Induction of mitochondrial changes in myeloma cells by imexon

Imexon is a cyanoaziridine derivative that has antitumor activity in multiple myeloma. Previous studies have shown that imexon induces oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This study reports that imexon has cytotoxic activity in other malignant cell lines including NCI-H929 myeloma cells and NB-4 acute promyelocytic leukemia cells, whereas normal lymphocytes and U266 myeloma cells are substantially less sensitive. Flow cytometric experiments have shown that imexon treatment is associated with the formation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Deltapsi(m)) in imexon-sensitive myeloma cell lines and NB-4 cells. In contrast, reduction of Deltapsi(m) and increased levels of ROS were not observed in imexon-resistant U266 cells. Treatment of imexon-sensitive RPMI 8226 cells with the antioxidant N-acetyl-L-cysteine (NAC) protects cells against these effects of imexon. Mitochondrial swelling was observed by electron microscopy in RPMI 8226 myeloma cells treated with 180 microM imexon as early as 4 hours. Damage to mitochondrial DNA was detected by a semiquantitative polymerase chain reaction assay in imexon-treated RPMI 8226 cells; however, nuclear DNA was not affected. Finally, partial protection of RPMI 8226 cells against the imexon effects was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex II. These changes are consistent with mitochondrial oxidation and apoptotic signaling as mediators of the growth inhibitory effects of imexon. Interestingly, oxidative damage and decrease of Deltapsi(m) induced by imexon highly correlates with sensitivity to imexon in several myeloma cell lines and an acute promyelocytic leukemia cell line. (Blood. 2001;97:3544-3551)     

Increasing the antioxidant defense in WEHI7.2 cells results in a more tumor-like metabolic profile

Chronic inflammation is often associated with increased cancer frequency. Continuous exposure to reactive oxygen species, as at the site of chronic inflammation, can result in cells with increased antioxidant defense enzymes. In WEHI7.2 cells, overexpression of catalase or thioredoxin by transfection or selection of a cell population resistant to hydrogen peroxide has resulted in WEHI7.2 variants with altered glucose and energy metabolism. This metabolic change would favor survival in a tumor environment. We conclude that metabolic alterations, due to increased antioxidant enzyme expression, may underlie the increased tumorigenicity seen previously in the variants and contribute to the increased tumor risk associated with chronic inflammation.     

Changes in phosphate metabolism in thymoma cells suggest mechanisms for resistance to dexamethasone-induced apoptosis. A 31P NMR spectroscopic study of cell extracts

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants.     

Molecular and cellular characterization of imexon-resistant RPMI8226/I myeloma cells

Imexon is an aziridine-containing iminopyrrolidone with selective growth-inhibitory potency for multiple myeloma. Our previous research indicates that imexon induces mitochondrial alterations, oxidative stress, and apoptosis. This drug represents an interesting model drug with a nonmyelosuppressive profile to study the basic mechanisms leading to antitumor activity and resistance. The major purpose of this study was to characterize an imexon-resistant RPMI8226/I cell line that was developed from RPMI8226 cells by continuous exposure to imexon. No significant differences were observed in the sensitivity to several cytotoxic drugs, including mitoxantrone, mitomycin C, melphalan, methotrexate, cytarabine, cisplatin, vincristine, and paclitaxel, in the imexon-resistant cells. However, RPMI8226/I cells were cross-resistant to arsenic trioxide, doxorubicin, fluorouracil, etoposide, irinotecan, and especially IFN-alpha. The data from DNA microarray and Western blot analyses indicated that the levels of antiapoptotic proteins Bcl-2 and thioredoxin-2, which reside mainly in the mitochondria, are increased in RPMI8226/I cells. In addition, increased levels of lung resistance protein were detected in imexon-resistant cells. Expression of P-glycoprotein was not detected in RPMI8226/I cells. No loss of mitochondrial membrane potential or increase in the levels of reactive oxygen species was observed in RPMI8226/I cells after exposure to imexon; however, the levels of glutathione are increased in the RPMI8226/I cells. Transmission electron microscopy revealed significant changes in the mitochondrial morphology of RPMI8226/I cells, whereas no ultrastructural changes were observed in other cellular compartments. Imexon-resistant RPMI8226/I myeloma cells appear to have a unique mechanism of resistance that is associated with morphological alterations of mitochondria, increased protection against oxidative stress, elevated levels of glutathione, and enhanced expression of antiapoptotic mitochondrial proteins.     

Tactoidal state and phase transitions in systems of linear polymers of variable length

The tactoidal state in systems containing long, rod-like molecules consists of partially aligned solute molecules in equilibrium with and at a concentration not much higher than that in the conjugate isotropic phase. Under the liquid lattice model of Flory [Proc. R. Soc. London Ser. A, (1956) 234, 73-89], as well as under other models, tactoid formation by molecules of fixed axial ratio depends on nonideality induced by excluded volumes; the process is wholly entropy driven and requires no direct interactions between rods.Many rod-like biological polymers exhibit reversible polymerization, so that axial ratio and length are not fixed. Polymerization and rod length will then not only induce nonideality, alignment, and phase separation, but will be affected by these. In this work these interrelations are treated under the model of Flory, modified to include a free energy of polymerization and to permit reversible changes in rod length. The primary conclusion is that, in contrast to the situation for fixed lengths, excluded volume-dependent nonideality alone does not suffice to induce a tactoidal phase separation. In the absence of attractions or repulsions between rods the anisotropic phase is highly concentrated. This phase only becomes tactoidal when a minimal level of repulsive interaction between rods is reached. Under this model, tactoid formation in systems such as deoxygenated hemoglobin S and tobacco mosaic virus depends on repulsive interactions or metastability or both. As a secondary result it is shown that rod length in the anisotropic phase is much greater than in the conjugate isotropic phase.     

Hemoglobin S polymerization and gelation under shear II. The joint concentration and shear dependence of kinetics

The kinetics of hemoglobin S gelation are critical in sickle disease because microvascular obstruction can be avoided if red blood cells pass these vessels during the delay time, before polymerization and gelation occur in sufficient degree to rigidify the cells. Kinetics, including the delay time and the closely related exponential progress rate, are highly sensitive to hemoglobin concentration and degree of deoxygenation. Kinetics are also greatly accelerated by shear, an effect that may contribute to pathogenesis, since red blood cells deform and can undergo shear in vivo. Here we examine the joint dependence of kinetics on shear and hemoglobin concentration. As shear rate increases, the concentration dependence of the exponential progress rate decreases. The large decrease in concentration dependence supports the conclusion that acceleration of gelation by shear is due to breakage and not to enhancement of heterogeneous nucleation. Under shear, new fibers are created by breakage of existing ones, as well as by heterogeneous nucleation. At high shear, the rate of new fiber creation by breakage is very great and dominates that by heterogeneous nucleation. Therefore, if breakage depended only on shear rate and were independent of the concentration of hemoglobin in solution, the concentration dependence of kinetics should vanish. Although it decreases, it does not disappear. The concentration dependence that remains at high shear arises from (1) the direct contribution of fiber growth rate to the exponential progress rate, (2) the dependence of breakage rate on fiber growth rate, and (3) the dependence of solution viscosity on hemoglobin concentration.     

Elevated basal reactive oxygen species and phospho-Akt in murine keratinocytes resistant to ultraviolet B-induced apoptosis

Resistance to apoptosis may be a critical phenotype that cells must acquire during skin carcinogenesis. The Akt kinase is a known upstream regulator of apoptosis in many cell types and has been shown to be activated by increased reactive oxygen species (ROS). We have previously demonstrated that two malignant variants (6M90 and 6R90) of the mouse keratinocyte 308 cell line have elevated ROS because of loss of catalase activity, and that this elevated ROS confers a growth advantage. We report here that in addition to a growth advantage, chronically increased ROS in the variants resulted in an increase in resistance to ultraviolet (UV) B-induced apoptosis. This resistance was due to basal increases of Akt phosphorylation in the malignant variants compared to the 308 cells. Modulation of ROS in 6M90 and 6R90 cells by catalase overexpression or antioxidant treatment resulted in decreased levels of Akt phosphorylation and subsequent loss of resistance to UVB-induced apoptosis. Conversely, treatment of 308 cells with hydrogen peroxide caused increases in Akt phosphorylation and increased apoptosis resistance. These results indicate that the chronically elevated ROS often observed in tumors may contribute to a malignant phenotype by keeping Akt in a phosphorylated state, resulting in increased apoptosis resistance.