A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5' ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized so far are composed of an RNA plus one or more proteins, and tRNA 5' end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

Overexpression of the tumor suppressor gene, wild-type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/cell of Adp53 (that caused the enforced overexpression of wtp53) was significantly enhanced by treatment with 2DG. Accumulation of glutathione disulfide was found to be significantly greater in both cell lines treated with 2DG+Adp53 and both cell lines treated with 2DG+Adp53 showed a approximately 2-fold increases in dihydroethidine (DHE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH(2)) oxidation, indicative of increased steady-state levels of O(2)(.-) and hydroperoxides, respectively. Finally, overexpression of catalase or glutathione peroxidase using adenoviral vectors partially, but significantly, protected DU-145 cells from the toxicity induced by 2DG+Adp53 treatment. These results show that treatment of human prostate cancer cells with the combination of 2DG (a nutrient stress) and overexpression of the tumor suppressor gene, wtp53, enhances clonogenic cell killing by a mechanism that involves oxidative stress as well as allowing for the speculation that inhibitors of glucose and hydroperoxide metabolism can be used in combination with Adp53 gene therapy to enhance therapeutic responses.     

Sensitivity to low-dose/low-LET ionizing radiation in mammalian cells harboring mutations in succinate dehydrogenase subunit C is governed by mitochondria-derived reactive oxygen species

It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O? metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O?(?U+2212)) and hydrogen peroxide (H?O?) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O??- and H?O?. When B9 cells were exposed to low-dose/low-LET radiation (5-50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5-50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O??- and H?O?. These results also support the hypothesis that mitochondrial O??- and H?O? originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.     

An inhibitor of K+ channels modulates human endometrial tumor-initiating cells

Background

Many potassium ion (K⁺) channels function as oncogenes to sustain growth of solid tumors, but their role in cancer progression is not well understood. Emerging evidence suggests that the early progenitor cancer cell subpopulation, termed tumor initiating cells (TIC), are critical to cancer progression.

Results

A non-selective antagonist of multiple types of K⁺ channels, tetraethylammonium (TEA), was found to suppress colony formation in endometrial cancer cells via inhibition of putative TIC. The data also indicated that withdrawal of TEA results in a significant enhancement of tumorigenesis. When the TIC-enriched subpopulation was isolated from the endometrial cancer cells, TEA was also found to inhibit growth in vitro.

Conclusions

These studies suggest that the activity of potassium channels significantly contributes to the progression of endometrial tumors, and the antagonists of potassium channels are candidate anti-cancer drugs to specifically target tumor initiating cells in endometrial cancer therapy.     

Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth

Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.     

2-deoxy-D-glucose causes cytotoxicity, oxidative stress, and radiosensitization in pancreatic cancer

Glucose metabolism as assessed by (18)FDG PET imaging provides prognostic information in patients with pancreatic cancer but the implications of manipulating glucose metabolism for therapeutic purposes are unknown. Based on previous results with other cancer cell types, we hypothesized that inhibition of glucose metabolism in pancreatic cancer cells would cause cell killing via oxidative stress resulting from disruptions in thiol metabolism. 2-Deoxy-D-glucose (2DG), a chemical inhibitor of glucose metabolism, and glucose deprivation induced cytotoxicity in human pancreatic cancer cells in a time-and dose-dependent manner as well as causing significant increases in metabolic oxidative stress as measured by increased glutathione disulfide accumulation and NADP(+)/NADPH ratios. Simultaneous administration of the thiol antioxidant N-acetylcysteine protected pancreatic cancer cells against the c-ytotoxic effects of 2DG as well as reversing 2DG-induced glutathione disulfide accumulation and augmenting intracellular cysteine pools. In nude mice with heterotopic pancreatic tumors, the combination of 2DG and ionizing radiation resulted in greater inhibition of tumor growth and increased survival, relative to either agent alone. These results support the hypothesis that inhibiting glucose metabolism causes cytotoxicity in human pancreatic cancer cells via metabolic oxidative stress and disruptions in thiol metabolism. These results also support the speculation that inhibitors of glucose metabolism can be used in combination with classical oxidative stress-inducing agents (such as ionizing radiation) to enhance therapeutic responses in pancreatic cancer.     

Oxidative stress in a phenylketonuria animal model

Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pah(enu2) animal model for PKU. Animals (14-24 weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals (n = 6) than in controls (n = 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including glucose-6-phosphate dehydrogenase (G6PD), which provides the RBC's main reducing power, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and catalase detoxifies H2O2 by catalyzing its reduction to O2 and H2O. Both catalase and G6PD were significantly increased in the RBCs of PKU animals.     

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2(-•) and H2O2

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.     

Cisplatin combined with zidovudine enhances cytotoxicity and oxidative stress in human head and neck cancer cells via a thiol-dependent mechanism

Oxidative stress and mitochondrial dysfunction in cancer cells represent features that may be exploited therapeutically. We determined whether agents that induce mitochondrial dysfunction, such as zidovudine (AZT) and cisplatin (CIS), could enhance killing of human head and neck cancer cells via oxidative stress. AZT- and/or CIS-induced cytotoxicity was determined using clonogenic survival, mitochondrial membrane potential was analyzed to investigate mitochondrial function, and glutathione was measured to determine thiol metabolism perturbations. AZT+CIS significantly increased toxicity and reduced mitochondrial membrane potential in FaDu, Cal-27, and SQ20B head and neck cancer cells while increasing the percentage of glutathione disulfide (%GSSG). Treatment with the thiol antioxidant N-acetylcysteine (NAC) reversed the loss of mitochondrial membrane potential and the increase in %GSSG and partially protected FaDu and Cal-27 cells from AZT+CIS. Finally, an inhibitor of glutathione synthesis, l-buthionine-[S,R]-sulfoximine, sensitized the cells to AZT+CIS-induced cytotoxicity, which was partially reversed by NAC. These results suggest that exposure of cancer cells to agents that induce mitochondrial dysfunction, such as AZT, causes significant sensitization to CIS-induced toxicity via disruptions in thiol metabolism and oxidative stress. These findings provide a biochemical rationale for evaluating agents that induce mitochondrial dysfunction in combination with chemotherapy and inhibitors of glutathione metabolism in head and neck cancer.