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Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

Ojini I, Gammie A - G3 (Bethesda) (2015)

Bottom Line: A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring.Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis.Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544.

No MeSH data available.


Related in: MedlinePlus

Verification of resistance discovery method using camptothecin. (A) Chemical structure of camptothecin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 9 μM camptothecin (Camp). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the TOP1 locus on chromosome XV (chrXV) conferring resistance to camptothecin. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in TOP1 conferring resistance to camptothecin. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because TOP1 is in the opposite orientation (chrXV:315387-313078) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run (indicated in red) is indicated in a format described in Figure 2.
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fig3: Verification of resistance discovery method using camptothecin. (A) Chemical structure of camptothecin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 9 μM camptothecin (Camp). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the TOP1 locus on chromosome XV (chrXV) conferring resistance to camptothecin. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in TOP1 conferring resistance to camptothecin. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because TOP1 is in the opposite orientation (chrXV:315387-313078) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run (indicated in red) is indicated in a format described in Figure 2.

Mentions: The anticancer compound camptothecin (Figure 3A) is a well-characterized topoisomerase I inhibitor that targets the TOP1 gene (Knab et al. 1993). Consistent with a mutational event resistance phenotype, the camptothecin resistance is characterized by a long lag phase (Figure 3B). As with canavanine, we identified six independent isolates with mutations in the TOP1 gene (Figure 3C). All six mutations represented frameshift mutations within homopolymer nucleotide stretches (Figure 3D). Taken together, this method proved to be a simple and rapid way to identify the drug resistance target for compounds when the loss of function of a single locus confers resistance.


Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

Ojini I, Gammie A - G3 (Bethesda) (2015)

Verification of resistance discovery method using camptothecin. (A) Chemical structure of camptothecin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 9 μM camptothecin (Camp). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the TOP1 locus on chromosome XV (chrXV) conferring resistance to camptothecin. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in TOP1 conferring resistance to camptothecin. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because TOP1 is in the opposite orientation (chrXV:315387-313078) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run (indicated in red) is indicated in a format described in Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4555229&req=5

fig3: Verification of resistance discovery method using camptothecin. (A) Chemical structure of camptothecin. The structure was rendered using ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence (No Drug) and presence of 9 μM camptothecin (Camp). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. (C) Schematic representation of the frameshift positions within the TOP1 locus on chromosome XV (chrXV) conferring resistance to camptothecin. The numbers indicate the chromosomal position. The mutations all resulted in frameshifts at homopolymers detailed in the bottom panel. (D) A table listing the mutations in TOP1 conferring resistance to camptothecin. The nucleotide position for each mutation is shown along with the region mutated. The sequence given corresponds to the strand in the W303 reference genome. Because TOP1 is in the opposite orientation (chrXV:315387-313078) within the W303 reference genome, the sequence shown is the reverse complement of the reference genome for the given interval. The nucleotide numbers differ slightly from the S288C draft genome. The specific insertion or deletion at the homopolymeric run (indicated in red) is indicated in a format described in Figure 2.
Mentions: The anticancer compound camptothecin (Figure 3A) is a well-characterized topoisomerase I inhibitor that targets the TOP1 gene (Knab et al. 1993). Consistent with a mutational event resistance phenotype, the camptothecin resistance is characterized by a long lag phase (Figure 3B). As with canavanine, we identified six independent isolates with mutations in the TOP1 gene (Figure 3C). All six mutations represented frameshift mutations within homopolymer nucleotide stretches (Figure 3D). Taken together, this method proved to be a simple and rapid way to identify the drug resistance target for compounds when the loss of function of a single locus confers resistance.

Bottom Line: A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring.Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis.Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544.

No MeSH data available.


Related in: MedlinePlus