Cultured Jurkat Cells Research Articles

Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis

Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus.

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC50 value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC50 values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H2O2 but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe3+), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe3+] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.

Multifunctional redox catalysts as selective enhancers of oxidative stress

Certain cancer cells proliferate under conditions of oxidative stress (OS) and might therefore be selectively targeted by redox catalysts. Among these catalysts, compounds containing a chalcogen and a quinone redox centre are particularly well suited to respond to the presence of OS. These catalysts combine the specific electrochemical features of quinones and chalcogens. They exhibit high selectivity and efficiency against oxidatively stressed rat PC12, human Jurkat and human Daudi cells in cell culture, where their mode of action most likely involves the catalytic activation of existent and the generation of new reactive oxygen species. The high efficiency and selectivity shown by these catalysts makes them interesting for the development of anti-cancer drugs.

Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: Role of glutamine and of cell shrinkage

Mammalian target of rapamycin (mTOR) mediates a signaling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth rate, participating to a versatile checkpoint that inspects the energy status of the cell. The pathway is activated by branched-chain amino acids (BCAA), leucine being the most effective, whereas amino acid dearth and ATP shortage lead to its deactivation. Glutamine- or amino acid-deprivation and hyperosmotic stress induce a fast cell shrinkage (with marked decrease of the intracellular water volume) associated to mTOR-dependent S6K1 dephosphorylation. Using cultured Jurkat cells, we have measured the changes of cell content and intracellular concentration of ATP, of relevant amino acids (BCAA) and of ninhydrin-positive substances (NPS, as measure of NH(2)-bearing organic osmolytes) under conditions that deactivate (leucine-deprivation, glutamine-deprivation, amino acid withdrawal, sorbitol-induced hyperosmotic stress) or reactivate a previously deactivated, mTOR-S6K1 pathway. We have also assessed the mitochondrial function by measurements of mitochondrial transmembrane potential in cells subjected to hypertonic stress. Our results indicate that diverse control signals converge on the mTOR-S6K1 signaling pathway. In the presence of adequate energy resources, the pathway senses the amino acid availability as inward transport of effective amino acids (as BCAA and especially leucine), but its activation occurs only in the presence of an extracellular amino acid complement, with glutamine as obligatory component, and does not tolerate decrements of cell water volume incapable of maintaining adequate intracellular physicochemical conditions.

Experimental and Theoretical Studies on the Pharmacodynamics of Cisplatin in Jurkat Cells

For Jurkat cells in culture exposed to cisplatin (1), we measured the number of platinum adducts on DNA and showed that it is proportional to the AUC, the area under the concentration vs time curve, for cisplatin. The number of platinum-DNA adducts is measured immediately following exposure to drug. The AUC is calculated either as the product of the initial cisplatin concentration and the exposure time or as the integral under the concentration vs time curve for the unreacted dichloro species, which decreases exponentially. We also show that the number of adducts correlates with decreases in respiration, with the amount of DNA fragmentation, and with cell viability, all measured 24 h after exposure to the drug. To study the reactions of cisplatin at concentrations approaching clinical relevance (65 microM), we use two-dimensional [1H15N]HSQC NMR and the 15N-labeled form of the drug, cis-Pt(15NH3)2Cl2, 1. In the absence of cells, 1 reacts with components of the growth medium and also transforms slowly (k(h) = 0.205 h-1 at 37 degrees C) into the chloro-aquo species, cis-[Pt(15NH3)2Cl(H2O)]+ (2), which at the pH of the medium (pH 7.15), is mainly in the deprotonated chloro-hydroxy form, cis-Pt(15NH3)2Cl(OH) (4). The concentration of 2 (4), as measured by HSQC NMR, decreases due to reaction with components of the medium. In the presence of 5 million or more cells, the concentration of 1 decreases with time, but the NMR signal for 2 (4) is not seen because it is rapidly removed from solution by the cells, keeping its concentration very low. These experiments confirm that the species preferentially removed from the medium by cells is 2 (4) and not 1. Our findings are discussed in the context of a kinetic model for platination of nuclear DNA by cisplatin, which includes aquation of cisplatin outside the cell, passage of 2 (4) through the cell membrane, reaction of reactive platinum species (RPS) in the cytosol with thiols, formation of adducts between RPS and accessible sites on genomic DNA, and removal of platinum from DNA by repair. Some of the rate constants involved are measured, but others can only be estimated. Calculations with this model show that little of the platinum reacts with intracellular thiols before reaching the nuclear DNA, indicating that binding to thiols is not important in cisplatin resistance. The model also predicts the circumstances under which the amount of platination of nuclear DNA is proportional to AUC.

External Bioenergy Increases Intracellular Free Calcium Concentration and Reduces Cellular Response to Heat Stress

BackgroundExternal bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and...

Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse development.

The caspase family of proteases represents the main machinery by which apoptosis occurs. In vitro studies have revealed that upstream caspases are activated in response to apoptotic stimuli, and the active caspases in turn process downstream effector caspases that are involved in the destruction of cellular structure. Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g. during development. We have identified vimentin as the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, showed that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. Our results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death.

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA Damage

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or “comet” assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1–5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H 2O 2-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H 2O 2. These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H 2O 2-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester), an intracellular Ca 2+-chelator, also protected against H 2O 2-induced DNA damage, it is likely that intracellular Ca 2+ changes are involved in this process as well. The exact role of Ca 2+ and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.

Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H 2O 2 from DNA damage

Trimetazidine is a well-established anti-ischemic drug, which has been used for long time in the treatment of pathological conditions related with the generation of reactive oxygen species. However, although extensively studied, its molecular mode of action remains largely unknown. In the present study, the ability of trimetazidine to protect low-density lipoproteins (LDL) from oxidation and cultured cells from H 2O 2-induced DNA damage was investigated. Trimetazidine, tested at concentrations 0.02 to 2.20 mM, was shown to offer significant protection to LDL exposed to three different oxidizing systems, namely copper, Fe/ascorbate, and met-myoglobin/H 2O 2. The oxidizability of LDL was estimated by measuring, (i) the lag period, (ii) the maximal rate of conjugated diene formation, (iii) the total amount of conjugated dienes formed, (iv) the electrophoretic migration of LDL protein in agarose gels (REM), and (v) the inactivation of the enzyme PAF-acetylhydrolase present in LDL. In addition, the presence of trimetazidine decreased considerably the DNA damage in H 2O 2-exposed Jurkat cells in culture. H 2O 2 was continuously generated by the action of glucose oxidase at a rate of 11.8 ± 1.5 μM per min (60 ng enzyme per 100 μl), and DNA damage was assessed by the single cell gel electrophoresis assay (also called comet assay). The protection offered by trimetazidine in this system (about 30% at best) was transient, indicating modification of this agent during its action. These results indicate that trimetazidine can modulate the action of oxidizing agents in different systems. Although its mode of action is not clarified, the possibility that it acts as a lipid barrier permeable transition metal chelator is considered.

Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (‘casein kinase-2’)

The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr- or Tyr-specific. In the presence of 10 μM TBB (and 100 μM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3β and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC 50 values one–two orders of magnitude higher than CK2 (IC 50=0.9 μM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays.

The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis.

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.

Role of Protein Kinase C in Signal Attenuation following T Cell Receptor Engagement

T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.

Cr(IV) causes activation of nuclear transcription factor-κB, DNA strand breaks and dG hydroxylation via free radical reactions

Electrophoretic mobility shift, DNA strand breakage assays and electron spin resonance (ESR) spin trapping were used to investigate the activation of nuclear transcription factor (NF)- κB, DNA strand breakage and 2′-deoxyguanosine hydroxylation induced by Cr(IV), as well the role of free radical reactions in these processes. Incubation of synthesized Cr(IV)-glutathione complex with cultured Jurkat cells resulted in activation of DNA binding activity of NF- κB. Cr(VI) is also able to induce NF- κB activation through Cr(V) and Cr(IV) intermediates generated during the reduction of Cr(VI) by the cells. Cr(III) did not cause observable NF- κB activation due to its inability to cross cell membranes. Cr(IV)-induced NF- κB activation is dose-dependent. Catalase inhibited the activation while superoxide dismutase enhanced it. The metal chelator, deferoxamine, and hydroxyl (OH) radical scavengers, sodium formate and aspirin, also inhibited the NF- κB activation. Electrophoretic assays using λ Hind III linear DNA showed that, in the presence of H 2O 2, Cr(IV) is capable of causing DNA strand breaks. Deferoxamine, sodium formate and aspirin inhibited the DNA strand breaks. HPLC measurements also show that OH radical generated by the Cr(IV)-mediated reaction with H 2O 2 was capable of causing 2′-deoxyguanosine (dG) hydroxylation to generate 8-hydroxyguanosine (8-OHdG). The relative magnitude of 8-OHdG formation correlated with the generation of OH radicals. ESR spin trapping measurements showed that reaction of Cr(IV) with H 2O 2 generated OH radicals, which were inhibited by deferoxamine, sodium formate and aspirin. The results show that Cr(IV) can cause NF- κB activation, DNA strand breaks and dG hydroxylation through OH radical-initiated reactions. This reactive chromium intermediate may play an important role in the mechanism of Cr(VI)-induced carcinogenesis. The results also suggest that the Cr(IV)-glutathione complex may be used as a model compound to study the role of Cr(IV) in Cr(VI) carcinogenicity.

Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells.

One-Electron Reduction of Chromium(VI) by α-Lipoic Acid and Related Hydroxyl Radical Generation, dG Hydroxylation and Nuclear Transcription Factor-κB Activation

Reaction of chromium(VI) with α-lipoic acid (reduced form, also called 1,2-dithiolane-3-pentanoic acid) generated Cr(V) and hydroxyl radical (•OH) as measured by electron spin resonance and ESR spin trapping. 5,5-Dimethyl-1-pyrroline was used as a spin trapping agent. Catalase inhibited the•OH generation and enhanced the Cr(V) formation. Superoxide dismutase had an opposite effect. H2O2enhanced the•OH generation and decreased the Cr(V) formation in a dose-dependent manner. Metal chelators, EDTA, diethylenetriaminepentaacetic acid, deferoxamine, and 1,10-phenanthroline inhibited•OH radical generation in the order of EDTA > 1,10-phenanthroline > DTPA > deferoxamine. Oxygen consumption measurements indicated that molecular oxygen was used to generate•OH radical in the mixture of Cr(VI) and α-lipoic acid. H2O2and superoxide radical (O2−) were involved as reactive intermediates. The•OH radical was generated via Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2→ Cr(VI) + OH−+•OH). HPLC measurements show that the•OH radical generated by this reaction is capable of generating 8-hydroxyl-2′-deoxyguanosine from 2-deoxyguanosine. Incubation of Cr(VI) with cultured Jurkat cells resulted in an activation of DNA binding activity of the nuclear factor (NF)-κB. Addition of α-lipoic acid enhanced the NF-κB activation, while the•OH radical scavenger, sodium formate, inhibited it, showing that α-lipoic acid enhanced Cr(VI)-induced NF-κB activation via free radical reactions. The results indicate that while α-lipoic acid is considered to be an antioxidant, it may be a cellular one-electron Cr(VI) reductant and could be involved in the mechanism of Cr(VI)-induced carcinogenesis.

Chromium(VI)-induced nuclear factor-kappa B activation in intact cells via free radical reactions.

Incubation of chromium(VI) [Cr(VI)] with cultured Jurkat cells resulted in activation of DNA binding activity of the nuclear factor (NF)-kappa B. In a combination with glutathione reductase, a Cr(VI) reducing agent, Cr(VI) expressed an enhanced activity in induction of NF-kappa B. This activation of NF-kappa B was decreased by a metal chelator, diethylene-triaminepentaacetic acid or catalase, but increased by superoxide dismutase. Addition of Mn2+, which reacts with Cr(IV) and inhibits Cr(IV)-mediated hydroxyl radical (.OH) generation via Fenton-like reaction, attenuated the activation of NF-kappa B. Sodium formate, an .OH radical scavenger, also inhibited the activation. Electron spin resonance measurements showed that the incubation of Cr(VI) with intact Jurkat cells generated reactive Cr(V) intermediate. Glutathione reductase and NADPH enhanced Cr(V) generation. Electron spin resonance spin trapping measurements using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent provided evidence that the incubation of Cr(VI) with the Jurkat cells in the presence of glutathione reductase generated .OH radicals. H2O2 enhanced .OH radical generation and also enhanced Cr(V) formation, indicating the role of Cr(IV) in .OH radical generation. We conclude that Cr(VI) can activate NF-kappa B in vitro via Cr(IV)-mediated free radical reactions. We hypothesize that Cr(VI)-mediated NF-kappa B activation may be involved in the mechanism of Cr(VI)-induced carcinogenicity.

The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitro cultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G 2/M arrest at low genistein concentrations (5–10 μg/ml), while at higher doses (20–30 μg/ml) there was also a perturbation in S-phase progression. The derangements in cell-cycle control were followed by apoptosic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 μ/ml genistein effectively inhibit tyrosine kinase activity in cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg ( N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12- O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.