Geraniol, a component of plant essential oils, sensitizes human colonic cancer cells to 5-Fluorouracil treatment.

Differentiation of human colonic cancer cells at confluency has been correlated to their increased resistance to chemotherapeutic agents. The aim of this study was to determine whether blocking Caco-2 cell differentiation could sensitize the cells to 5-fluorouracil (5-FU) treatment. We show that in cells at confluency, geraniol (400 microM) prevented the formation of brush-border membranes and inhibited the expression of intestinal hydrolases (sucrase, lactase, alkaline phosphatase). The antiproliferative effect of geraniol (400 microM) together with 5-FU (5 microM) was twice that of 5-FU alone. The cytotoxicity induced by 5-FU was enhanced in the presence of geraniol, as shown by a 50% increase of lactate dehydrogenase release in the culture medium. These effects are related to enhanced intracellular accumulation of 5-FU in the presence of geraniol as shown by a 2-fold increase in intracellular 5-[6-(3)H]FU (1.5 microCi/ml). It is concluded that geraniol sensitizes colonic cancer cells to 5-FU treatment, by increasing the cytotoxicity of the drug, and that this results from the facilitated transport of 5-FU and the blockade of the morphological and functional differentiation of the cancer cells.

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.

New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism.

Cytidine metabolism in the yeast Saccharomyces cerevisiae was analyzed by genetic and biochemical approaches. Disruption of a unique ORF (Genbank accession No. U 20865) bearing homology with eucaryotic or bacterial cytidine deaminases abolished cytidine deaminase activity and resulted in 5-fluorocytidine resistance. The gene encoding cytidine deaminase will be referred to as CDD1 (Genbank accession number AF080089). The ability to isolate mutants resistant to 5-fluorocytidine which mapped to five other loci demonstrated the existence of a complex cytidine metabolic network. Deciphering this network revealed several original features:(1) cytidine entry is mediated by the purine-cytosine transporter (Fcy2p),(2) cytidine is cleaved into cytosine by the uridine nucleosidase (Urh1p),(3) cytidine is phosphorylated into CMP by the uridine kinase (Urk1p),(4) a block in cytosine deaminase (Fcy1p), but not in cytidine deaminase (Cdd1p), constitutes a limiting step in cytidine utilisation as a UMP precursor.

Dihydroorotate (dhout) and orotate (orout) utilizer mutants in yeast: identification of the dhout mutation and allelism of the DHO and URE2 genes.

We induced by UV mutagenesis a series of yeast mutants that were able to utilize dihydroorotic (dhout) and orotic acid (orout) as precursors for pyrimidine biosynthesis. These recessive mutations defined three complementation groups named dhout, orout1 and orout2. The wild-type allele of the gene responsible for dihydroorotate utilization was cloned using the sensitivity of the dhout mutant to 5-fluoroorotate. The DHO gene was sequenced and found to be identical to the URE2 gene. The dhout mutation resulted from the introduction of a stop codon instead of a glutamine at position 59, which led to the production of a truncated Ure2p. Therefore, the URE2 and DHO genes are alleles in yeast.

Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans.

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10(-2) M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected.

6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae.

The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae. In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceding effect. PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wild-type, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.

cis- and trans-acting regulatory elements of the yeast URA3 promoter.

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

[Study of several parameters of magnesium metabolism in spontaneously hypertensive rats]. / Etude de quelques paramètres du métabolisme du magnésium chez le rat spontanément hypertendu.

We have compared a few parameters of magnesium metabolism in spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). The results show a decrease of plasma magnesium and urinary magnesium. There is no difference in fecal magnesium and in balance of magnesium. These results have raised a question as to the importance of changes in magnesium metabolism in the mechanism of hypertension.

Complete sequence of a eukaryotic regulatory gene.

Dihydroorotase, the third enzymatic activity of the pyrimidine pathway, is encoded in Saccharomyces cerevisiae by a single gene URA4, which is induced at the transcriptional level by accumulation of ureidosuccinic acid. A regulatory gene PPR2 (pyrimidine pathway regulatory 2) acting specifically on this step, has been characterized, cloned and sequenced. The main open reading frame is 384 nucleotides long and potentially codes for a basic protein, favoring a molecular mechanism involving direct binding of a regulatory protein to DNA. The short length of the PPR2 polypeptide chain and the presence of seven cysteine residues suggest that the active form of the protein is an oligomer assembled through disulphide bonds. An uninducible allele has been cloned and sequenced. The mutation corresponds to an A leads to T transversion changing a lysine triplet into an ochre codon. The uninducible phenotype of this mutant is completely suppressed by an ochre suppressor, strengthening the hypothesis that PPR2 acts on URA4 transcription through the synthesis of a regulatory protein.

Genetic evidence for the creation of a reinitiation site by mutation inside the yeast ura 2 gene.

The ura 2 gene of yeast codes for two enzymatic activities which are translated from a unique messenger RNA in the order carbamoyl-phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) (Lacroute, 1968; Denis-Duphil and Kaplan, 1976). Nonsense mutations in the CPSPase region cause a complete loss in ATCase activity by a total polar effect, characteristic of eukaryotic mRNA translation, and due to the unique site of protein initiation present on each messenger (Shaffer et al., 1969). A triple nonsense mutant in the CPSase has been constructed by recombination and ATCase+ revertants have been selected from it. Among seventeen revertants obtained, three had a deletion covering the three nonsense mutations relieving thus the polar effect (Fink and Styles, 1974) but fourteen others examined had retained all the CPSase DNA including the three nonsense mutations; this can be explained in the present state of knowledge only by the creation by mutation of reinitiation site either for transcription or for translation in the region of the ura 2 gene distal to the last nonsense mutation.

Dominant and semidominant mutations leading to thermosensitivity of ribonucleic acid biosynthesis in Saccharomyces cerevisiae.

Different dominant thermosensitive mutations affecting the same gene were selected in Saccharomyces cerevisiae. Ribonucleic acid (RNA) synthesis decreased rapidly and markedly at 37 C in all the mutants whether they were in a homozygous or a heterozygous state. Protein biosynthesis was at first unaffected and then decreased slowly, stopping after 5 h. Measurements of RNA biosynthesis in isolated nuclei as well as in vitro activities of RNA polymerases A and B at 22 and 37 C failed to reveal any difference between mutants and the wild type. Analysis of the nature of the residual RNAs synthesized at the high temperature in the mutants showed a small relative increase in the messenger RNA fraction, but it was not sufficient to indicate a specific inactivation of RNA polymerase A activity. The results suggest an impairment in a common regulatory element for all RNA polymerases acting at the level of the initiation of transcription. Similar mutants with a semidominant phenotype were obtained in which the lesions were in two other unlinked loci.