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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

Mutations in the endocytosis pathway confer resistance to bactobolin. (A) Chemical structure of bactobolin. 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 100 μM bactobolin (Bact). Optical density readings at 600 nm (OD600) were taken every 15 min for 72 hr. (C) The table lists the genes and mutations conferring resistance to bactobolin. The coding strand nucleotide sequence and mutation for each isolate is shown. Five caused inactivating frameshifts and one (within YSC84) resulted in a mutation in the splice donor consensus sequence (SDC). (D) A schematic drawing of the clathrin-dependent endocytic pathway is adapted from a previously published model (Weinberg and Drubin 2012). The black line represents a membrane undergoing endocytosis. The endocytosis components that were identified are represented with colors: Ede1 (red); Ysc84 (green); Vrp1 (orange); Sla2 (purple); and Inp52 (blue). Other endocytosis components are in gray.
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fig5: Mutations in the endocytosis pathway confer resistance to bactobolin. (A) Chemical structure of bactobolin. 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 100 μM bactobolin (Bact). Optical density readings at 600 nm (OD600) were taken every 15 min for 72 hr. (C) The table lists the genes and mutations conferring resistance to bactobolin. The coding strand nucleotide sequence and mutation for each isolate is shown. Five caused inactivating frameshifts and one (within YSC84) resulted in a mutation in the splice donor consensus sequence (SDC). (D) A schematic drawing of the clathrin-dependent endocytic pathway is adapted from a previously published model (Weinberg and Drubin 2012). The black line represents a membrane undergoing endocytosis. The endocytosis components that were identified are represented with colors: Ede1 (red); Ysc84 (green); Vrp1 (orange); Sla2 (purple); and Inp52 (blue). Other endocytosis components are in gray.

Mentions: Bactobolin (Figure 5A), an anticancer compound with protein synthesis inhibitory activity, has demonstrated in vitro cytotoxicity against the human melanoma cell line B16 and has also prolonged the survival time of mice with leukemia L-1210 (Ishizuka et al. 1980; Kawada et al. 1999). Therefore, the potent anticancer activity of bactobolin necessitates further investigation into possible modes of resistance, allowing for the design of bactobolin-based combination therapies. As with the other compounds used in this analysis, the resistance profile for bactobolin is consistent with a mutational event (Figure 5B). The results from the bactobolin resistance experiments failed to reveal a single resistance locus; however, a gene ontology analysis showed there was a significant enrichment of genes involved in endocytosis. Each isolate contained an inactivating mutation within a gene in the endocytosis pathway; five were frameshift events and one destroyed a splice donor consensus sequence (Figure 5, C and D). Taken together, the results suggest that the endocytosis pathway is important for the entry of bactobolin into the cells. Furthermore, the results show that a pathway is also easily identified by using this simple platform.


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

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

Mutations in the endocytosis pathway confer resistance to bactobolin. (A) Chemical structure of bactobolin. 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 100 μM bactobolin (Bact). Optical density readings at 600 nm (OD600) were taken every 15 min for 72 hr. (C) The table lists the genes and mutations conferring resistance to bactobolin. The coding strand nucleotide sequence and mutation for each isolate is shown. Five caused inactivating frameshifts and one (within YSC84) resulted in a mutation in the splice donor consensus sequence (SDC). (D) A schematic drawing of the clathrin-dependent endocytic pathway is adapted from a previously published model (Weinberg and Drubin 2012). The black line represents a membrane undergoing endocytosis. The endocytosis components that were identified are represented with colors: Ede1 (red); Ysc84 (green); Vrp1 (orange); Sla2 (purple); and Inp52 (blue). Other endocytosis components are in gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Mutations in the endocytosis pathway confer resistance to bactobolin. (A) Chemical structure of bactobolin. 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 100 μM bactobolin (Bact). Optical density readings at 600 nm (OD600) were taken every 15 min for 72 hr. (C) The table lists the genes and mutations conferring resistance to bactobolin. The coding strand nucleotide sequence and mutation for each isolate is shown. Five caused inactivating frameshifts and one (within YSC84) resulted in a mutation in the splice donor consensus sequence (SDC). (D) A schematic drawing of the clathrin-dependent endocytic pathway is adapted from a previously published model (Weinberg and Drubin 2012). The black line represents a membrane undergoing endocytosis. The endocytosis components that were identified are represented with colors: Ede1 (red); Ysc84 (green); Vrp1 (orange); Sla2 (purple); and Inp52 (blue). Other endocytosis components are in gray.
Mentions: Bactobolin (Figure 5A), an anticancer compound with protein synthesis inhibitory activity, has demonstrated in vitro cytotoxicity against the human melanoma cell line B16 and has also prolonged the survival time of mice with leukemia L-1210 (Ishizuka et al. 1980; Kawada et al. 1999). Therefore, the potent anticancer activity of bactobolin necessitates further investigation into possible modes of resistance, allowing for the design of bactobolin-based combination therapies. As with the other compounds used in this analysis, the resistance profile for bactobolin is consistent with a mutational event (Figure 5B). The results from the bactobolin resistance experiments failed to reveal a single resistance locus; however, a gene ontology analysis showed there was a significant enrichment of genes involved in endocytosis. Each isolate contained an inactivating mutation within a gene in the endocytosis pathway; five were frameshift events and one destroyed a splice donor consensus sequence (Figure 5, C and D). Taken together, the results suggest that the endocytosis pathway is important for the entry of bactobolin into the cells. Furthermore, the results show that a pathway is also easily identified by using this simple platform.

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