The antitumor activity of Meconopsis horridula Hook, a traditional Tibetan medical plant, in murine leukemia L1210 cells.

BACKGROUND: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. METHODS AND RESULTS: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. CONCLUSION: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process.

Formulation, antileukemia mechanism, pharmacokinetics, and biodistribution of a novel liposomal emodin.

Emodin is a multifunctional Chinese traditional medicine with poor water solubility. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a pegylated vitamin E derivate. In this study, a novel liposomal-emodin-conjugating TPGS was formulated and compared with methoxypolyethyleneglycol 2000-derivatized distearoyl-phosphatidylethanolamine (mPEG2000-DSPE) liposomal emodin. TPGS improved the encapsulation efficiency and stability of emodin egg phosphatidylcholine/cholesterol liposomes. A high encapsulation efficiency of 95.2% ± 3.0%, particle size of 121.1 ± 44.9 nm, spherical ultrastructure, and sustained in vitro release of TPGS liposomal emodin were observed; these were similar to mPEG2000-DSPE liposomes. Only the zeta potential of -13.1 ± 2.7 mV was significantly different to that for mPEG2000-DSPE liposomes. Compared to mPEG2000-DSPE liposomes, TPGS liposomes improved the cytotoxicity of emodin on leukemia cells by regulating the protein levels of myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein, which was further enhanced by transferrin. TPGS liposomes prolonged the circulation time of emodin in the blood, with the area under the concentration-time curve (AUC) 1.7 times larger than for free emodin and 0.91 times larger than for mPEG2000-DSPE liposomes. In addition, TPGS liposomes showed higher AUC for emodin in the lung and kidney than for mPEG2000-DSPE liposomes, and both liposomes elevated the amount of emodin in the heart. Overall, TPGS is a pegylated agent that could potentially be used to compose a stable liposomal emodin with enhanced therapeutics.

Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3.

Lunasin is a novel chemopreventive peptide featuring a cell adhesion motif composed of arginine-glycine-aspartate (RGD) which has been associated to cytotoxicity to established cell lines. The objectives of this study were to determine the effect of lunasin on the viability of L1210 leukemia cells and to understand the underlying mechanisms involved. Pure lunasin and lunasin enriched soy flour (LES) caused cytotoxicity to L1210 leukemia cells with IC(50) of 14 and 16 microM (lunasin equivalent), respectively. Simulated gastrointestinal digestion showed that 25% of the original amount of lunasin survived 3 h of pepsin digestion and 3% of lunasin remained after sequential pepsin-pancreatin digestion for a total of 6 h. Cell cycle analysis showed that lunasin caused a dose-dependent G2 cell cycle arrest and apoptosis. Treatment of L1210 leukemia cells with 1 mg/mL of LES for 18 h led to an increase in the amount of apoptotic cells from 2 to 40%. Compared to untreated cells, treatment with 1 mg/mL LES showed a 6-fold increase on the expressions of caspases-8 and -9, and and a 12-fold increase on the expression of caspase-3. These results showed for the first time that lunasin, a naturally occurring peptide containing an RGD motif, caused apoptosis to L1210 leukemia cells through caspase-3 activation.

[Activity of neurohormonal hypothalamus-adrenal cortex axis in the course of transplantable mouse leukemia L1210]. / Aktywnosc osi neurohormonalnej podwzgórze-kora nadnerczy w przebiegu przeszczepialnej bialaczki mysiej L1210.

The activity of the neurohormonal hypothalamus-adrenal cortex axis in the course of transplantable mouse leukemia L1210 was investigated. The secretory activity of the arcuate nucleus producing CRF, the fascicular zone of adrenal cortex secreting glicocorticosteroids and nucleus supraopticus hypothalami producing vasopressin was estimated with the karyometric method. Four groups of mice killed in various stages of cancerous process's advance were used. It was demonstrated that rapidly developing cancer is a considerable stress, which is proved by the increase in the neurohormonal axis activity in comparison to the control groups. The activity of the hypothalamo-adrenal axis was typical for a stress reaction: a two-phase process with a considerable increase in activity in the introductory phase of the developing leukemia and a visible decrease in the last phase, typical for an unspecific reaction, where no more effective mobilization for adaptation and activation of mechanisms is possible. The production of vasopressin has similar progress to other glands investigated, which suggests its stimulating influence on activation of the pituitary-adrenal axis. The statistical analysis of karyometric measurements was carried out by the Student's t test.

Sensitivity to 5,10-dideazatetrahydrofolate is fully conserved in a murine leukemia cell line highly resistant to methotrexate due to impaired transport mediated by the reduced folate carrier.

A murine leukemia cell line was identified that is highly resistant to methotrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dideazatetrahydrofolate (DDATHF). A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in drug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell line markedly increased DDATHF influx (32x) but only modestly increased influx of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively). The growth inhibition or growth requirements for these folates fell by factors of 18, 2, and 4, respectively, in the transfectant. Preservation of DDATHF influx in cells with V104M RFC1 resulted in even greater preservation (60%) of the exchangeable drug level. Another major element in the preservation of DDATHF activity was the impact of the mutated carrier on cellular folate pools. For folic acid, folate pools were essentially unchanged but DDATHF polyglutamate levels decreased in lines that express the V104M carrier. However, with 5-formyltetrahydrofolate as the growth source, there was a marked decrease in folate pools in the lines carrying the mutated carrier, and DDATHF polyglutamate levels were unchanged. Hence, DDATHF activity was preserved in cells with V104M RFC1 due to (a) relative conservation of DDATHF transport, and (b) depletion of cellular THF cofactors with diminishing folate cofactor competition at folylpolyglutamate synthetase and possibly glycinamide ribonucleotide formyltransferase. Hence, resistance to one antifolate, in this case MTX, because of a loss of RFC1 transport activity need not exclude the subsequent utility of another antifolate that uses the same carrier.

Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells.

Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3'-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating approximately 65-90 kDa native reduced folate carrier and a approximately 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed.

Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier.

A phenylalanine substitution for serine in the reduced folate carrier at residue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cell line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folate source. The transport characteristics of the mutated carrier were studied by transfection into the MTXrA line, which lacks endogenous RFC1 function. The level of expression of carrier in the cell lines studied was determined by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF). Influx of 5-CH3-THF and 5-CHO-THF mediated by RFC1-S309F was 20- and 7-fold greater than that of MTX, respectively. Consistent with the influx difference between 5-CHO-THF and MTX, the growth requirement (EC50) for 5-CHO-THF in MTXrA-S309F cells was decreased by a factor of 9, while the MTX IC50 was reduced by a factor of only approximately 2 as compared with the recipient MTXrA cells. The decrease in 5-CH3-THF influx mediated by the mutated carrier was attributed to a decrease in the mobility of the 5-CH3-THF-carrier complex, since the influx Kt was essentially unchanged. However, the reduction in 5-CHO-THF and MTX influx was attributed to decreases in both carrier affinity and Vmax, although the decline in the MTX influx Vmax appeared to be much greater than for 5-CHO-THF. The inhibitory effect of chloride on 5-CHO-THF influx observed for L1210 cells was eliminated in the MTXrA-S309F line. This study represents another example of a single mutation in RFC1 that markedly impairs MTX influx but partially preserves transport of reduced folates when cells are selected with 5-CHO-THF as the available folate substrate. The data indicate that residues in the predicted eighth transmembrane domain of RFC1 can play an important role in the selectivity of folate binding and the mobility of the carrier-substrate complex.

The potentiated antileukemic effects of doxorubicin and interleukin-12 combination are not dependent on nitric oxide production.

In our recent study we described a significant antileukemic efficacy of a combination therapy with interleukin-12 (IL-12) and doxorubicin (DOX) in the L1210 leukemia model. This therapeutic effect was abrogated by elimination of activated macrophages. Activated macrophages produce a variety of factors that can contribute to the elimination of tumor cells in vivo, including proteases, TNF, reactive oxygen intermediates, and nitric oxide (NO). Based on the results of previous reports, the contribution of NO in potentiated antileukemic effects of IL-12 + DOX combination seemed to be highly possible. Both DOX and IL-12 given alone increased the production of NO by peritoneal macrophages, however, macrophages derived from the mice treated with the combination of those agents produced significantly less NO than macrophages from IL-12-alone-treated mice. Production of NO by spleen macrophages after IL-12 + DOX treatment was higher than it was in controls, IL-12-alone or DOX-alone-treated groups. In serum, concentrations of NOx- in IL-12- or IL-12 + DOX-treated mice were significantly higher in comparison with controls, however not significantly different from each other. Addition of L-NAME treatment to the IL-12 + DOX therapy in leukemia-bearing mice did not significantly change the antileukemic efficacy of this therapy. Thus, our results indicate that the augmented antileukemic effects of IL-12 + DOX combination therapy in L1210 model are NO-independent. Therefore, further studies on the possible mechanisms of potentiated antileukemic activity of combination of IL-12 and DOX would be worth pursuing.

Singlet oxygen toxicity is cell line-dependent: a study of lipid peroxidation in nine leukemia cell lines.

Singlet oxygen (1O2) can be quenched by water, lipids, proteins, nucleic acids and other small molecules. Polyunsaturated fatty acids (PUFA) of cells principally quench 1O2 by chemical mechanisms, producing lipid hydroperoxides, while proteins physically and chemically quench 1O2. Because cell lines can have different PUFA and protein levels, we hypothesized that 1O2 toxicity will vary between cell lines. We used Photofrin as a source of 1O2. Exposure of nine different leukemia cell lines (CEM, HEL, HL-60, K-562, KG-1, L1210, Molt-4, THP-1 and U-937) to Photofrin and light results in changes in membrane permeability (trypan blue) that vary with cell line. The greater the lipid content of the cell line, the less susceptible they are to membrane damage. When the cell media was supplemented with docosahexaenoic acid (DHA, 22:6), the overall unsaturation of cellular lipids increased. Photofrin and light resulted in increased radical formation in these supplemented cells compared to controls; however, there was no difference in membrane permeability between DHA-supplemented and control cells. Lipid-derived radical formation (electron paramagnetic resonance spin trapping) was cell line dependent; but no correlation between lipid content of cells and radical formation was found. However, we found that the greater the protein content of cells the more they were protected against membrane damage induced by Photofrin photosensitization. This suggests that cellular proteins are a key target for 1O2-mediated toxicity. A remarkable observation is that cell size correlates inversely with ability of cells to cope with a given flux of 1O2.

[Scavenging effect of total flavonoids of lycium barbarum L on active oxygen radicals and inhibitory effects on heat output from L1210 cells].

The scavenging effects of total flavonoids of Lycium barbarum L. (TFL) were studied by using ESR-spin trapping technique and the inhibitory effects on heat output of both polymorphonuclear leukocyte(PMN) respiration burst and L1210 cells were measured by using microcalorimetric technique. TFL (0-217 mg/L) could scavenge O2-. in xanthine/xanthine oxidase (Xan/XO)system, with scavenging rate of 0-51%. TFL(7.5-200 mg/L)could scavenge OH. produced in Fenton reaction and the scavenging rate is between 20% to 72%. Those effects were concentration-dependent. Furthermore, TFL(0.56 g/L)could completely inhibit the heat output from PMA-stimulated PMN and TFL(1.0-5.0 g/L)could inhibit the heat output from L1210 cells.

Vitamin E slows the rate of free radical-mediated lipid peroxidation in cells.

Much of what is known about the antioxidant mechanism of vitamin E has been learned from studies of lipid dispersions, solutions, or subcellular organelles. We have investigated the effect of vitamin E supplementation on intact live eucaryotic cells. L1210 murine leukemia cells were exposed to an oxidative stress induced by 20 microM Fe2+ and 100 microM ascorbic acid introduced immediately before oxidative measurements were begun, and the kinetics of the generation of lipid-derived free radicals, as measured by EPR spin trapping (a product) and O2 consumption (a reactant) were measured. Cells grown for 24 h with supplemental (5-100 microM) vitamin E in their media had a slower rate of lipid radical generation compared to cells grown without vitamin E supplementation; this inhibition in the rate of oxidation was generally dependent upon the amount of vitamin E supplementation. In complementary studies measuring O2 consumption, 5-100 microM vitamin E slowed the rate of oxidation (10-fold with 100 microM supplemental vitamin E) consistent with the EPR studies. The membrane active drug edelfosine accentuated the vitamin E effects; vitamin E introduced a discernible lag phase (time delay) in both lipid radical generation and O2 consumption that was not seen in the absence of edelfosine. Vitamin E supplementation of cells also altered the kinetics of ascorbate free radical formation. We conclude that vitamin E inhibits lipid peroxidation in cells by slowing the rate of lipid peroxidation; but with iron/ascorbate as the initiating system, vitamin E does not delay the onset of peroxidation. Of special interest is that these free radical peroxidation events parallel cell membrane damage as detected using trypan blue exclusion. These observations are consistent with the free radical events preceding and causing the observed membrane damage.

Role of hydrogen peroxide in the cytotoxic effects of UVA/B radiation on mammalian cells.

Effects of selenium (Se) deficiency on the sensitivity of murine leukemia L1210 cells to broad band UVA/B radiation (310-400 nm) have been investigated. Cells rendered glutathione peroxidase (GPX) deficient by shortterm (2-3 week) growth in 1%, serum/RPMI medium without added Se [L.Se(-) cells] were found to be much less resistant to clonally assessed UVA/B lethality than Se-supplemented controls [L.Se(+) cells]. By contrast, long-term ( > 20 week) Se-deprived [L'.Se(-)] cells whose catalase (CAT) activity was elevated > 100-fold were far more resistant to UVA/B than L.Se(+) cells. Similar trends were observed for cells irradiated in 1% serum/RPMI or Hank's medium. Whereas the CAT inhibitor 3-amino-1,2,4-triazole had no effect on L.Se(+) photosensitivity, it produced a large increase in L'.Se(-) photosensitivity. These findings are consistent with H2O2 intermediacy in photokilling and suggest that L1210 cells depend mainly on GPX for protection against this species but switch to overexpressed CAT after chronic Se deprivation. In agreement with this, steady-state H2O2 levels measured by H2O2 electrode during UVA/B exposure were higher in L.Se(-) than L.Se(+) suspensions but much lower (barely detectable) in L'.Se(-) suspensions. Cytotoxic effects of UVA/B and variations thereof resulting from Se manipulation could be mimicked by treating cells with glucose oxidase in the presence of D-glucose, providing further support for H2O2 involvement. Whether UVA/B-generated H2O2 is directly cytotoxic or gives rise to a more damaging species such as hydroxyl radical (HO) is presently unknown.

Omega-3 polyunsaturated fatty acids increase purine but not pyrimidine transport in L1210 leukaemia cells.

Here we show that in vitro supplementation of L1210 murine lymphoblastic leukaemia cells with n-3 polyunsaturated fatty acids results in considerable changes in the fatty acid composition of membrane phospholipids. Incubations for 48 h with 30 microM eicosapentaenoic acid (20:5, n-3; EPA) or docosahexaenoic acid (22:6, n-3; DHA) results primarily in substitution of long chain n-6 fatty acids with long-chain n-3 fatty acids. This results in a decrease in the n-6/n-3 ratio from 6.9 in unsupplemented cultures to 1.2 or 1.6 for EPA and DHA supplemented cultures, respectively. Coincident with these changes in membrane fatty acid composition, we observed a 5-fold increase in the rate of adenosine (5 microM) uptake via a nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter in EPA- and DHA-supplemented L1210 cells, relative to unsupplemented cells. This seemed to result from a decrease in the Km for adenosine from 12.5 microM in unsupplemented cultures to 5.1 microM in DHA-treated cultures. Guanosine (50 microM) transport was similarly affected by DHA with a 3.5-fold increase in the initial rate of uptake. In contrast, pyrimidine transport, as measured by uptake of thymidine and cytidine, was not similarly affected, suggesting that substrate recognition had been altered by fatty acid supplementation. Studies using [(3)H]NBMPR showed that there was no effect of EPA or DHA on either the number of NBMPR-binding sites or the affinity of these sites for NBMPR. This observation suggests that the increases in adenosine and guanosine transport were not due to increases in the number of transported sites but rather that EPA and DHA directly or indirectly modulate transporter function.

Selenoperoxidase-dependent glutathione cycle activity in peroxide-challenged leukemia cells.

Murine leukemia L1210 cells rendered deficient in glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) by Se deprivation (L.Se(-) cells) were found to be more sensitive to tert-butyl hydroperoxide (t-BuOOH) cytotoxicity than Se-replete controls (L.Se(+) cells). Human K562 cells, which express PHGPX, but not GPX, were also more sensitive to t-BuOOH in the Se-deficient (K.Se(-)) than Se-satisfied (K.Se(+)) condition. In examining the metabolic basis for selenoperoxidase-dependent resistance, we found that glucose-replete Se(-) cells reduce t-BuOOH to t-butanol far more slowly than Se(+) cells, the ratio of the first-order rate constants approximating that of the GPX activities (L1210 cells) or PHGPX activities (K562 cells). Monitoring peroxide-induced changes in GSH and GSSG gave consistent results; e.g., glucose-depleted L.Se(+) cells exhibited a first order loss of GSH that was substantially faster than that of glucose-depleted L.Se(-) cells. Under the conditions used, peroxide-induced conversion of GSH to GSSG could be stoichiometrically reversed by resupplying D-glucose, indicating that no significant lysis or GSSG efflux and/or interchange had taken place. The apparent first-order rate constant for GSH decay increased progressively for L1210 cells expressing a range of GPX activities from approximately 5% to 100%, demonstrating that peroxide detoxification is strictly dependent on enzyme content. The initial rate of 14CO2 release from D-[1-14C]glucose supplied in the medium was much greater for L.Se(+) or K.Se(+) cells than for their respective Se(-) counterparts, consistent with greater hexose monophosphate shunt activity in the former. These results highlight the importance of selenoperoxidase action in the glutathione cycle as a means by which tumor cells cope with hydroperoxide stress.

Relative alpha-tocopherol deficiency in cultured cells: free radical-mediated lipid peroxidation, lipid oxidizability, and cellular polyunsaturated fatty acid content.

We propose that most cultured cells are deficient in vitamin E. Using our optimized assay for tocopherol, we find that L1210 lymphoblastic leukemia cells, cultured in standard growth media, contain only 2.3 +/- 0.03 micrograms of tocopherol/10(8) cells, whereas when they are transplanted and grown for the same time in the ascites fluid of mice fed standard diets, this increases to 5.8 +/- 0.6 micrograms of tocopherol/10(8) cells. This apparent tocopherol deficiency in cultured cells is likely due to the low concentrations of tocopherol contained in most tissue culture media, even with the addition of serum. To further study this apparent deficiency and the relationship of cellular tocopherol to membrane lipid bis-allylic hydrogen positions, we supplemented the growth media of L1210 lymphoblastic leukemia cells with alpha-tocopherol and compared the resultant cellular tocopherol content to the degree of unsaturation of cellular lipids, alpha-Tocopherol was incorporated by cells in a time- and concentration-dependent manner with plateaus at 24 h and 100 microM, respectively. A maximum 400% increase in cellular tocopherol was easily achieved. By experimentally modifying the fatty acid content of cellular lipids, we were able to determine that cellular tocopherol uptake and content is not a function of cellular lipid composition; cells enriched with polyunsaturated lipids incorporated tocopherol to the same extent as those enriched with more saturated lipids. Thus, as the cellular polyunsaturated fatty acid content increases, the tocopherol:bis-allylic position ratio in the cells decreases, resulting in less antioxidant protection for each lipid double bond. Consequently, when polyunsaturated fatty acid-enriched cells are exposed to an oxidative stress, such as Fe2+, their tocopherol levels decline much faster than cells enriched with saturated fatty acids. This decline is consistent with their respective tocopherol:bis-allylic position ratio. These results provide a basis, at the cellular level, for investigators to consider vitamin E when studying cell response to oxidative stress.

Cytoprotection against merocyanine 540-sensitized photoinactivation of the Na+,K(+)-adenosine triphosphatase in leukemia cells: glutathione and selenoperoxidase involvement.

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na+,K(+)-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na+,K(+)-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photo-inactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na+,K(+)-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na+,K(+)-ATPase, but not AChE. This is consistent with the fact that Na+,K(+)-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.

Membrane lipid free radicals produced from L1210 murine leukemia cells by photofrin photosensitization: an electron paramagnetic resonance spin trapping study.

We have detected membrane lipid-derived free radicals from neoplastic cells subjected to Photofrin photosensitization. The presence of the prooxidants iron or iron plus ascorbate in the L1210 cell system increased the intensity of the spin-trapped lipid radical electron paramagnetic resonance spectra and correspondingly decreased cell survival. In addition, raising the proportion of unsaturated lipids in the cell membranes by supplementation of the growth medium with docosahexaenoic acid increased lipid radical formation and decreased cell survival when the L1210 cells were subjected to Photofrin and light. These data educe the hypothesis that the extent of radical generation as well as the efficacy of photodynamic therapy can be increased when prooxidant conditions, which enhance free radical processes, are present in conjunction with photosensitizers that target membrane lipids.

Selenoperoxidase-mediated cytoprotection against the damaging effects of tert-butyl hydroperoxide on leukemia cells.

Murine leukemia L1210 cells grown for 5-7 d in the presence of 1% serum without added selenium [Se(-) cells] expressed < 5% of the glutathione peroxidase (GPX) activity of selenium-supplemented controls [Se(+) cells]. Clonogenic survival assays indicated that t-butyl hydroperoxide (t-BuOOH) is much more toxic to Se(-) cells (LC50 approximately 10 microM) than to Se(+) or selenium-repleted [Se(-/+)] cells (LC50 approximately 250 microM). Hypersensitivity of Se(-) cells to t-BuOOH was partially reversed by treating them with Ebselen, a selenoperoxidase mimetic; thus, selenoperoxidase insufficiency was probably the most serious defect of Se deprivation. Cytotoxicity of t-BuOOH was inhibited by desferrioxamine and by alpha-tocopherol, indicating that redox iron and free radical intermediates are involved. Elevated sensitivity of Se(-) cells to t-BuOOH was accompanied by an increased susceptibility to free radical lipid peroxidation, which became even more pronounced in cells that had been grown in arachidonate (20:4, n-6) supplemented media. That glutathione (GSH) is required for cytoprotection was established by showing that Se(+) cells are less resistant to t-BuOOH after exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. Coupled enzymatic assays indicated that Se(+) or Se(-/+) cells metabolize t-BuOOH 20-25 times more rapidly than Se(-), consistent with the measured difference in GPX activities of these cells. Correspondingly, when challenged with t-BuOOH, Se(+) cells showed an initial loss of GSH and elevation of GSSG that exceeded that of Se(-) cells. It was further shown that like Se(-) cells, BSO- or BCNU-treated Se(+) cells metabolize t-BuOOH more slowly than nontreated controls. These results clearly indicate that selenoperoxidase action in the glutathione cycle is a vital element in cellular defense against toxic hydroperoxides.

Alkaline elution analysis of DNA fragmentation induced during apoptosis.

We report that alkaline elution analysis fails to provide a complete profile of DNA fragmentation induced by some genotoxic agents, particularly if these agents induce apoptotic cell death resulting in fragmentation of DNA into oligonucleosomal length multimers. It was questioned whether these oligonucleosome fragments are responsible for the more rapidly eluting component of DNA that is observed as a biphasic elution profile when cells treated with certain genotoxic agents are subjected to alkaline elution. The results of this study indicate that DNA fragmented during apoptotic cell death is eluted in fractions before alkaline denaturation that are normally discarded. Collection of fractions prior to alkaline denaturation is recommended for complete evaluation of the total spectrum of damage induced by genotoxic agents, especially when the compound is suspected of inducing cell death by apoptosis.