Amanda L. Kalen

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells’ transitions into quiescence. MnSOD (+/–) and (–/–) MEFs demonstrated increased superoxide steady‐state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD (+/–) MEFs exhibited an increase in the percentage of G2 cells compared to MnSOD (+/+) MEFs. Overexpression of MnSOD in MnSOD (+/–) MEFs suppressed superoxide levels and G2 accumulation, decreased cyclin B1 protein levels, and facilitated cells’ transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria‐derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial ‘ROS‐switch’ favoring a superoxide‐signaling regulating proliferation and a hydrogen peroxide‐signaling supporting quiescence.

Superoxide Signaling Mediates N-acetyl-l-cysteine–Induced G1 Arrest: Regulatory Role of Cyclin D1 and Manganese Superoxide Dismutase

Thiol antioxidants, including N-acetyl-L-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn-superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G(1) arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G(1) arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G(1) arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G(1) arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O(2)(*-)) in NAC-treated cells compared with control. Scavenging of O(2)(*-) with Tiron reversed the NAC-induced G(1) arrest. These results show that an O(2)(*-) signaling pathway regulates NAC-induced G(1) arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity.

Erlotinib-mediated Inhibition of EGFR Signaling Induces Metabolic Oxidative Stress through NOX4

Redox regulation of epidermal growth factor receptor (EGFR) signaling helps protect cells against oxidative stress. In this study, we investigated whether the cytotoxicity of an EGFR tyrosine kinase inhibitor, erlotinib (ERL), was mediated by induction of oxidative stress in human head and neck cancer (HNSCC) cells. ERL elicited cytotoxicity in vitro and in vivo while increasing a panel of oxidative stress parameters which were all reversible by the antioxidant N-acetyl cysteine. Knockdown of EGFR by using siRNA similarly increased these oxidative stress parameters. Overexpression of mitochondrial targeted catalase but not superoxide dismutase reversed ERL-induced cytotoxicity. Consistent with a general role for NADPH oxidase (NOX) enzymes in ERL-induced oxidative stress, ERL-induced cytotoxicity was reversed by diphenylene iodonium, a NOX complex inhibitor. ERL reduced the expression of NOX1, NOX2, and NOX5 but induced the expression of NOX4. Knockdown of NOX4 by using siRNA protected HNSCC cells from ERL-induced cytotoxicity and oxidative stress. Our findings support the concept that ERL-induced cytotoxicity is based on a specific mechanism of oxidative stress mediated by hydrogen peroxide production through NOX4 signaling.

Mitochondrial ROS and radiation induced transformation in mouse embryonic fibroblasts

Manganese superoxide dismutase (SOD2) is a nuclear encoded and mitochondria localized antioxidant enzyme that converts mitochondria derived superoxide to hydrogen peroxide. This study investigates the hypothesis that mitochondria derived reactive oxygen species (ROS) regulate ionizing radiation (IR) induced transformation in normal cells. Mouse embryonic fibroblasts (MEFs) with wild type SOD2 (+/+), heterozygous SOD2 (+/-), and homozygous SOD2 (-/-) genotypes were irradiated with equitoxic doses of IR, and assayed for transformation frequency, cellular redox environment, DNA damage, and cell cycle checkpoint activation. Transformation frequency increased (~ 5-fold) in SOD2 (-/-) compared to SOD2 (+/+) MEFs. Cellular redox environment (GSH, GSSG, DHE, and DCFH-oxidation) did not show any significant change within 24h post-IR. However, a significant increase in cellular ROS levels was observed at 72h post-IR in SOD2 (-/-) compared to SOD2 (+/+) MEFs, which was consistent with an increase in GSSG in SOD2 (-/-) MEFs. Late ROS accumulation was associated with an increase in micronuclei frequency in SOD2 (-/-) MEFs. Exit from G2 was accelerated in irradiated SOD2 (+/-) and SOD2 (-/-) compared to SOD2 (+/+) MEFs. These results support the hypothesis that SOD2 activity and mitochondria generated ROS regulate IR induced transformation in mouse embryonic fibroblasts.

Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer

The toxicity of pharmacologic ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Because pancreatic cancer cells are sensitive to H2O2 generated by ascorbate, they would also be expected to become sensitized to agents that increase oxidative damage such as ionizing radiation. The current study demonstrates that pharmacologic ascorbate enhances the cytotoxic effects of ionizing radiation as seen by decreased cell viability and clonogenic survival in all pancreatic cancer cell lines examined, but not in nontumorigenic pancreatic ductal epithelial cells. Ascorbate radiosensitization was associated with an increase in oxidative stress-induced DNA damage, which was reversed by catalase. In mice with established heterotopic and orthotopic pancreatic tumor xenografts, pharmacologic ascorbate combined with ionizing radiation decreased tumor growth and increased survival, without damaging the gastrointestinal tract or increasing systemic changes in parameters indicative of oxidative stress. Our results demonstrate the potential clinical utility of pharmacologic ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

Mitochondrial DNA Depletion Induces Radioresistance by Suppressing G2 Checkpoint Activation in Human Pancreatic Cancer Cells

Abstract Cloos, C. R., Daniels, D. H., Kalen, A., Matthews, K., Du, J., Goswami, P. C. and Cullen, J. J. Mitochondrial DNA Depletion Induces Radioresistance by Suppressing G2 Checkpoint Activation in Human Pancreatic Cancer Cells. Radiat. Res. 171, 581–587 (2009). We hypothesized that mitochondrial function regulates cell cycle checkpoint activation and radiosensitivity. Human pancreatic tumor cells (MiaPaCa-2, rho+) were depleted of mitochondrial DNA (rho°) by culturing cells in the presence of ethidium bromide. Depletion of mitochondrial DNA was verified by PCR amplification of total DNA using primer pairs specific for mitochondrial DNA. Loss of mitochondrial DNA decreased plating efficiency and the percentage of cells in S phase. Exponential cultures were irradiated with 2, 4 and 6 Gy (dose rate: 0.83 Gy/min) of ionizing radiation and harvested for determination of cell viability, growth and cell cycle phase distributions. Rho° cells were radioresistant compared to rho+ cells, with a dose-modifying factor (DMF) of 1.6. Although cell growth was significantly inhibited in irradiated rho+ cells compared to unirradiated control cells, the inhibition in Rho° cells was minimal. In addition, mitochondrial DNA depletion suppressed radiation-induced G2 checkpoint activation, which was accompanied by increases in both cyclin B1 and CDK1. These results suggest that mitochondrial function may regulate cell cycle checkpoint activation and radiosensitivity in pancreatic cancer cells.

Enhancing the Antitumor Activity of Adriamycin and Ionizing Radiation

Overexpression of manganese superoxide dismutase (MnSOD), when combined with certain chemicals that inhibit peroxide removal, increases cancer cell cytotoxicity. Elevating MnSOD levels in cells enhances the conversion of superoxide (O(2)(*-)) to hydrogen peroxide (H(2)O(2)), combined with inhibiting the removal of H(2)O(2), further increases H(2)O(2) levels, leading to increased cytotoxicity. We hypothesized that increasing endogenous O(2)(*-) production in cells that were pretreated with adenoviral MnSOD (AdMnSOD) plus 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) would lead to an increased level of intracellular H(2)O(2) accumulation and increased cell killing. The cytotoxic effects of Adriamycin or radiation, agents known to produce O(2)(*-), were determined in MDA-MB-231 breast cancer cells pretreated with AdMnSOD plus BCNU both in vitro and in vivo. In vitro, AdMnSOD plus BCNU sensitized cells to the cytotoxicity of Adriamycin or radiation. In vivo, AdMnSOD, BCNU, and Adriamycin or ionizing radiation inhibited tumor growth and prolonged survival. The results suggest that agents that produce O(2)(*-) in combination with AdMnSOD plus BCNU may represent a powerful new antitumor regimen against breast cancer.

MnSOD activity protects mitochondrial morphology of quiescent fibroblasts from age associated abnormalities.

Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54h in 21% compared to 44h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.

Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes.

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.

Preferential selection of MnSOD transcripts in proliferating normal and cancer cells

Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix-localized redox enzyme that is known to regulate the cellular redox environment. Cellular redox environment changes during transitions between quiescent and proliferative cycles. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. The present study investigates if the 3′-untranslated region (UTR) of MnSOD regulates its expression during transitions between quiescent and proliferating cycles, and in response to radiation. A preferential increase in the levels of the 1.5-kb MnSOD transcript was observed in quiescent cells, whereas the abundance of the longer transcript showed a direct correlation with the percentage of S-phase cells. The log-transformed expression ratio of the longer to the shorter transcript was also higher in proliferating normal and cancer cells. Deletion and reporter assays showed a significant decrease in reporter activity in constructs carrying multiple AU-rich sequence that are present in the 3′-UTR of the longer MnSOD transcript. Overexpression of the MnSOD 3′-UTR representing the longer transcript enhanced endogenous MnSOD mRNA levels, which was associated with an increase in MnSOD protein levels and a decrease in the percentage of S-phase cells. Irradiation increases the mRNA levels of the 1.5-kb MnSOD transcript, which was consistent with a significant increase in the reporter activity of the construct carrying the 3′-UTR of the shorter transcript. We conclude that the 3′-UTR of MnSOD regulates MnSOD expression in response to different growth states and radiation.