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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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Mitochondrial inhibitors rescue wild-type N2 worms from the hemiasterlin analog. About 400 synchronized N2 L1 larvae were grown in liquid culture in the presence of increasing concentrations of the mitochondrial inhibitor alone or mitochondrial inhibitor plus 1.3 μM hemiasterlin analog. After 3 d of exposure healthy, morphologically normal and mobile L4 and adult worms were counted. Worms exposed to 1.3 μM hemiasterlin analog alone die or develop as paralyzed and dumpy animals. In contrast, when exposed to 1.3 μM hemiasterlin analog in the presence of an inhibitor of mitochondrial respiration, N2 worms grow as healthy, mobile, and morphologically normal animals. TTFA, rotenone, FCCP, oligomycin, and antimycin eventually killed worms at higher concentrations, but were protective at lower dosages. Rotenone, oligomycin, and antimycin also slightly delayed worm growth comparable to untreated animals. Stigmatellin, myxothiazol, and FCCP protected worms from hemiasterlin analog and did not affect animal growth. Experiments with each drug were independently reproduced three times, and average values are graphed ± SEM. For most concentrations tested, the error bars are smaller than the symbol that indicates the data point.
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Figure 7: Mitochondrial inhibitors rescue wild-type N2 worms from the hemiasterlin analog. About 400 synchronized N2 L1 larvae were grown in liquid culture in the presence of increasing concentrations of the mitochondrial inhibitor alone or mitochondrial inhibitor plus 1.3 μM hemiasterlin analog. After 3 d of exposure healthy, morphologically normal and mobile L4 and adult worms were counted. Worms exposed to 1.3 μM hemiasterlin analog alone die or develop as paralyzed and dumpy animals. In contrast, when exposed to 1.3 μM hemiasterlin analog in the presence of an inhibitor of mitochondrial respiration, N2 worms grow as healthy, mobile, and morphologically normal animals. TTFA, rotenone, FCCP, oligomycin, and antimycin eventually killed worms at higher concentrations, but were protective at lower dosages. Rotenone, oligomycin, and antimycin also slightly delayed worm growth comparable to untreated animals. Stigmatellin, myxothiazol, and FCCP protected worms from hemiasterlin analog and did not affect animal growth. Experiments with each drug were independently reproduced three times, and average values are graphed ± SEM. For most concentrations tested, the error bars are smaller than the symbol that indicates the data point.

Mentions: We hypothesized that if certain mitochondrial defects cause drug resistance in our mutants, then partially inhibiting mitochondrial respiration in wild-type N2 worms might confer the same phenotype. If true, growing wild-type worms in the presence of inhibitors that disrupt individual mitochondrial respiration complexes would show the contribution of each complex to hemiasterlin resistance. Inhibitors of respiration complexes I and II represented our special interest, because gas-1 and mev-1 mutants with mutations in those complexes were sensitive to the hemiasterlin analog. However, we found that rotenone, which inhibits complex I, TTFA (complex II inhibitor), antimycin, myxothiazol, and stigmatellin (complex III inhibitors) all rescue wild-type worms from the hemiasterlin analog (Figure 7). FCCP, a potent uncoupler of oxidative phosphorylation, conferred strong resistance to the drug. Oligomycin, an inhibitor of mitochondrial ATP synthase, also protected N2 worms from hemiasterlin, although less efficiently than respiration inhibitors.


Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans.

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

Mitochondrial inhibitors rescue wild-type N2 worms from the hemiasterlin analog. About 400 synchronized N2 L1 larvae were grown in liquid culture in the presence of increasing concentrations of the mitochondrial inhibitor alone or mitochondrial inhibitor plus 1.3 μM hemiasterlin analog. After 3 d of exposure healthy, morphologically normal and mobile L4 and adult worms were counted. Worms exposed to 1.3 μM hemiasterlin analog alone die or develop as paralyzed and dumpy animals. In contrast, when exposed to 1.3 μM hemiasterlin analog in the presence of an inhibitor of mitochondrial respiration, N2 worms grow as healthy, mobile, and morphologically normal animals. TTFA, rotenone, FCCP, oligomycin, and antimycin eventually killed worms at higher concentrations, but were protective at lower dosages. Rotenone, oligomycin, and antimycin also slightly delayed worm growth comparable to untreated animals. Stigmatellin, myxothiazol, and FCCP protected worms from hemiasterlin analog and did not affect animal growth. Experiments with each drug were independently reproduced three times, and average values are graphed ± SEM. For most concentrations tested, the error bars are smaller than the symbol that indicates the data point.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2836976&req=5

Figure 7: Mitochondrial inhibitors rescue wild-type N2 worms from the hemiasterlin analog. About 400 synchronized N2 L1 larvae were grown in liquid culture in the presence of increasing concentrations of the mitochondrial inhibitor alone or mitochondrial inhibitor plus 1.3 μM hemiasterlin analog. After 3 d of exposure healthy, morphologically normal and mobile L4 and adult worms were counted. Worms exposed to 1.3 μM hemiasterlin analog alone die or develop as paralyzed and dumpy animals. In contrast, when exposed to 1.3 μM hemiasterlin analog in the presence of an inhibitor of mitochondrial respiration, N2 worms grow as healthy, mobile, and morphologically normal animals. TTFA, rotenone, FCCP, oligomycin, and antimycin eventually killed worms at higher concentrations, but were protective at lower dosages. Rotenone, oligomycin, and antimycin also slightly delayed worm growth comparable to untreated animals. Stigmatellin, myxothiazol, and FCCP protected worms from hemiasterlin analog and did not affect animal growth. Experiments with each drug were independently reproduced three times, and average values are graphed ± SEM. For most concentrations tested, the error bars are smaller than the symbol that indicates the data point.
Mentions: We hypothesized that if certain mitochondrial defects cause drug resistance in our mutants, then partially inhibiting mitochondrial respiration in wild-type N2 worms might confer the same phenotype. If true, growing wild-type worms in the presence of inhibitors that disrupt individual mitochondrial respiration complexes would show the contribution of each complex to hemiasterlin resistance. Inhibitors of respiration complexes I and II represented our special interest, because gas-1 and mev-1 mutants with mutations in those complexes were sensitive to the hemiasterlin analog. However, we found that rotenone, which inhibits complex I, TTFA (complex II inhibitor), antimycin, myxothiazol, and stigmatellin (complex III inhibitors) all rescue wild-type worms from the hemiasterlin analog (Figure 7). FCCP, a potent uncoupler of oxidative phosphorylation, conferred strong resistance to the drug. Oligomycin, an inhibitor of mitochondrial ATP synthase, also protected N2 worms from hemiasterlin, although less efficiently than respiration inhibitors.

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