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Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans.

Zubovych IO, Straud S, Roth MG - Mol. Biol. Cell (2010)

Bottom Line: Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2.Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant.Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA.

ABSTRACT
In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

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Not all mutations in mitochondrial proteins confer increased resistance to the hemiasterlin analog. Synchronized L1 larvae of each strain were incubated at the indicated hemiasterlin concentrations for 4–5 d, and healthy mobile adults with eggs were counted. For each concentration, two wells were scored in an experiment. Average values are graphed ± SEM. Note that data for N2 and mev-1 superimpose. Similar results were reproduced in at least three independent experiments.
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Figure 5: Not all mutations in mitochondrial proteins confer increased resistance to the hemiasterlin analog. Synchronized L1 larvae of each strain were incubated at the indicated hemiasterlin concentrations for 4–5 d, and healthy mobile adults with eggs were counted. For each concentration, two wells were scored in an experiment. Average values are graphed ± SEM. Note that data for N2 and mev-1 superimpose. Similar results were reproduced in at least three independent experiments.

Mentions: Because all three of the mutations we had identified so far as conferring drug resistance were found in mitochondrial proteins, we asked if other known mitochondrial mutants would show a similar phenotype. We tested four well-studied mutants: gas-1(fc21), mev-1(kn1), isp-1(qm150), and two alleles of clk-1, (e2519) and (qm30), for resistance to the hemiasterlin analog. The first three of these mutants contain missense mutations in complexes I (NADH-ubiquinone oxidoreductase 49-kDa subunit), II (succinate dehydrogenase cytochrome b subunit), and III (Rieske iron sulfur protein), respectively. The two clk-1 mutants possess either a missense mutation (e2519) or deletion (qm30) in the gene encoding the demethoxyubiquinone hydroxylase, an enzyme required for the biosynthesis of ubiquinone. Ubiquinone accepts electrons from both complex I and II in the electron transport chain. All four mutants are defective in oxidative phosphorylation (Feng et al., 2001; Kayser et al., 2001, 2004; Senoo-Matsuda et al., 2001). isp-1 and clk-1 were resistant to hemiasterlin, and gas-1 and mev-1 had wild-type sensitivity (Figure 5). Both isp-1 and clk-1 worms grow slowly and are long lived; in contrast, gas-1 and mev-1 have a slight growth delay and are short lived. Thus, we tested if drug resistance correlates with growth rates and the life span. We found that har-1 and spg-7 mutants grow slightly slower than N2 worms, but phb-2 has wild-type growth, indicating that resistance to the hemiasterlin analog is not correlated with slow growth.


Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans.

Zubovych IO, Straud S, Roth MG - Mol. Biol. Cell (2010)

Not all mutations in mitochondrial proteins confer increased resistance to the hemiasterlin analog. Synchronized L1 larvae of each strain were incubated at the indicated hemiasterlin concentrations for 4–5 d, and healthy mobile adults with eggs were counted. For each concentration, two wells were scored in an experiment. Average values are graphed ± SEM. Note that data for N2 and mev-1 superimpose. Similar results were reproduced in at least three independent experiments.
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Related In: Results  -  Collection

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Figure 5: Not all mutations in mitochondrial proteins confer increased resistance to the hemiasterlin analog. Synchronized L1 larvae of each strain were incubated at the indicated hemiasterlin concentrations for 4–5 d, and healthy mobile adults with eggs were counted. For each concentration, two wells were scored in an experiment. Average values are graphed ± SEM. Note that data for N2 and mev-1 superimpose. Similar results were reproduced in at least three independent experiments.
Mentions: Because all three of the mutations we had identified so far as conferring drug resistance were found in mitochondrial proteins, we asked if other known mitochondrial mutants would show a similar phenotype. We tested four well-studied mutants: gas-1(fc21), mev-1(kn1), isp-1(qm150), and two alleles of clk-1, (e2519) and (qm30), for resistance to the hemiasterlin analog. The first three of these mutants contain missense mutations in complexes I (NADH-ubiquinone oxidoreductase 49-kDa subunit), II (succinate dehydrogenase cytochrome b subunit), and III (Rieske iron sulfur protein), respectively. The two clk-1 mutants possess either a missense mutation (e2519) or deletion (qm30) in the gene encoding the demethoxyubiquinone hydroxylase, an enzyme required for the biosynthesis of ubiquinone. Ubiquinone accepts electrons from both complex I and II in the electron transport chain. All four mutants are defective in oxidative phosphorylation (Feng et al., 2001; Kayser et al., 2001, 2004; Senoo-Matsuda et al., 2001). isp-1 and clk-1 were resistant to hemiasterlin, and gas-1 and mev-1 had wild-type sensitivity (Figure 5). Both isp-1 and clk-1 worms grow slowly and are long lived; in contrast, gas-1 and mev-1 have a slight growth delay and are short lived. Thus, we tested if drug resistance correlates with growth rates and the life span. We found that har-1 and spg-7 mutants grow slightly slower than N2 worms, but phb-2 has wild-type growth, indicating that resistance to the hemiasterlin analog is not correlated with slow growth.

Bottom Line: Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2.Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant.Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA.

ABSTRACT
In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

Show MeSH
Related in: MedlinePlus