Kelley K. Kiningham

Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death

Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesize that mitochondria‐derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated murine fibrosarcoma cell lines, which expressed a range of mitochondrial manganese superoxide dis‐mutase (MnSOD) activities, with respiratory chain inhibitors. Apoptosis was confirmed by DNA fragmentation analysis and electron microscopy. MnSOD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl‐xL, p53 and poly(ADP‐ribose) polymerase (PARP) to identify specific cellular pathways that might contribute to the mitochondrial‐initiated ROS‐mediated cell death. Cells overexpressing Mn‐SOD contained less bcl‐xL within the mitochondria compared to control (NEO) cells, therefore excluding the role of bcl‐xL. p53 was undetectable by Western analysis and examination of the proapo‐ptotic protein bax, a p53 target gene, did not increase with treatment. Activation of caspase‐3 (CPP‐32) occurred in the NEO cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP‐32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fragments. Taken together, these findings suggest that mitochondrial‐mediated ROS generation is a key event by which inhibition of respiration causes cell death, and identifies CPP‐32 and the PARP‐linked pathway as targets of mitochon‐drial‐derived ROS‐induced cell death.—Kiningham, K. K., Oberley, T. D., Lin, S.‐M., Mattingly, C. A., St. Clair, D. K. Overexpression of manganese superoxide dismutase protects against mitochondrial‐initi‐ated poly(ADP‐ribose) polymerase‐mediated cell death. FASEB J. 13, 1601–1610 (1999)

Transcription regulation of human manganese superoxide dismutase gene.

The human MnSOD gene has a typical housekeeping gene promoter, but is highly inducible by various physical, chemical, and biological agents. Transcription factors SP-1 and AP-2 seem to have opposite roles in the transcriptional activity of the basal promoter. Whereas SP-1 plays a positive role, which is absolutely essential for transcription from the human MnSOD promoter, AP-2 appears to play a negative role in this process. An enhancer element is found in the promoter region of the human MnSOD gene. Several important enhancer elements are located in the second intron. The NF-kappa B site in the second intron is essential but not sufficient for high-level induction of MnSOD by cytokines. Although mutations in the regulatory elements may be partially responsible for the lack of induction of MnSOD in some cell types, differences in the degree of induction exist that cannot be accounted for by the defect in the DNA sequence. It is highly likely that this difference is due to the presence or absence of coactivator or suppressor proteins in the cells and may have a physiological role in the defense against oxidative stress.

Overexpression of MnSOD Protects Murine Fibrosarcoma Cells (FSa-II) from Apoptosis and Promotes a Differentiation Program upon Treatment with 5-Azacytidine: Involvement of MAPK and NFκB Pathways

Stable transfection of neomycin and human manganese superoxide dismutase (MnSOD2) expression plasmids into a murine fibrosarcoma cell line (FSa-II) was previously done in our laboratory. Treatment with 10 microM 5-azacytidine induced apoptosis in the control cell line (NEO), whereas the MnSOD-overexpressing cell line (SOD-H) demonstrated differentiated-appearing morphology. The levels of the myogenic transcription factor, MyoD, and the muscle-specific marker, alpha-actin, were increased over time with 5-azacytidine treatment in the SOD-H cell line. Nuclear transcription factor NFkappaB was activated in the SOD-H cell line, whereas inhibition of NFkappaB activation reduced the levels of MyoD and alpha-actin. Members of mitogen-activated protein kinase pathway and the Raf1/MEK/ERK cascade were shown to play a positive role in this event. Overexpression of MnSOD not only can protect cells from the toxic effects of 5-azacytidine, but can also promote the fibrosarcoma cells to enter a differentiation program.

TPA-Activated Transcription of the Human MnSOD Gene: Role of Transcription Factors SP-1 and Egr-1

Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription.

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose-responsive manner at physiological concentrations, estradiol-17beta surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 microM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.

KEAP1 Is a Redox Sensitive Target That Arbitrates the Opposing Radiosensitive Effects of Parthenolide in Normal and Cancer Cells

Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells toward death, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. We previously showed that parthenolide enhances ROS production in prostate cancer cells through activation of NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes to parthenolides radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury. Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Furthermore, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective properties in normal cells.

Tamoxifen enhancement of TNF-α induced MnSOD expression: modulation of NF-κB dimerization

Manganese superoxide dismutase (MnSOD) has been shown to suppress the development of cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen that is widely used in chemotherapy, is known to be a modulator of antioxidant status. However, the mechanism by which TAM mediates antioxidant enzyme induction remains unclear. In this study we investigated TAM enhancement of MnSOD induction by TNF-α. The results show that co-treatment with TAM and TNF-α increases the MnSOD promoter/enhancer driven luciferase activity, MnSOD mRNA and protein levels. Interestingly, co-treatment with TAM and TNF-α drastically decreases the binding activity of the p50/p50 homodimer and increases that of the p50/p65 heterodimer compared to TNF-α alone. This change in DNA binding could not be attributed to a decrease in the level of p50, its precursor, p105, or its inhibitors. Furthermore, TAM did not enhance degradation of IκB-α. These results suggest that p50/p50 homodimer may act as an inhibitory complex of MnSOD expression. Modulation of the DNA binding activity in favor of the p50/p65 complex may enhance NF-κB mediated induction of MnSOD by TAM. These findings reveal a potential novel mechanism for the induction of the human MnSOD gene.

Antioxidant function of metallothioneins

Since the discovery of metallothionein in 1957, it has been accepted that the primary roles of this protein are: 1) detoxification of heavy metals, and 2) regulation of the metabolism of essential metals. Recently, studies have suggested an additional role for metallothionein as a free radical scavenger. This article reviews in vitro and in vivo evidence which supports the role for metallothionein in modulation of reactive oxygen species. J. Trace Elem. Exp. Med. 11:219–226, 1998.

IκBα (inhibitory κBα) identified as labile repressor of MnSOD (manganese superoxide dismutase) expression

Cytokines, phorbol esters, radiation and chemotherapeutic drugs up-regulate the expression of MnSOD (manganese superoxide dismutase). Using the VA-13 cell line, we studied the regulation of SOD2 upon treatment with PMA. Pre-treatment with CHX (cycloheximide) followed by PMA led to significantly higher levels of MnSOD mRNA compared with those with either agent alone, suggesting de novo synthesis of an inhibitory protein. PMA treatment modulates redox-sensitive transcription factors, therefore we evaluated the effects of this combination treatment upon AP-1 (activator protein 1) and NF-κB (nuclear factor κB), two trans-acting factors suggested to play a role in SOD2 regulation. Co-administration of CHX and PMA led to a time-dependent increase in the binding activity of NF-κB. Therefore we evaluated IκBα (inhibitory κBα) and found that co-administration decreased its steady-state level compared with either agent alone, suggesting that enhanced NF-κB activation is due to inhibition of IκBα synthesis. PMA activates PKC (protein kinase C) enzymes which phosphorylate IκBα, leading to its degradation, therefore we used GF109203X to inhibit PKC activity. Stable transfection utilizing a PMA-responsive element in the human SOD2 gene, showed a concentration-dependent decrease in luciferase and NF-κB-binding activity with GF109203X. Western blot analysis indicated the presence of several PKC isoforms in the VA-13 cell line; however, PMA pre-treatment specifically down-regulated α and βI, suggesting a role for one or more of these proteins in SOD2 induction. Taken together, these results indicate that the PKC pathway leading to SOD2 induction proceeds at least in part through NF-κB and that inhibition of IκBα synthesis might serve as a potential pharmacological approach to up-regulate MnSOD.

In Situ Reduction of Oxidative Damage, Increased Cell Turnover, and Delay of Mitochondrial Injury by Overexpression of Manganese Superoxide Dismutase in a Multistage Skin Carcinogenesis Model

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.