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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

Examples of the effects of compounds of interest from the small molecule screening. Representative growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence of a drug (DMSO control, No Drug), in the presence of a drug that inhibited the growth of both strains (sensitive), in the presence of 400 μM etoposide (resistant, short lag) or 100 μM camptothecin (resistant, long lag), or in the presence of 60 μM bactobolin (synthetic growth defect). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr using a Biotek Plate Reader. The number of compounds remaining in each category after the screening process is indicated in the upper left of each panel of the msh2Δ curves. The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference.
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fig1: Examples of the effects of compounds of interest from the small molecule screening. Representative growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence of a drug (DMSO control, No Drug), in the presence of a drug that inhibited the growth of both strains (sensitive), in the presence of 400 μM etoposide (resistant, short lag) or 100 μM camptothecin (resistant, long lag), or in the presence of 60 μM bactobolin (synthetic growth defect). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr using a Biotek Plate Reader. The number of compounds remaining in each category after the screening process is indicated in the upper left of each panel of the msh2Δ curves. The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference.

Mentions: To identify drugs with high susceptibility to resistance formation, we screened compounds provided by the National Institutes of Health National Cancer Institute Developmental Therapeutics Program (NCI/DTP) Open Chemical Repository. Because yeast cells are often refractory to the effects of small molecules due to the presence of multidrug transporters (encoded by the pleiotropic drug resistance, PDR, gene family) and the abundance of ergosterol (ERG) in the yeast plasma membrane, an erg6∆ pdr5∆ knockout strain and an erg6∆ pdr5∆ msh2∆ triple knockout strain were constructed allowing for enhanced sensitivity to small molecules. The compounds were characterized according to their ability to inhibit growth in the mismatch repair proficient strain, the mismatch repair deficient strain or both. Figure 1 summarizes the results of the screening. The leftmost panel shows that in the absence of drug (the DMSO control), there is no difference between the two strains in terms of lag phase, growth rate, or saturation point. The panel second from the left is representative of the 88 compounds that inhibited growth, irrespective of the mismatch repair status (sensitive). The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference. One of the two compounds causing the msh2∆ specific synthetic growth defect is shown in the rightmost panel.


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

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

Examples of the effects of compounds of interest from the small molecule screening. Representative growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence of a drug (DMSO control, No Drug), in the presence of a drug that inhibited the growth of both strains (sensitive), in the presence of 400 μM etoposide (resistant, short lag) or 100 μM camptothecin (resistant, long lag), or in the presence of 60 μM bactobolin (synthetic growth defect). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr using a Biotek Plate Reader. The number of compounds remaining in each category after the screening process is indicated in the upper left of each panel of the msh2Δ curves. The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Examples of the effects of compounds of interest from the small molecule screening. Representative growth curves of erg6∆ pdr5∆ wild-type (WT) and msh2Δ erg6∆ pdr5∆ (msh2Δ) strains in the absence of a drug (DMSO control, No Drug), in the presence of a drug that inhibited the growth of both strains (sensitive), in the presence of 400 μM etoposide (resistant, short lag) or 100 μM camptothecin (resistant, long lag), or in the presence of 60 μM bactobolin (synthetic growth defect). Optical density readings at 600 nm (OD600) were taken every 15 min for 48 hr using a Biotek Plate Reader. The number of compounds remaining in each category after the screening process is indicated in the upper left of each panel of the msh2Δ curves. The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference.
Mentions: To identify drugs with high susceptibility to resistance formation, we screened compounds provided by the National Institutes of Health National Cancer Institute Developmental Therapeutics Program (NCI/DTP) Open Chemical Repository. Because yeast cells are often refractory to the effects of small molecules due to the presence of multidrug transporters (encoded by the pleiotropic drug resistance, PDR, gene family) and the abundance of ergosterol (ERG) in the yeast plasma membrane, an erg6∆ pdr5∆ knockout strain and an erg6∆ pdr5∆ msh2∆ triple knockout strain were constructed allowing for enhanced sensitivity to small molecules. The compounds were characterized according to their ability to inhibit growth in the mismatch repair proficient strain, the mismatch repair deficient strain or both. Figure 1 summarizes the results of the screening. The leftmost panel shows that in the absence of drug (the DMSO control), there is no difference between the two strains in terms of lag phase, growth rate, or saturation point. The panel second from the left is representative of the 88 compounds that inhibited growth, irrespective of the mismatch repair status (sensitive). The three rightmost panels are representative of molecules resulting in an msh2∆-specific growth difference. One of the two compounds causing the msh2∆ specific synthetic growth defect is shown in the rightmost panel.

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