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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

Inactivation of Ipt1 is the major cause of resistance to mitoxantrone. (A) Chemical structure of mitoxantrone. The structure was rendered with ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence and presence of mitoxantrone (50 μM). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. The OD600 offset observed in the presence of the drug is due to the colored nature of mitoxantrone. (C) Schematic representation of the frameshift positions within the IPT1 locus on chromosome IV (chrIV) conferring resistance to mitoxantrone. 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 IPT1 conferring resistance to mitoxantrone. 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 IPT1 is in the opposite orientation (chrIV: 591344–589761) 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 is indicated in a format described in Figure 2.
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fig6: Inactivation of Ipt1 is the major cause of resistance to mitoxantrone. (A) Chemical structure of mitoxantrone. The structure was rendered with ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence and presence of mitoxantrone (50 μM). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. The OD600 offset observed in the presence of the drug is due to the colored nature of mitoxantrone. (C) Schematic representation of the frameshift positions within the IPT1 locus on chromosome IV (chrIV) conferring resistance to mitoxantrone. 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 IPT1 conferring resistance to mitoxantrone. 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 IPT1 is in the opposite orientation (chrIV: 591344–589761) 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 is indicated in a format described in Figure 2.

Mentions: Mitoxantrone (Figure 6A) is a commonly prescribed drug used to treat a wide variety of cancers and autoimmune diseases (Hande 1998). Mitoxantrone inhibits rapidly dividing cells by targeting topoisomerase II during DNA synthesis (Bellosillo et al. 1998; Fox and Smith 1990). The mitoxantrone resistance phenotype is consistent with a mutational event resistance (Figure 6B). We identified the mutations within six isolates and found that the IPT1 gene is the major target for mitoxantrone resistance (Figure 6C). All of the isolates had frameshift mutations at homopolymeric repeats within the IPT1 gene (Figure 6D). IPT1 encodes an inositolphosphotransferase involved in synthesis of mannose-(inositol-phosphate)2-ceramide, with the most abundant sphingolipid in yeast (Dickson et al. 1997). These data suggest that entry of mitoxantrone may depend on lipid rafts or essential transporters within lipid rafts. Interestingly, in human cell lines, mitoxantrone resistance has been linked to differences in plasma membrane permeability (Breuzard et al. 2005).


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

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

Inactivation of Ipt1 is the major cause of resistance to mitoxantrone. (A) Chemical structure of mitoxantrone. The structure was rendered with ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence and presence of mitoxantrone (50 μM). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. The OD600 offset observed in the presence of the drug is due to the colored nature of mitoxantrone. (C) Schematic representation of the frameshift positions within the IPT1 locus on chromosome IV (chrIV) conferring resistance to mitoxantrone. 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 IPT1 conferring resistance to mitoxantrone. 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 IPT1 is in the opposite orientation (chrIV: 591344–589761) 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 is indicated in a format described in Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Inactivation of Ipt1 is the major cause of resistance to mitoxantrone. (A) Chemical structure of mitoxantrone. The structure was rendered with ChemDraw. (B) Growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence and presence of mitoxantrone (50 μM). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr. The OD600 offset observed in the presence of the drug is due to the colored nature of mitoxantrone. (C) Schematic representation of the frameshift positions within the IPT1 locus on chromosome IV (chrIV) conferring resistance to mitoxantrone. 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 IPT1 conferring resistance to mitoxantrone. 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 IPT1 is in the opposite orientation (chrIV: 591344–589761) 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 is indicated in a format described in Figure 2.
Mentions: Mitoxantrone (Figure 6A) is a commonly prescribed drug used to treat a wide variety of cancers and autoimmune diseases (Hande 1998). Mitoxantrone inhibits rapidly dividing cells by targeting topoisomerase II during DNA synthesis (Bellosillo et al. 1998; Fox and Smith 1990). The mitoxantrone resistance phenotype is consistent with a mutational event resistance (Figure 6B). We identified the mutations within six isolates and found that the IPT1 gene is the major target for mitoxantrone resistance (Figure 6C). All of the isolates had frameshift mutations at homopolymeric repeats within the IPT1 gene (Figure 6D). IPT1 encodes an inositolphosphotransferase involved in synthesis of mannose-(inositol-phosphate)2-ceramide, with the most abundant sphingolipid in yeast (Dickson et al. 1997). These data suggest that entry of mitoxantrone may depend on lipid rafts or essential transporters within lipid rafts. Interestingly, in human cell lines, mitoxantrone resistance has been linked to differences in plasma membrane permeability (Breuzard et al. 2005).

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