In a genetic screen for C. elegans
mutants resistant to the antimitotic toxin, hemiasterlin, we obtained eight mutants (Zubovych et al., 2006
). We have now identified the genetic lesions in three of these, and all reside in mitochondrial proteins. Two of these, PHB-2 and SPG-7, the worm ortholog of a mitochondrial AAA-metalloprotease, paraplegin, are known to interact physically (Merkwirth and Langer, 2009
). In addition, we show that other mitochondrial mutations can produce drug resistance in worms. eat-3
, encoding a mitochondrial dynamin; isp-1
, with a mutation in the Rieske iron sulfur protein; and clk-1
, with a mutation in the ubiquinone biosynthesis protein, were all resistant to the hemiasterlin analog. However, not all mutations in mitochondria result in drug resistance. gas-1
, with point mutations in complex I and II proteins, respectively, were as sensitive to the hemiasterlin analog as wild-type worms. It is possible that only certain, fairly specific mutations can provide worms with drug resistance. Alternatively, it is the degree of impairment that may be important. Genome-wide microarray profiling of gas-1, mev-1
, and isp-1
have shown that mitochondrial respiratory chain mutants differentially up-regulate basic cellular metabolic pathways, implying numerous possibilities for compensatory adaptation (Falk et al., 2008
). For example, drug-sensitive gas-1
mutants have compensated decreased complex I capacity by increasing complex II–dependent oxidative phosphorylation and drug-sensitive mev-1
mutants balance complex II deficiency by increasing activity of complex I, but isp-1
, which is drug-resistant, has deficits in both complex I and II (Falk et al., 2008
). Likewise, each mitochondrial mutant strain is unique, with distinct metabolic rates, life span and drug sensitivity. In agreement with this, phb-2, har-1
, and spg-7
also show different sensitivities to some drugs.
Consistent with the interpretation that impairment of respiration is the key to drug resistance in our mutant worms, we found that exogenous inhibitors of mitochondrial respiration rescue wild-type worms from the hemiasterlin analog. In fact, the rescue was striking, with nontoxic concentrations of the respiration inhibitors completely protecting wild-type worms from a normally lethal dose of the hemiasterlin analog. Addition of the antioxidant NAC fully blocked the protection by mitochondrial inhibitors and by the mutations in phb-2, har-1, and spg-7, indicating that hemiasterlin resistance in mutants, or resistance caused by respiration inhibitors in the wild type, requires a mechanism sensitive to ROS. Furthermore, sod-2;sod-3, mutant worms lacking both MnSODs, which should result in increased mitochondrial ROS, tolerated the hemiasterlin analog better than wild-type worms. When crossed with our phb-2, har-1, or spg-7 mutants, the resulting triple mutants lacking MnSODs were more resistant to the hemiasterlin analog than were single phb-2, har-1, or spg-7 mutants.
Although treating wild-type worms with mitochondrial inhibitors reproducibly stimulated H2O2 production, in resting conditions without inhibitors the Amplex Red assay could not detect differences between the mutants and the wild-type worms. This could simply mean that the level of ROS production required for a drug-resistant phenotype is below the sensitivity range of the assay, which requires that H2O2 be secreted from the animal. Because Amplex Red measures only H2O2, it is also possible that some other ROS species might be necessary for drug resistance. If so, sensing of ROS is likely to be confined to the mitochondria, because the more reactive oxygen species have short half-lives and do not diffuse far. Our data suggest that an increase in superoxide is enough to promote resistance, as paraquat produces superoxide and the absence of MnSOD enzymes would enhance superoxide rather than H2O2 levels. Alternatively, drug resistance might be due to a mechanism that is sensitive to ROS, but the mutants activate that mechanism through a different process independent of ROS.
Interestingly, in mammalian cell culture mtDNA mutations resulted in respiration-deficient ρ0 cells with altered drug responses (Singh et al., 1999
; Pelicano et al., 2006
). Despite mitochondrial metabolic defects, in one study ρ0 cells demonstrated resistance to common anticancer agents (Pelicano et al., 2006
). In another study ρ0 were resistant to cell death induced by adriamycin and photodynamic therapy, but were equally sensitive to alkylating agents and gamma-radiation compared with their respiration-competent parental cells (Singh et al., 1999
). Obviously, in mammalian cells mitochondrial defects also influence drug sensitivity in complex ways.
Although some mitochondrial mutants are multidrug-resistant, it is unlikely that resistance is due to overexpression of P-glycoprotein or other drug efflux pumps. In a microarray experiment comparing har-1 to N2 wild-type worms, none of the genes encoding potential efflux pumps was overexpressed in the mutant (Wu and Roth, data not shown). Typically P-glycoprotein results in a very high resistance, such as a thousand fold. In our mutants we observed small resistance, only 2.5–5-fold, depending on the compound. Also, taxol and vinblastine are excellent substrates for P-glycoprotein and hemiasterlin is not, yet resistance to those drugs was equivalent. The fact that our mitochondrial mutants were also resistant to 2-deoxyglucose makes the drug efflux possibility extremely unlikely, because sugars are not transported by P-glycoprotein and any unknown pump exporting 2-deoxyglucose would likely export glucose.
It is possible that either chronic (mutants) or acute (N2 worms grown in mitochondrial inhibitors) mitochondrial dysfunction may serve to “precondition” worms to hemiasterlin analog by activating survival pathways. If so, our results showing that mutants with an activated AKT pathway are not protected from hemiasterlin analog and that loss of HIF-1 does not block resistance in our mitochondrial mutants suggests that these pathways are not necessary for hemiasterlin resistance. In contrast, a pkc-1 mutation suppressed the drug resistance of our mitochondrial mutants and showed reduced protection by FCCP from hemiasterlin, consistent with functioning downstream from the defects or inhibitors. PKC-1 is the kinase most closely related by sequence to the mammalian PKC isoform reported to respond to ROS by preconditioning cells to hypoxic shock. In our “acute” assay, N2 L1 larvae are exposed to hemiasterlin and the mitochondrial inhibitor simultaneously, so the protective effect of the mitochondrial inhibitor must act faster than the toxic effects of the drug. One possibility is that the protective effect occurs at the mitochondria itself, making the mitochondria less able to participate in events leading to death.