SOD2-mediated adaptive responses induced by low-dose ionizing radiation via TNF signaling and amifostine.

Manganese superoxide dismutase (SOD2)-mediated adaptive processes that protect against radiation-induced micronucleus formation can be induced in cells after a 2-Gy exposure by previously exposing them to either low-dose ionizing radiation (10cGy) or WR1065 (40µM), the active thiol form of amifostine. Although both adaptive processes culminate in elevated levels of SOD2 enzymatic activity, the underlying pathways differ in complexity, with the tumor necrosis factor α (TNFα) signaling pathway implicated in the low-dose radiation-induced response, but not in the thiol-induced pathway. The goal of this study was the characterization of the effects of TNFα receptors 1 and 2 (TNFR1, TNFR2) on the adaptive responses induced by low-dose irradiation or thiol exposure using micronucleus formation as an endpoint. BFS-1 wild-type cells with functional TNFR1 and 2 were exposed 24h before a 2-Gy dose of ionizing radiation to either 10cGy or a 40µM dose of WR1065. BFS2C-SH02 cells, defective in TNFR1, and BFS2C-SH22 cells, defective in both TNFR1 and TNFR2 and generated from BFS2C-SH02 cells by transfection with a murine TNFR2-targeting vector and confirmed to be TNFR2 defective by quantitative PCR, were also exposed under similar conditions for comparison. A 10-cGy dose of radiation induced a significant elevation in SOD2 activity in BFS-1 (P<0.001) and BFS2C-SH02 (P=0.005) but not BFS2C-SH22 cells (P=0.433), compared to their respective untreated controls. In contrast, WR1065 significantly induced elevations in SOD2 activity in all three cell lines (P=0.001, P=0.007, P=0.020, respectively). A significant reduction in the frequency of radiation-induced micronuclei was observed in each cell line when exposure to a 2-Gy challenge dose of radiation occurred during the period of maximal elevation in SOD2 activity. However, this adaptive effect was completely inhibited if the cells were transfected 24h before low-dose radiation or thiol exposure with SOD2 siRNA. Under the conditions tested, TNFR1 and 2 inhibition negatively affected the low-dose radiation-induced but not the thiol-induced adaptive responses observed to be mediated by elevations in SOD2 activity.

Amifostine reduces lung vascular permeability via suppression of inflammatory signalling.

Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria wall-lipopolysaccharide (LPS). Endothelial permeability was monitored by changes in transendothelial electrical resistance. Cytoskeletal remodelling and reactive oxygen species (ROS) production was examined by immunofluorescence. Cell signalling was assessed by Western blot. Measurements of Evans blue extravasation, cell count and protein content in bronchoalveolar lavage fluid were used as in vivo parameters of lung vascular permeability. Hydrogen peroxide, LPS and interleukin-6 caused cytoskeletal reorganisation and increased permeability in the pulmonary endothelial cells, reflecting endothelial barrier dysfunction. These disruptive effects were inhibited by pre-treatment with amifostine and linked to the amifostine-mediated abrogation of ROS production and redox-sensitive signalling cascades, including p38, extracellular signal regulated kinase 1/2, mitogen-activated protein kinases and the nuclear factor-kappaB pathway. In vivo, concurrent amifostine administration inhibited LPS-induced oxidative stress and p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and neutrophil recruitment to the lungs. The present study demonstrates, for the first time, protective effects of amifostine against lipopolysaccharide-induced lung vascular leak in vitro and in animal models of lipopolysaccharide-induced acute lung injury.

Activation of NFkappaB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells.

The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFkappaB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFalpha. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFkappaB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFkappaB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50-p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFkappaB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFkappaB activation by SH. Thus, while oxidative damage is known to activate NFkappaB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFkappaB. There does not appear to be a significant role, if any, for the production of H(2)O(2) as an intermediate step in the activation of NFkappaB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFkappaB. In contrast to TNFalpha, exposure of cells to either 40 microM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.

Cytoprotection by WR-1065, the active form of amifostine, is independent of p53 status in human malignant glioma cell lines.

PURPOSE: This study tests the hypothesis that p53 status, i.e. wild type versus mutant form, is a determinant in radiation protection of human glioma cells by WR-1065, the active thiol form of amifostine (WR-2721). MATERIALS AND METHODS: The cytoprotective effectiveness of WR-1065 when present during irradiation was investigated using four well-characterized human glioma cell lines. The p53 positive lines were U87 and D54, and the mutant p53 lines were U251 (mutant at codon 273; CGT/CAT; Arg/His) and A172 (mutant at codon 242; TGC/TTC; Cys/Phe). Treatment conditions included exposure of cells to a range of doses (0-10Gy) alone or in combination with 4mM of WR-1065 added 30min prior to irradiation. Resultant survival curves were obtained using a clonogenic assay and protection factors, the ratio of terminal slopes +/- WR-1065, were determined for each glioma cell line. RESULTS: The Do values of wild-type U87 and D54 were 1.62 and 1.89Gy while those of p53 mutants U251 and A172 were 1.64 and 1.68 Gy, respectively. Protection factors were determined to be 2.4 and 1.9 for U87 and D54, and 2.6 and 2.8 for U251 and A172, respectively. CONCLUSIONS: The p53 status of the four human glioma cell lines tested was not a predictor for either their relative sensitivity to ionizing radiation or ability to be protected by WR-1065. It is concluded that cytoprotection exhibited by cells exposed to WR-1065 during irradiation is independent of their p53 status.

Amifostine: mechanisms of action underlying cytoprotection and chemoprevention.

Amifostine is an important drug in the new field of cytoprotection. It was developed by the Antiradiation Drug Development Program of the US Army Medical Research and Development Command as a radioprotective compound and was the first drug from that Program to be approved for clinical use in the protection of dose limiting normal tissues in patients against the damaging effects of radiation and chemotherapy. Its unique polyamine-like structure and attached sulfhydryl group give it the potential to participate in a range of cellular processes that make it an exciting candidate for use in both cytoprotection and chemoprevention. Amifostine protects against the DNA damaging effects of ionizing radiation and chemotherapy drug associated reactive species. It possesses anti-mutagenic and anti-carcinogenic properties. At the molecular level, it has been demonstrated to affect redox sensitive transcription factors, gene expression, chromatin stability, and enzymatic activity. At the cellular level it has important effects on growth and cell cycle progression. This review focuses on relating its unique chemical design to mechanisms of action that underlie its broad usefulness as both a cytoprotective and chemopreventive agent for use in cancer therapy.

Chemopreventive doses of amifostine confer no cytoprotection to tumor nodules growing in the lungs of mice treated with cyclophosphamide.

In addition to the cytoprotective benefits of amifostine (Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) to normal cells, it also prevents the induction of somatic mutations that can lead to therapy-induced second cancers. The mutagenic effects of cyclophosphamide, an agent that is known to be mutagenic to normal cells, were determined in mouse splenocytes using a mutational assay system. Cyclophosphamide 100 mg/kg increased mutant frequencies 10-fold. In contrast, amifostine 100 mg/kg, whether administered 30 minutes before or 2 hours after cyclophosphamide administration, resulted in eightfold lowered mutant frequencies. To address potential cytoprotective effects on tumors exposed to this dose, amifostine was administered to tumor-bearing mice either 30 minutes before or 2 hours after the administration of cyclophosphamide. Cyclophosphamide (range, 10 to 100 mg/kg) was administered intraperitoneally into mice 4 days following the injection of 3.5 x 10(5) viable fibrosarcoma (FSa) cells. At this time, microcolonies of FSa tumors containing 50 to 200 cells were present in the lung. The number of FSa lung nodules formed at the end of 14 days in control animals was compared with that of animals treated with cyclophosphamide +/- amifostine. No cytoprotection of murine FSa tumors by amifostine was observed across the entire cyclophosphamide dose range tested, regardless of time of administration, demonstrating the utility of amifostine as a chemopreventive drug under conditions that do not allow cytoprotection for tumor cells.

Effects of thiols on topoisomerase-II alpha activity and cell cycle progression.

Thiol containing compounds exhibiting antioxidant properties are currently being evaluated for use in cytoprotection and chemoprevention. Many of these have also been found to be effective in inhibiting cell cycle progression and cellular proliferation. N-Acetyl-L-cysteine (L-NAC), along with its nonmetabolically active stereoisomer N-acetyl-D-cysteine (D-NAC), together with captopril and dithiothreitol (DTT) were investigated to assess their effects on cell cycle progression as determined by flow cytometry. Topoisomerase-IIa (topo-II alpha) activity, an enzyme involved in DNA synthesis, was also monitored as a function of drug dose using a kinetoplast DNA (kDNA) decatenation assay. Chinese hamster ovary (CHO) AA8 cells were exposed to each thiol at concentrations ranging from 4 microM to 4 mM for a period of 3 h. Following the removal of the thiols, cell cultures were followed for an additional 5 h to assess changes in cell cycle progression. L-NAC, which also serves as a precursor for glutathione (GSH) synthesis, effectively inhibited topo-IIa activity by at least 50% at all concentrations tested. Associated with this reduction in enzyme activity was a sixfold increase in the relative number of cells accumulating in G2phase. D-NAC, which is unable to participate in GSH synthesis, was only half as effective as L-NAC at each concentration tested in inhibiting topo-IIa activity as well as perturbing cell progression through G2. In comparison, captopril, an inhibitor of angiotensin converting enzyme (ACE), had little effect on the progression of cells into G2 phase. In contrast to the repressive effects of L-NAC and D-NAC, it enhanced topo-IIa activity over control values by approximately 20%. DTT, a well characterized thiol known to be capable of reducing disulphides in proteins, was observed to be relatively ineffective in either perturbing cell cycle progression or affecting topo-IIa activity. This suggests an involvement of a mechanism(s) in addition to thiol mediated affects on reduction/oxidation processes. The inhibitory effects of L-NAC and D-NAC on topo-IIa activity, in contrast to the other two thiols, may be due in part to the presence of amine groups which could allow for their participation in polyamine related processes. The difference in the magnitude of the effect exhibited by L-NAC, as compared to D-NAC, on the repression topo-IIa activity also suggests a role for GSH in this process. Inhibition of cellular progression and proliferation by thiols can therefore be mediated by diverse mechanisms which include both cycle-phase specific (i.e. L-NAC and D-NAC) and non cell cycle specific (i.e. captopril) processes.

Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinoma MCF-7 cells.

The aim of this study was to identify the molecular mechanism of action of the isoflavone, genistein. Genistein at 0.15 mM caused MCF-7 apoptotic cell death, which was accompanied by cell cycle delay in the G2/M phase. Twenty-four hours post-treatment, 47.3% of the MCF-7 cells accumulated at G2/M, compared with 19.9% in the untreated controls. At 0.15 mM, genistein caused an increase in the steady-state levels of the wild-type tumour suppressor p53, which was attributed to stabilising the tumour suppressor protein, since p53 mRNA levels did not increase. Prior to the upregulation of p53, which became evident within 6 h of genistein treatment, there was increased bcl-2 phosphorylation at 30 min post-treatment. Although early changes (30-120 min) in the phosphotyrosine peptide patterns were not detected, after 24h, genistein inhibited phosphorylation of several peptides. These results suggest that genistein's dual roles of protein tyrosine kinase inhibitor and topoisomerase II inhibitor are essential for the initiation of apoptosis.

WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase II alpha leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells.

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) II alpha activity, and topo II alpha phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 microM of WR-1065 for 30 min did not effect cell cycle progression nor topo II alpha activity and phosphorylation status. However, concentrations ranging from 4 microM to 4 mM were equally effective in significantly altering these three end points. Cell cycle progression was analysed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G2. Topo II alpha activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 microM to 4 mM dose range tested. Likewise, topo II alpha phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo II alpha activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G2 phase of the cell cycle.

Repression of c-myc gene expression by the thiol and disulfide forms of the cytoprotector amifostine.

The clinically approved cytoprotector amifostine, designated WR-2721, [S-2-(3-aminopropylamino)ethylphosphorothioic acid], protects against both radiation and drug-induced mutagenesis in animal systems. These effects extend over a wide concentration range making amifostine a strong candidate for evaluation as a possible cancer chemopreventive agent. To better identify and develop potential intermediate biomarkers for chemoprevention at the molecular level we applied the technique of differential display RT-PCR to assess the effects of both the thiol (SH), i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of amifostine on gene expression in CHO-AA8 cells. Cells were exposed to either 40 microM or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in corresponding untreated control cells. One band that showed a differential response was sequenced and was found to have 78% homology with a segment of the human pHL-1 cDNA clone contained in GenBank. This clone contains a COX III mitochondrial DNA insert and two exons of human c-myc. Northern blot analyses were performed by using the cloned human c-myc exon 1 probe to confirm whether c-myc gene expression was affected. Repression of c-myc expression was observed under all of the conditions evaluated. An exposure of cells to 40 microM of the disulfide form of amifostine was the most effective in repressing c-myc, i.e. 27% of control level. A concentration of 4 mM of the disulfide form reduced gene expression to 45% of the control level, while the thiol form was less effective, with 4 mM and 40 microM concentrations reducing c-myc gene expression to 65% and 46% of control levels, respectively.

The effects of cycloheximide and WR-1065 on radiation-induced repair processes: a mechanism for chemoprevention.

The effects of cycloheximide (CHX) and 2-[(aminopropyl)-amino]ethanethiol (WR-1065), each alone or in combination, on radiation-induced mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus and cell killing were investigated using a Chinese hamster ovary (CHO) AA8 cell system. Treatment with CHX, a potent inhibitor of protein synthesis, at a concentration of 10 micrograms/ml administered 30 min prior to irradiation with 7.5 Gy had no effect on cell survival but did reduce the radiation-induced mutation frequency (per 10(6) survivors) from 106.5 +/- 8.8 (SEM) to 36.2 +/- 5.6 (SEM). Exposure of cells to 4 mM WR-1065 reduced the mutation frequency to 44.8 +/- 4.2 (SEM), but the combination of agents afforded no additional protection, that is 41.1 +/- 3.3 (SEM). The mechanism of action attributed to CHX in reducing mutation frequency is its ability to prevent the induction of an error-prone repair system. Split-dose radiation experiments, that is 8 Gy versus 4 Gy + 4 Gy separated by 3 h, were performed to evaluate and contrast the relative abilities of CHX and WR-1065, each alone or in combination, in affecting cell survival. Cycloheximide administered to cells 30 min before the first radiation dose and present throughout the 3 h incubation time prior to the second dose inhibited split-dose repair as evidence by a reduction in surviving fraction by 60% as compared with the value obtained for non-CHX-treated cells that were exposed to two equal doses of 4 Gy. Cells exposed to 4 mM WR-1065 immediately following the first 4 Gy radiation dose and then washed free 2.5 h before exposure to a second Gy dose, which was also followed by a 30 min exposure to WR-1065, increased the surviving fraction by 80% over the value obtained for cells not exposed to WR-1065 during their split-dose radiation treatment. When CHX treatment was combined with WR-1065 was abolished, that is surviving cell fraction was again reduced by approximately 60% as compared with untreated control groups. These results indicate that protein synthesis is required for WR-1065 to affect split-dose related repair processes. Presumably, the inhibition of the induction of an error-phone repair system by CHX would account for its effects on both resultant decreases in mutation frequency and cell survival. In contrast, WR-1065 and/or its disulfide metabolite appear to facilitate the efficacy and fidelity of such a repair system once it is induced.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of topoisomerase II alpha activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065).

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector when it is administered 30 min prior to exposure of Chinese hamster ovary K1 cells to radiation (i.e., a dose modification factor of 1.4) at a concentration of 4 mM. Under these exposure conditions, topoisomerase (Topo) I and II alpha activities and associated protein contents were measured in cells of the K1 cell line using the DNA relaxation assay, the P4 unknotting assay and immunoblotting, respectively. WR-1065 was ineffective in modifying Topo I activity, but it did reduce Topo II alpha activity by an average of 50%. The magnitude of Topo II alpha protein content, however, was not affected by these exposure conditions. The effects on the cell cycle were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for up to 6 h resulted in a build-up of cells in the G2/M-phase compartment. However, under these conditions and in contrast to Topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotective agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of action attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis and repair and progression of cells through the phases of the cell cycle. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by WR-1065 on Topo II, which is involved in DNA synthesis.

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065.

WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G2 in WR-1065 treated Chinese hamster ovary cells grown in alpha-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with 137Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in alpha-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G2 population and a decrease in the G1 population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.

Accumulation of CHO cells in G2 phase following exposure to WR-1065.

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.