolated from spontaneous fermentations.

A study of 26 killer-resistant wine strains of Saccharomyces cerevisiae, isolated during spontaneous fermentations in three vineyards in NW Spain, was carried out employing several methods that included a spheroplast-killing assay and analysis of chromosomal DNA patterns by pulse-field agarose electrophoresis. The results showed that 92% of the strains were derivatives of K2 killer toxin producing wine strains isolated from the same fermentations, and that they could be grouped into four different karyotypes. The remaining strains were killer-resistant at cell-wall level and were not related to the others, as was demonstrated by the absence of L and M ds-RNAs and by their different karyotypes.

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe.

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.

Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae.

The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1. Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11. The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3) that, however, was unable to suppress other FLO1 genes in other flocculent strains.

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations.

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe.

The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.

[Yeasts associated with spontaneous fermentation processes in wines from Ribeiro. Analysis of homo/heterothallism and the killer system of S. cerevisiae strains]. / Levaduras asociadas a procesos de fermentación espontánea en vinos de Ribeiro. Análisis del homo/heterotalismo y sistema killer de las cepas de S. cerevisiae.

A taxonomic analysis has been carried out concerning the different yeast species involved in spontaneous fermentations of Ribeiro wine. The results revealed the presence of 15 species belonging to 7 genera: Saccharomyces, Torulaspora, Dekkera, Saccharomycodes, Debaryomyces, Kloeckera and Candida. Within the genus Candida a high number of species was detected. The 28 isolated strains of S. cerevisiae showed with some exceptions, a remarkable sporulation ability and viability of the meiotic products. All the strains were homothallic. Among all S. cerevisiae strains, 6 exhibited killer K2 activity with higher isolation percentages in intermediate fermentations than in late fermentations.

Improvement of the alcoholic fermentation of grape juice with mixed cultures of Saccharomyces cerevisiae wild strains. Negative effect of Kloeckera apiculata.

Four assays were designed to study the fermentative behaviour of two selected wild strains of Saccharomyces cerevislae when inoculated in sterilized grape juice either alone or in association. In addition, a wild strain of Kloeckera apiculata was employed for studying and characterizing the influence of this micro-organism on the fermentations when associated with Sacch. cerevisiae. Fermentation was improved when the two strains of Sacch. cerevisiae were grown together since the residual sugar fell to 0.12% and ethanol increased up to more than 11% (v/v). In this case, a proliferation of the two strains with successive exponential phases was observed. On the other hand, when Kloeckera apiculata was incorporated into the fermentations, these were incomplete with a residual sugar level of 1.64% and a final ethanol concentration of 6.4%.