Terbinafine resistance in a pleiotropic yeast mutant is caused by a single point mutation in the ERG1 gene

Klobučnı́ková, Vlasta; Kohút, P; Leber, Regina; Fuchsbichler, Sandra; Schweighofer, Natascha; Turnowsky, Friederike; Hapala, Ivan

doi:10.1016/j.bbrc.2003.08.051

Cited by 43 publications

(38 citation statements)

References 20 publications

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“…The wild-type ERG1 gene was randomly mutagenized in vitro by PCR amplification, as described previously (13). The PCR fragments were digested with PstI, cloned into the centromere vector pRS315, and transformed into E. coli XL1.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid DNA was isolated from approximately 1,000 E. coli transformants by standard procedures (29), and the combined recombinant plasmids were transformed into S. cerevisiae KLN1 (14) by the protocol of Gietz et al (9). Transformants expressing functional SE were selected by complementation of the aerobic growth defect of KLN1 on YPD agar plates (13). The transformants were tested for terbinafine sensitivity on YPD plates containing 10 g/ml of terbinafine.…”

Section: Methodsmentioning

confidence: 99%

“…After 8 h (exponential phase) and 24 h (stationary phase), cells were collected by centrifugation, washed twice in distilled water, and diluted in water to a final concentration of 1 ϫ 10 9 cells/ml; 1 ml of this suspension was taken for lipid extraction. Extraction and detection of lipids were performed as described previously (13). Thin-layer chromatography (TLC) plates were analyzed by using a personal molecular imager (FX; Bio-Rad) and were quantified by the use of QuantityOne software (Bio-Rad) or, alternatively, by determining the radioactivity in individual lipid spots by liquid scintillation counting.…”

Section: Methodsmentioning

confidence: 99%

“…The amplified PCR fragments were used to replace the corresponding wild-type sequence in the ERG1 gene contained in recombinant plasmid pNS1 (13). The correct sequences were confirmed by DNA sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently reported on terbinafine-resistant S. cerevisiae mutants, all of which carry unique amino acid substitutions in the Erg1 protein (13,15). Interestingly, the amino acid residues affected are conserved in the squalene epoxidases of various origins and are distributed along the sequence of the Erg1 protein (25).…”

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confidence: 99%

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Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

Ruckenstuhl

Lang

Poschenel

et al. 2007

Antimicrob Agents Chemother

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Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G 25 S, D 335 X (W, F, P), and G 210 A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G 30 S and L 37 P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R 269 G variant exhibited moderately reduced SE activity and a 5-to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

Ruckenstuhl

Lang

Poschenel

et al. 2007

Antimicrob Agents Chemother

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Inhibition of human vascular endothelial cells proliferation by terbinafine

Liang

et al. 2004

Intl Journal of Cancer

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Terbinafine inhibits oral squamous cell carcinoma growth through anti‐cancer cell proliferation and anti‐angiogenesis

Chien

Lee

Kao

et al. 2011

Molecular Carcinogenesis

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Terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, has been reported to exert an anti-tumor effect in various cancer cells. However, the effect of TB on oral cancer has not been evaluated. Herein we demonstrate that TB (0-60 µM) concentration-dependently decreased cell number in cultured human oral squamous cell carcinoma (OSCC), KB cells. The anti-proliferation effect of TB was also observed in two other OSCC cell lines, SAS and SCC 15. TB (60 µM) was not cytotoxic and its inhibition on KB cell growth was reversible. [(3) H]thymidine incorporation and flow cytometric analyses revealed that TB-inhibited DNA synthesis and induced the G0/G1 cell-cycle arrest. The TB-induced cell-cycle arrest occurred when the cyclin-dependent kinase 2 activity was inhibited just as the protein levels of p21(cip1) and p27(kip1) were increased. The TB-induced G0/G1 cell-cycle arrest was completely blocked when the expressions of p21(cip1) and p27(kip1) were knocked-down together. Taken together, these results suggest that the p21(cip1) - and p27(kip1) -associated signaling pathways might be involved in the TB-induced anti-proliferation in KB cells. In vivo, TB (50 mg/kg, i.p.) significantly inhibited the KB tumor size. In these TB-treated tumors, increases in the levels of p21(cip1) and p27(kip1) protein and decreases in the number of proliferating cell nuclear antigen-positive cells and the microvessel density were observed. These findings demonstrate for the first time that TB might have potential to serve as a therapeutic tool in the treatment of oral cancer.

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Terbinafine resistance in a pleiotropic yeast mutant is caused by a single point mutation in the ERG1 gene

Cited by 43 publications

References 20 publications

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

Inhibition of human vascular endothelial cells proliferation by terbinafine

Terbinafine inhibits oral squamous cell carcinoma growth through anti‐cancer cell proliferation and anti‐angiogenesis

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