Differential activation of nuclear transcription factor κB, gene expression, and proteins by amifostine's free thiol in human microvascular endothelial and glioma cells

Differential Activation of Nuclear Transcription Factor JoB, Gene Expression, and Proteins by Amifostine's Free Thiol in Human Microvascular Endothelial and Glioma Cells DavidJ. Grdina, Jeffrey S. Murley, Yasushi Kataoka, and Douglas P. Calvin The effects of WR1065 (SH), the free thiol form of amifostine, on nuclear transcription factor KB (NF~B) activation, manganese superoxide dismutase (MnSOD) gene expression, and secretion of human vascular endothelial cell growth factor (hVEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-~ (TNF-~), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, and interleukins IL-I~, IL-6, and IL-8 were investigated and compared in human microvascular endothelial (HIVIEC) and human glioma cells. WR1065 was evaluated at 2 concentrations, 4 mmol/L, ie, its most effective cytoprotective dose, and 40/~mol/L, a noncytoprotective but highly effective dose capable of preventing radiation and chemotherapeutic drug-induced mutations in exposed cells. A 30-minute exposure of HMEC and glioma cell lines U87 and U251 to WR1065 at either of the concentrations resulted in a marked activation of

NFKB as determined by a gel shift assay, with the maximum effect observed between 30 minutes and 1 hour after treatment. Using a supershift assay, WR1065 exposure was observed to affect only the p50-p65 heterodimer, and not the homodimers or heterodimers containing p52 or c-Rel subunits of NFKB. WR1065 was also found to enhance MnSOD gene expression in both HMEC and glioma cells. Gene expression was enhanced 1.8-fold over control levels in HMEC over a period ranging from 12 to 24 hours after the time of maximum activation of NFKB. In contrast, MnSOD gene expression in U87 cells rose 3.5 times above control levels over this same period. WR1065 had no effect on the levels of adhesion molecules, cytokines, and growth factors secreted by cells exposed for up to 24 hours as measured by enzyme-linked immunosorbent assay.

mifostine along with other thiol-containing drugs such as N-acetylcysteine (NAC), mesna (sodium 2-mercaptoethane sulfonate), and oltipraz (5- (2-pyrazinyl) -4-methyl- 1,2-dithiol-3-thione) have been used in a variety of clinical settings to protect against oxidative damage. 1-3 Each is capable of not only scavenging highly reactive free radicals that are produced by oxidative stress, but also of participating in intracellular reductive/ oxidative processes that have broad biological implications. These include the modulation of redox-sensitive transcription factors, alteration of gene expression, and modification of protein activity. 4-6 These reducing agents are known to directly activate the redox-sensitive transcription

factor NF•B. 7-L~NFKB is an inducible transcription factor that plays an essential role in the expression of a number of gene families that include cytokines and their receptors, cell adhesion molecules, growth factors, and antioxidant genes. ~ DNA-binding motifs for NFKB are found in the promoter regions of a large n u m b e r of genes involved in inflammatory processes and apoptosis, t2 Activation of NFKB usually is, therefore, related to a damage response by the cell to oxidative stress-inducing agents. Such responses are linked to a n u m b e r of disease states including enhanced h u m a n immunodeficiency virus (HIV) gene expression, L3 atherogenesis, 14 diabetic vasculopathy, ~5 and cancer. 16 Antioxidants such as NAC were identified initially as being potentially effective therapeutics because of their ability to suppress NFKB activation by deleterious agents such as ionizing radiation and the subsequent activation of genes involved in inflammatory processes. 17.18The demonstration that NFKB also could be activated after the exposure of cells to antioxidant agents such as amifostine, NAC, and oltipraz in the absence of an oxidative damage-inducing insult appears to be paradoxical. Such a finding suggests that amifostine along with other thiol-con-

A

From the Department of Radiation and Cellular Oncology, University of Chicago, Chicago, [L and the Interdisciplinary Oncology Program, H. Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL. This work was supported by NIH/NCI R01 grant CA37435 to D.J. Grdina. Address reprint requests to David J. Grdina, The University of Chicago, Depar(ment of Radiation and Cellular Oncology, MCl105, Rm. E-SB-11B, 5841 S. Maryland Ave, Chicago, [L 6063Z Copyright'2002, Elsevier Science (USA). All rights" reserved. 1053-4296/02/1201-0019535.00/0 doi:l O.lO53/srao.2002.31383

Copyright 2002, Elsevier Science (USA). All rights reserved.

Seminars in Radiation Oncology, Vol 12, No 1, Suppl 1 (JanuaF), 2002: pp 103-111

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raining antioxidants such as NAC, mesna, oltipraz, and captopril might be effective inducers of inflammatory responses when administered to patients. Over 150 genes have been identified thus far as being affected by NFJ
expression of MnSOD in both nonmalignant and malignant h u m a n cells.

Materials and M e t h o d s Cells, Culture, and Radiation Conditions 9 Endothelial cells from h u m a n dermis immortalized with SV40 (HMEC) were obtained from Dr T J . Lawley, Biological Products Branch, Centers for Disease Control. They were maintained in endothelial basal medium MCDB131 (Gibco/ BRL, Grand Island, NY) and were grown under conditions described in detail elsewhere. 1~ Hum a n glioma cell lines U87, p53 wild type, and U251,p53 mutation at codon 272, were obtained from the American Type Culture Collection and grown in 75% Dulbecco's modified Eagle's medium and 25% F12 medium (Gibeo) according to methods described in detail elsewhere. 22 All experiments were performed using cells grown to confluency. Cells were irradiated at room temperature with a 250 kVp GE Maxitron x-ray generator operating at 26 mA (HVL, 0.5 m m Cu) with a dose rate of 1.91 Gy/min.

Drugs WR1065 (SH) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer T r e a t m e n t , National Cancer Institute. Immediately before use, it was dissolved at a concentration of 1 mol/L in phosphate-buffered saline (PBS; 8.1 mmol/L Na~HPO4, 1.5 mmol/L Kn2PO4, 140 mmol/L NaC1; Gibco/BRL). Appropriate dilutions then were made in PBS for immediate exposure of cells in their respective culture medium. Both the L- and D-isomeric forms of NAC were used. L-NAC was obtained commercially from Sigma (St Louis, MO). D-NAC was a gift from Dr J e a n e t t e Roberts, who synthesized it in the D e p a r t m e n t of Medicinal Chemistry at the University of Utah. Appropriate concentrations of NAC also were made up in PBS just before use. 1~ TNFc~ (1 m g / m L in PBS) was purchased from Sigma (St Louis, MO). Cells were exposed to a final concentration of l0 ng/mL for 30 minutes in growth medium.

Preparation of Nuclear Extracts Nuclear extracts were prepared from confluent cultures as previously described, w Briefly, nuclear protein was isolated at selected times after

DifferentiM Activation of Genes by Amifostine

exposure of cells to either 40-/xmol/L or 4-mmol/L doses of WR1065. Cells were washed in ice-cold PBS and then removed from plates by gentle scraping. Cells were lysed and nuclei isolated at 4~ The isolated nuclei were resuspended in a high-salt buffer, and after a 15-minute incubation on ice, the nuclear suspension was centrifuged to pellet the nuclei. The resultant supernatant containing the nuclear proteins was removed, and protein concentrations were determined. 23

Electrophoretic Mobility Shift Assays Assays were performed using the Promega (Madison, WI) gel shift assay system following a method described in detail elsewhere. 1~ Briefly, a NFKB consensus oligonucleotide was end labeled with -/YP Adenosine 5'-triphosphate.,Nonspecific and specific competitor reactions were run to show binding specificity using nuclear extracts of H e L a cells as controls, t~ Competitor reactions contained a 50-fold excess of unlabeled SP1 consensus sequence oligonucleotide (nonspecific competitor) or unlabeled NF•B consensus sequence oligonucleotide (specific competitor). All reactions were run at room temperature. The binding reactions were loaded on an 8% nondenaturing polyacrylamide gel and electrophoresed at 5 V/cm for 3 hours. Gels were dried, and binding activ{ty was quantitated by laser densitometric scanning of the resulting autoradiographs. Supershift assays were performed in the same m a n n e r with the exception that subsequent to incubation with the NFKB oligonucleotide, affinity-purified rabbit or goat polyclonal antibodies raised against h u m a n NFKB pS0, p52, p65, and c-Rel (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) were added to the reaction mixture and then incubated at room t e m p e r a t u r e for 30 minutes.

RNA Isolation and Northern Blot Analysis Total RNA was isolated from HMEC and U87 cells using the QIAGEN RNeasy kit (Santa Valencia, CA) and quantified by spectrophotometry. Following a method described in detail elsewhere, RNA was transferred to BrightStar-Plus positively charged nylon m e m b r a n e s (Ambion, Austin, TX). 1~ After transfer, the m e m b r a n e s were washed and then air dried and baked under a vacuum for 2 hours at 80~ The h u m a n MnSOD clone was obtained from the American

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Type Culture Collection (Bethesda, MD). The plasmid was amplified and then purified using a QIAfilter plasmid kit (QIAGEN). The cDNA was isolated from the vector by restriction enzyme digestion and then gel purified. A 7S rRNA probe was kindly supplied by Dr Alan Diamond, Department of Nutrition, University of Illinois at Chicago. It was used as a loading control in all Northern blot experiments. Probes were labeled overnight and unincorporated nucleotides were removed by column chromatography. Following a standard hybridization procedure, 10 blots were air dried and exposed to film at -80~ Band intensities were quantified by laser densitometry.

ELISA Assay Measurements of human vascular endothelial cell growth factor (hVEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNF-c~), interleukin 1 alpha (IL-la), IL-6, IL-8, E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) levels in the supernatant of cell cultures after exposure of cells to 40/xmol/L and 4-mmol/L concentrations of WR1065 for either 6 hours or 24 hours were determined using Quantikine ELISA kits from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions.

Results Presented in Fig 1 are representative gel shifts describing the kinetics of NF•B activation in HMEC after their exposure to either 40-/xmol/L or 4-mmol/L concentrations of WR1065 for 30 minutes. Both concentrations of WR1065 were effective in activating NFKB. The time course of activation was similar for the 2 concentrations tested, with the greatest effect observed between 30 minutes and 1 hour after removal of the drug. To further characterize the effect of WR1065 on the activation of NF•B, supershift gels were run using antibodies to the p50, p65, p52, and c-Rel subunits. Cells were exposed to 4 mmol/L of WR1065 for 30 minutes and then evaluated 30 minutes later. A representative supershift is presented in Fig 2, demonstrating that both the pS0 and p65 subunits are involved. No supershift was observed using antibodies to either p52 or c-Rel. The effect of WR1065 at a concentration of 4 mmol/L on MnSOD gene expression was evalu-

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HMEC

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Figure 1. Time course of NFKB activation in HMEC exposed to 40/xmol/L and 4 mmol/L WR1065 for 30 minutes. Nuclear protein extracts were prepared immediately after drug exposure and at 30 minutes, 1, 2, 3, and 4 hours after drug removal. ated by Northern blot (Fig 3). The relative intensities of the M n S O D bands were measured by densitometric scanning and then compared and normalized with the corresponding 7S rRNA loading controls. A plot of the kinetics of M n S O D expression levels from 3 separate experiments is presented in Fig 4. M n S O D mRNA levels steadily increased to a maximum of about 1.8-fold at 20 hours after the beginning of exposure of cells to WR 1065. HMEC

Nonspecific Competitor Specific Competitor

Representative gel shifts presented in Fig 5 describe the effects of WR1065 and TNF-c~ exposure on the activation of NF~B in human glioma cells. As described earlier for HMEC, both 40/xmol/L and 4-mmol/L concentrations of WR1065 were effective in activating NFKB. The greatest activation of NFKB was observed 1 hour after removal of the drug (data not shown). Cells also were exposed to TNF-c~, a known activator of NFKB, as a positive control. TNF-c~ was much more effective in activating NFKB than was WR1065. A Northern blot along with a plot of the kinetics of M n S O D expression levels in U87 glioma cells exposed to 4 mmol/L of WR1065 is shown in Fig 6. In contrast to HMEC cells,

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Figure 2. Effect of antibodies to p50, p52, p65, and c-Rel on the DNA binding of NFKB induced in HMEC 30 minutes after exposure to 4 mmol/L WR1065 (SH) (supershift assay).

Figure 3. Representative Northern blot describes the time course of elevation in MnSOD gene expression in HMEC exposed to 4 mmol/L WR1065 (SH).

Differential Activation o/ Genes by Amifostine

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WR1065 induced more than a 3-fold increase in

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Figure 4. Bar graph describing the tim~ course of elevation of MnSOD gcne expression in HMEC exposed to 4 mmol/L WRt065 (SH) from 3 separate experiments. Error bars represent the standard error of the mean (SEM).

pression was elevated from 12 to 24 hours after the initial exposure of cells to WR1065, with the maximum expression level appearing at 18 hours. The secretion of hVEGF, bFGF, TNF-a, IL-la, IL-6, IL-8, E-selectin, P-selectin, ICAM-1, and VCAM-1 by HMEC and h u m a n glioma cells was evaluated using enzyme-linked immunosorbent assay (ELISA). No changes were observed for any of these proteins after either a 6-hour or 24-hour treatment with WR1065, regardless of the concentration used. Because the data for each of these proteins were negative, only representative results for E-selectin and hVEGF are presented for illustrative purposes in Figs 7 and 8, respectively.

Discussion The activation of NFKB by thiol-containing reducing agents is not a novel observation. NAC, dithiothreitol (DTT), 2-mercaptoethanol (2-ME),

Nonspecific competitor

Specific competitor Control 4mrvl SH-30min,wash,30min

40p~I SH-30min, wash, lh 10ng/ml TNFa-30min 4ram SH-30mtn~10ng/ml TNFa -30rain 40~M SH-30min,10ng/mi TNFa-30rain

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r NFK:B Figure 5. The comparative effects of a 30-minute exposure of 40 ~mol/L and 4 mmol/L WR1065 (SH) and 10 ~g/mL TNF-a alone or in combination on NFKB activation in U87 and U251 human glioma cells. Nuclear protein extracts were prepared 30 minutes after exposure to 4 mmol/L WR1065 (SH) and TNF-c~alone or in combination and 1 hour after exposure to 40/xmol/L WR1065 (SH).

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U87 Glioma Cells

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Figure 6. Time course of elevation in MnSOD gene expression in U87 glioma cells exposed to 4 mmol/L WR1065 (SH). (Inset) Northern blot showing MnSOD gene expression in cells exposed to 4 mmol/L WR1065 (SH) at times of 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours. oltipraz, and amifostine all have been reported to be effective in activating NFKB. 7,8,~~ It has been suggested that this activation is caused by the generation of reactive oxygen intermediates such as hydrogen peroxide that are formed during an auto-oxidation process. 7 This, however, is unlikely given the observations by several investigators that the addition of hydrogen peroxide scavenging agents such as catalase and pyruvate during exposure of cells to these thiols is ineffective in inhibiting NFKB activation. 7,m Although the mechanism underlying activation of NF~
h u m a n glioma cells (Fig 5) and it was independent of the dose tested. The magnitude of the changes observed in the gel shifts appeared, however, to be slightly greater after an exposure of cells to 40/xmol/L as compared with 4 mmol/L of WR1065 in both HMEC and U251 cells. WR1065 appeared to affect only the p50 and p65 subunits that comprise the principal active form of NF•B (Fig 2). TNF-o~, a known activator of NFJ
Differential Activation of Genes by Amifostine

Human Soluble E-selectin in HMEC Cells

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Figure 7. The effect of 6- and 24-hour exposures of HMEC cells to 40 /xmol/L and 4 mmol/L WR1065 (WR) and 10 mmol/L L-NAC and D-NAC as well as irradiation with 8 Gy and sham treatment on levels of E-selectin measured by ELISA. Experiments were repeated 3 times, and error bars represent the SEM.

MnSOD and those coding for proteins involved in the inflammatory process. 12 Although TNF-c~ activation of NFKB leads to enhanced expression of ICAM-1, WR1065 exposure does not affect its

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expression, l~ WR1065, like the reducing agents NAC, DTT, 2-ME, and oltipraz, induces an enhanced expression of MnSOD (Figs 3, 4, and 6). Its overexpression has been shown to protect against proapoptotic stimuli and ischemia-induced damage, whereas a reduction in its activity has been linked to the progression of malignant disease and deleterious processes related to aging. 19 Transfection of MnSOD into either rodent or human cancer cells that results in enhanced gene expression and subsequent protein activity has been associated with a reduction of the malignant phenotype and a loss of metastatic ability. 16,2~ For these reasons, we evaluated the effects of WR1065 on MnSOD gene expression in both HMEC and human glioma cells. Although the kinetics of enhanced gene expression appeared to be similar in both HMEC and U87 cells, ie, increased levels of expression between 12 and 24 hours, the magnitude of the increased MnSOD expression was almost twice that in glioma cells as compared with HMEC cells (Figs 3, 4, and 6). This finding is consistent with the generalization that baseline MnSOD expression may be lower in most malignant compared with nonmalignant cells. The differential effects of WR1065 on MnSOD and ICAM-1, each known to have NF~B responsive elements in their promoter regions, is consistent with the general observation that activation of NF~B by reducing agents has not been

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Figure 8. The effect of 24-hour exposure of HMEC cells to 40/xmol/L and 4 mmol/L WR1065 (WR) after sham irradiation or irradiation with 750 cGy on levels of hVEGF. Also presented for comparison are hVEGF levels secreted by U87 glioma cells sham irradiated or after exposure to 1,000 cGy. These results are the average from 3 separate experiments. Error bars represent the SEM.

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linked to the induction of inflammatory processes. More than 150 genes are now known to be responsive to NFKB activation. 12 Activation of NF~B, however, will not lead to changes in expression of all genes having NFKB responsive elements in their promoter regions. Promoter and enhancer regions of most genes contain response elements for more than a single transcription factor, suggesting that more than one transcription factor may be required to induce the transcription of a given gene. A mechanism of combinatorial regulation of transcription factors provides the selectivity or specificity of subsequent gene responses. 12 This may account for the lack of an effect on ICAM-1 expression after exposure of cells to WR1065 compared with TNF-cr even though both are capable of activating NFKB and inducing MnSOD gene expression. Because activation of NFKB can lead to the expression of a number of gene families that include cytokines, cell adhesion molecules, and growth factors, we investigated the effects of 6and 24-hour exposures of WR1065 on the secretion of these proteins by H M E C and U87 glioma cells. Specifically, WR1065 was ineffective in enhancing the levels of hVEGF, bFGF, TNF-c~, ILl c~, IL-6, IL-8, E-selectin, P-selectin, ICAM-1, and VCAM-1 secreted by exposed cells. Because the effects were negative for all of the experimental conditions evaluated, only the data for hVEGF and E-selectin were presented as representative results in Figs 7 a n d 8, respectively. These observations are consistent with clinical findings where it has been reported that t r e a t m e n t with amifostine had no affect on serum levels of interleukins I L - l r a and IL-1/3 and cytokines TNF-c~, granulocyte macrophage colony stimulating factor (GMCSF), and stem cell factor (SCF) in patients with acute myelogenous leukemia. 24 Conventional wisdom dictates that because oxidative damage can activate NFKB and enhance the expression of genes involved in inflammatory and damage-related responses that are linked to a number of disease states, there is a need to develop therapeutic inhibitors rather than inducers of NFKB. The observation that reducing agents such as NAC, oltipraz, and amifostine also can activate NFKB suggests that any change/ in the redox environment of the cell may signal the activation of NFKB. However, only oxidative stress will lead to the full range of inflammatory responses attributed to this activation.

Otherwise, it is difficult to reconcile the known protective effects of amifostine, which include its ability to protect against mitochondrial lipid peroxidation, 25 lipid peroxidation in endothelial cells during hypoxia and reoxygenation, 26 and damage to endothelial cells induced by lipopolysaccharides 27 with its ability to activate NFKB. It is proposed that activation of NFKB by reducing agents such as amifostine in the absence of oxidative damage will lead not only to an enhancement in the antioxidant status of the cell, but also to t h e selective activation of antioxidant genes such as MnSOD.

Conclusions NF•B is a redox-sensitive transcription factor whose activation, although usually associated with the exposure of cells to oxidative damage, also can be induced by reducing agents. WR1065, the free thiol form of amifostine, can activate NFKB in both h u m a n microvascular endothelial and h u m a n glioma cells lines. Only the p50-p65 heterodimer compared with the homodimers or heterodimers containing p52 or cRel is affected. Unlike activation of NFKB by oxidative damaging agents that leads to the generalized expression of inflammatory genes and protein products, WR1065 activation of NFKB does not lead to changes in the levels of adhesion molecules, cytokines, and growth factors secreted by either normal or malignant cells. However, exposure of cells to WR1065 does result in the enhanced expression of the antioxidant gene MnSOD, a well-characterized NFKB responsive g e n e that possesses m a n y properties of a tumor suppressor gene. Activation of NFKB in cells by amifostine does not lead to the development of inflammatory processes but rather is related to its cytoprotective and antitumor properties.

Acknowledgments The authors acknowledge helpful discussions with Dr David Rubin during the preparation of this manuscript. This work was supported by N I H / N C I RO1 grant CA37435 to D J . Grdina.

References 1. Clapper ML: Chemopreventive activity of oltipraz. Pharmacol Ther 78:17-27, 1998

Differential Activation of Genes by Amifostine

2. Hensley ML, Schuchter LM, Lindley C, et al: American Society of Clinical Oncology clinical practice guidelines for the use of chemotherapy and radiotherapy protectants. J Clin Oncol 17:3333-3355, 1999 3. Sriswasdi C, Jootar S, Giles FJ: Amifostine and hematologic effects. J Med Assoc Thai 83:374-382, 2000 4. Flohe L, Brigelius-Flohe R, Saliou C, et al: Redox regulation of NF-kappa B activation. Free Radical Biol Med 22:1115-1126, 1997 5. MurleyJ, Constantinou A, Kamath NS, et al: WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase II~x leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells. Cell Prolif 30:283-294, 1997 6. Sen CK, Packer L: Antioxidant and redox regulation of gene transcription. FASEBJ 10:709-720, 1996 7. Das KC, Lewis-Molock Y, White CW: Activation of NF-•B and elevation ofMnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. Am J Physiol 269:L588-L602, 1995 8. Antras-Ferry J, Maheo K, Chevanne M, et al: Oltipraz stimulates the transcription of the manganese superoxide dismutase gene in rat hepatocytes. Carcinogenesis I8: 2113-2117, 1997 9. Romano MF, Lamberti A, Bisogni R, et al: Amifostine inhibits hematopoietic progenitor cell apoptosis by activating NF-KB/ReI transcription factors. Blood 94:40604066, 1999 10. MurleyJS, Kataoka Y, Hallahan DE, et al: Activation of NFKB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells. Free Radical Biol Med 30:1426-1439, 2001 11. Baeuerle PA, Henkel T: Function and activation of NF-~B in the immune system. Annu Rev Immunol 12:141-179, 1994 12. Pahl HL: Activators and target genes of Rel/NFKB transcription factors. Oncogene 18:6853-6866, 1999 13. Griffen GE, Leung K, Folks TM, et al: Activation of HIV gene expression during monocyte differentiation by induction of NF~B. Nature 339:70-73, 1989 14. Parhami F, Fanf ZT, Fogelman AM, et al: Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest 92:471-478, 1993 15. Schmidt AM, Hori O, ChenJX, et ah Advanced glycation endproducts interacting with their endothelial rficeptor induce expression of vascular adhesion molecule-1

16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

111

(VCAM-1) in cultured human endothelial cells and in mice. A potential mechanism for accelerated vasculopathy of diabetes. J Clin Invest 96:1395-1403, 1995 Safford SE, Oberley TD, Urano M, et al: Suppression of fibrosarcoma metastasis by elevated expression of manganese superoxide disnmtase. Cancer Res 54:4261-4265, 1994 Baeuerle PA, Baichwal VR: NF-KB as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol 65:111-137, 1997 Das KC, White CW: Activation of NF-~cB by antineoplastic agents. J Biol Chem 272:14914-14920, 1997 Macmillan-Crow LA, Cruthirds DL: Invited review: Manganese superoxide dismutase in disease. Free Radical Res 34:325-336, 2001 Liu R, Oberley TD, Oberley LW: Transfection and expression ofMnSOD cDNA decreases tumor malignancy of human oral squamous carcinoma SCC-25 cells. Human Gene Ther 8:535-595, 1997 Li N, Oberley TD, Oberley LW, et al: Overexpression of manganese superoxide dismutase in DUI45 human prostate carcinoma cells has multiple effects on cell phenotype. Prostate 35:221-233, 1998 Kataoka Y, MurleyJS, Patel R, et al: Cytoprotection by WR1065, the active form of amifostine, is independent of p53 status in human malignant glioma cell lines. Int J Radiat Biol 76:633-639, 2000 Brahford MM: A rapid and sensitive method for the quant!tation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976 Tao M, Li B, NayiniJ, et ah In vivo effects of IL-4, IL-10, and amifostine on cytokine production in patients with myelogenous leukemia. Leuk Lymphoma 41:161-168, 2001 Tretter L, Ronai E, Szabados G, et aI: The effect of the radioprotector WR-2721 and WR-1065 on mitochondrial lipid peroxidation. Int J Radiat Biol 57:467-478, 1990 Mertsch K, Grune T, Kunstmann S, et al: Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation. Biochem Pharmacol 56:945-954, 1998 Keshavarzian A, Haydek J, Zabihi R, et al: Agents capable of eliminating reactive oxygen species. Catalase, WR2721, or Cu(II)2(3,5-DIPS)4 decrease experimental colitis. Dig Dis Sci 37:1866-1873, 1992