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After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca 2+ , an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca 2+ sensitive K + channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca 2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca 2+ -dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.

Extremely-low-frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic" to humans on the grounds of an epidemiological association of ELF-MF exposure with an increased risk of childhood leukaemia. Yet, underlying mechanisms have remained obscure. Genome instability seems an unlikely reason as the energy transmitted by ELF-MF is too low to damage DNA and induce cancer-promoting mutations. ELF-MF, however, may perturb the epigenetic code of genomes, which is well-known to be sensitive to environmental conditions and generally deranged in cancers, including leukaemia. We examined the potential of ELF-MF to influence key epigenetic modifications in leukaemic Jurkat cells and in human CD34+ haematopoietic stem cells undergoing in vitro differentiation into the neutrophilic lineage. During granulopoiesis, sensitive genome-wide profiling of multiple replicate experiments did not reveal any statistically significant, ELF-MF-dependent alterations in the patterns of active (H3K4me2) and repressive (H3K27me3) histone marks nor in DNA methylation. However, ELF-MF exposure showed consistent effects on the reproducibility of these histone and DNA modification profiles (replicate variability), which appear to be of a stochastic nature but show preferences for the genomic context. The data indicate that ELF-MF exposure stabilizes active chromatin, particularly during the transition from a repressive to an active state during cell differentiation.

The radioprotective capacity of a rationally-designed Mn2+-decapeptide complex (MDP), based on Mn antioxidants in the bacterium Deinococcus radiodurans, was investigated in a mouse model of radiation injury. MDP was previously reported to be extraordinarily radioprotective of proteins in the setting of vaccine development. The peptide-component (DEHGTAVMLK) of MDP applied here was selected from a group of synthetic peptides screened in vitro for their ability to protect cultured human cells and purified enzymes from extreme damage caused by ionizing radiation (IR). We show that the peptides accumulated in Jurkat T-cells and protected them from 100 Gy. MDP preserved the activity of T4 DNA ligase exposed to 60,000 Gy. In vivo, MDP was nontoxic and protected B6D2F1/J (female) mice from acute radiation syndrome. All irradiated mice treated with MDP survived exposure to 9.5 Gy (LD70/30) in comparison to the untreated mice, which displayed 63% lethality after 30 days. Our results show that MDP provides early protection of white blood cells, and attenuates IR-induced damage to bone marrow and hematopoietic stem cells via G-CSF and GM-CSF modulation. Moreover, MDP mediated the immunomodulation of several cytokine concentrations in serum including G-CSF, GM-CSF, IL-3 and IL-10 during early recovery. Our results present the necessary prelude for future efforts towards clinical application of MDP as a promising IR countermeasure. Further investigation of MDP as a pre-exposure prophylactic and post-exposure therapeutic in radiotherapy and radiation emergencies is warranted.

Ionizing radiation (IR)-induced oxidative stress in tumor cells is effectively managed by constitutive and inducible antioxidant defense systems. This study was initiated to understand the relative contribution of different redox regulatory systems in determining the tumor radio-resistance. In this study, human T-cell lymphoma (Jurkat) cells were exposed to IR (4 Gy) and monitored for the spatio-temporal changes in cellular redox regulatory parameters. We monitored the changes in the levels of reactive oxygen species (ROS) (total, mitochondrial, primary, and secondary), thiols (total, surface, and intracellular), GSH/GSSG ratio, antioxidant enzyme activity viz. thioredoxin (Trx), Trx reductase (TrxR), glutathione peroxidase, and glutathione reductase with respect to time. We have also measured protein glutathionylation. We observed that tumor cells mount a biphasic response after IR exposure which can be divided into early (0-6 h) and late (16-48 h) responses in terms of changes in cellular redox parameters. During early response, constitutively active GSH and Trx systems respond to restore cellular redox balance to pre-exposure levels and help in activation of redox-sensitive transcription factor Nrf-2. During late response, increase in the levels of antioxidants GSH and Trx rescue cells against IR-mediated damage. We observed that disruption of either glutathione or thioredoxin metabolism led to partial impairment of ability of cells to survive against IR-induced damage. But simultaneous disruption of both the pathways significantly increased radio sensitivity of Jurkat cells. This highlighted the importance of these two antioxidant pathways in regulating redox homeostasis under conditions of IR-induced oxidative stress.

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4-1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. The laser technology targets the global mechanical properties of the viral protein shell, making it relatively insensitive to the local genetic mutation in the target viruses. As a result, the approach can inactivate both the wild and mutated strains of viruses. This intriguing advantage is particularly important in the treatment of diseases involving rapidly mutating viral species such as HIV. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.

Purpose: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to gamma-ray exposure., Methods and Materials: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy., Results: Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [(3)H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle., Conclusion: These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to gamma-rays.

Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines., Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to gamma-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells., Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells., Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.

Zn-protoporphyrin (ZnPP) is a promising candidate for cancer therapy. It is known to inhibit heme-oxygenase-1 (HO-1), resulting in suppressed biliverdin/bilirubin production accompanying lowered antioxidative capacity. As a consequence, a significant suppression of tumor growth in vivo was reported. Recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light. In the present report, we describe the photosensitizing capabilities of water-soluble polymer conjugates of ZnPP as novel compounds for photodynamic therapy against solid tumors. The polymer conjugation made ZnPP water-soluble, thus possible for injection for its aqueous solution. The cellular uptake and photobiological activity of ZnPP derivatives have been tested using a human T-cell leukemia cell line in vitro and demonstrated most potent phototoxic effects of SMA-ZnPP followed by PEG-ZnPP under aerobic conditions.

Background: It has been suggested that defective handling of apoptotic cells by macrophages plays a key role in the development of systemic lupus erythematosus (SLE). The relative contribution of intrinsic defects and serum factors remains controversial., Objective: To compare monocytes from SLE patients, patients with rheumatoid arthritis, and healthy controls for their ability to differentiate in vitro into macrophages and to bind/engulf apoptotic cells., Methods: Peripheral blood derived monocytes from healthy donors or from patients with SLE or rheumatoid arthritis were allowed to differentiate into macrophages. The in vitro uptake of apoptotic cells by macrophages was evaluated by a flow cytometry assay that allowed discrimination between binding and internalisation., Results: Monocytes from SLE and rheumatoid patients showed a striking defect in adherence to plastic compared with healthy donors. Absence or heat inactivation of serum resulted in a reduction in the binding and engulfment of apoptotic cells by macrophages. Macrophages from rheumatoid and SLE patients had similar percentages of apoptotic cells bound to their surface compared with normal controls. However, macrophages from SLE patients showed a significant defect in the internalisation of apoptotic cells compared with those from healthy controls, even in the presence of normal human serum., Conclusions: Monocytes from patients with SLE and rheumatoid arthritis have a similar defect in their capacity to adhere to plastic. However, only macrophages from SLE patients showed an impaired ability to engulf apoptotic cells, which indicates that an intrinsic cellular defect may be responsible for this phenomenon.

DCB, 3,3'-dichlorobenzidine, is used primarily as an intermediate in the manufacture of diarylide yellow or azo red pigments for printing ink, textile, paint, and plastics. It is also used in tattoo inks. In this article, we investigate light-induced toxicity of DCB in both bacteria and human Jurkat T-cells. DCB itself is not toxic or mutagenic to Salmonella typhimurium TA102, but is photomutagenic at concentrations as low as 2 microM and phototoxic at concentrations >100 microM when bacteria are exposed to DCB and light at the same time (1.2 J/cm2 of UVA and 2.1 J/cm2 of visible light). Furthermore, DCB is both photocytotoxic and photogenotoxic to human Jurkat T-cells. Under a light irradiation dose of 2.3 J/cm2 of UVA and 4.2 J/cm2 of visible light, it causes the Jurkat T-cells to become nonviable in a DCB dose-dependent manner and the nonviable cells reaches 60% at DCB concentrations higher than 50 microM. At the same time, DNA fragmentation is observed for cells exposed to both DCB and light, determined by single cell gel electrophoresis (alkaline comet assay). As much as 5% (average) DNA fragmentation was observed when exposed to 200 microM DCB and light irradiation. This suggests that DCB can penetrate the cell membrane and enter the cell. Upon light activation, DCB in the cells can cause various cellular damages, leading to nonviable Jurkat T-cells. It appears, the nonviable cells are not caused solely by fragmentation of cellular DNA, but by other damages such as to proteins and cell membranes, or DNA alkylation. Therefore, persons exposed to DCB through environmental contamination or through tattoo piercing using DCB-containing inks must not only concern about its toxicity without exposing to light, but also its phototoxicity.

Background: Ultraviolet-A1 (340-400 nm) (UVA1) radiation causes singlet-oxygen damage that depolarizes mitochondrial membranes triggering immediate apoptosis (T < or = 4 h), while it also causes oxidative damage to DNA inducing delayed apoptosis (T > or = 24 h). In this study, we examined some potential therapeutic endpoints associated with UVA1-mediated immediate and delayed apoptosis, such as receptor and cytokine changes., Methods: We quantified the number of membrane-bound CD3 receptors on transformed T lymphocytes (Jurkat) and the number of membrane-bound CD19 receptors on transformed B lymphocytes (Daudi) using flow cytometry. We also quantified the release of the cytokines interferon gamma (IFN-gamma) and interleukin-2 (IL-2) using enzyme-linked immunosorbent assays., Results: Out of the entire population of cells, only the apoptotic Daudi cells immediately decreased CD19 expression via capping, while only the apoptotic Jurkat cells increased CD3 receptor expression 24 h post-exposure. Both receptor changes occurred in a UVA1 dose-dependent manner. We also examined other T-cell receptors, such as CD4, CD25, and CD69, but they did not change for up to 24 h following exposure. During UVA1-triggered immediate apoptosis of Jurkat T cells, IFN-gamma levels increased in a dose-dependent manner at 4 h, but returned to baseline levels at 24 h post-exposure, whereas, there was no significant change in IL-2 at 4 or 24 h., Conclusion: Thus, UVA1-triggered immediate apoptosis causes a rapid decrease in the number of CD19 receptors on Daudi B cells and release of IFN-gamma from Jurkat T cells at 4 h, and UVA1-mediated delayed apoptosis causes an increase in the number of CD3 receptors on Jurkat T cells.

Nonylphenol polyethoxylates (NPEOs) are widely used as non-ionic surfactants and their biodegradation products such as 4-n-nonylphenol are stable and have been demonstrated to be cytotoxic. In the aquatic environment, these compounds are usually exposed to sunlight, and while the correlation between the biodegradation of NPEOs and changes in cytotoxicity has been reported, the relationship between the photodegradation of NPEOs and cytotoxicity has not. In this study, we investigated the degradation of NPEO by ultraviolet (UV) irradiation, especially UVB irradiation, and the effects on mammalian cell lines. NPEO with a smaller number of ethylene oxide (EO) units showed greater cytotoxicity. Although NPEO (10) completely inhibited the proliferation of the cells, NPEO (70) showed no toxicity. UVB irradiation significantly induced a shortening of the side chain, which was due to the production of ROS. The EO side chain of NPEO (10), was gradually degradated, but that of NPEO (70) was degradated near the benzene ring. Furthermore, the degradation of the benzene ring was more effective in NPEO (70) than NPEO (10). The toxicity of NPEO (10) in cultured cells decreased following UVB irradiation, whereas that of NPEO (70) was induced after UVB irradiation at 500 J/cm2 and disappeared at 1000 J/cm2. This might be due to the production of NPEO with a short side chain and 4-n-nonylphenol by the degradation of EO and due to the degradation of the benzene ring at higher doses of UVB irradiation. This study shows the significance of UV exposure to the degradation of alkylphenol polyethoxylates in the environment.

Purpose: Tumor hypoxia reduces the efficacy of radiotherapy, many types of chemotherapy, and tumor necrosis factor-alpha (TNF-alpha). TRAIL (TNF-alpha-related apoptosis-inducing ligand) is a ligand for death receptors of the TNF superfamily shown to be selectively toxic for tumor cells and thereby a promising antineoplastic tool. The impact of hypoxia on TRAIL-induced apoptosis was examined in this study., Methods and Materials: Apoptosis induction and growth rates of various tumor cell lines under hypoxia were evaluated in vitro. Biologically effective induction of hypoxia was verified by determination of hypoxia-inducible factor-1 (HIF-1) activation. The efficacy of TRAIL- and radiation-induced apoptosis under different oxygen conditions was quantified in vitro. The impact of Bcl-2 on TRAIL-induced apoptosis under hypoxia or normoxia was evaluated by comparing cells expressing Bcl-2 with a vector control., Results: Moderate hypoxia caused no growth retardation or apoptosis, but led to activation of HIF-1 as a prerequisite of hypoxic gene induction. Cellular responses to TRAIL differed considerably among the cell lines tested. Hypoxia reduced radiation-induced, but not TRAIL-induced, apoptosis in the tested cell lines. Hypoxia did not induce Bcl-2 expression. Bcl-2 had a minor impact on the efficacy of TRAIL-induced apoptosis., Conclusion: Taken together, the data indicate that TRAIL is clearly effective under conditions of proven hypoxia.

The human leukemic T-cell line Jurkat was used to define the role of the cellular stress pathway with its key player kinase JNK in cancer therapy-induced apoptosis. JNK activity was inhibited by stable transfection with a dominant negative mutant of the upstream kinase JNKK/MKK4 or with the novel, potent and selective JNK1, -2 and -3 inhibitor SP600125. Inhibition of JNK activity delayed the onset of apoptosis induced by cisplatin, doxorubicin, gamma-irradiation and CD95-L but did not prevent apoptosis per se. Early events during apoptosis such as induction of CD95-L, activation of caspase-8 and exposure of phosphatidylserine on the cell surface were strongly inhibited. Also, at early time points of apoptosis, loss of the mitochondrial membrane potential and release of cytochrome c were markedly impaired. However, late signaling events during apoptosis such as cleavage of PARP and DNA fragmentation apoptosis were only marginally affected. These findings are in accordance with the activity of initiator and effector caspases. Whereas activity of the initiator caspase-8 was strongly inhibited early and late after induction, an inhibition of caspase-3 activity was only observed early after induction of apoptosis. We therefore suggest that cellular stress signaling contributes to the initiation of apoptosis, whereas it might be dispensable for the progression of apoptosis. Dysfunction of this pathway under pathological conditions might contribute to therapy resistance of cancer cells., (Copyright 2003 Wiley-Liss, Inc.)

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase-3 activation, we paid attention to the influence exerted by Protein kinase C delta on transcription factor NF-kappaB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC delta/NF-kappaB pathway activation, probably mediated by novel IKKs and a role for PI-3-kinase in activating PKC delta at Thr 505 by PDK-1 phosphorylation is suggested. In addition, since caspase-3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF-kappaB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF-kappaB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings., (Copyright 2003 Wiley-Liss, Inc.)

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be "entrapped" in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using "lab-on-a-chip." In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 474-479, 2003.)

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation., (Copyright 2003 Wiley-Liss, Inc.)

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiation-induced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptor-mediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lck-deficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as deltapsi(m) breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations.

It was recently shown that antibodies catalyze a reaction between water and ultraviolet light (UV) creating singlet oxygen and ultimately H2O2. Although the in vivo relevance of these antibody reactions is unclear, it is interesting that among a wide variety of non-antibody proteins tested, the T cell receptor is the only protein with similar capabilities. In clinical settings UV is believed to exert therapeutic effects by eliminating inflammatory epidermal T cells and we hypothesized that UV-triggered H2O2 production is involved in this process. To test the hypothesis we developed tools to study production of H2O2 by T cell receptors with the long-term goal of understanding, and improving, UV phototherapy. Here, we report the development of an inexpensive, real time H2O2 monitoring system having broad applicability. The detector is a Clark oxygen electrode (Pt, Ag/AgCl) modified to detect UV-driven H2O2 production. Modifications include painting the electrode black to minimize UV effects on the Ag/AgCl electrode and the use of hydrophilic, large pore Gelnots electrode membranes. Electrode current was converted to voltage and then amplified and recorded using a digital multimeter coupled to a PC. A reaction vessel with a quartz window was developed to maintain constant temperature while permitting UV irradiation of the samples. The sensitivity and specificity of the system and its use in cell-free and cell-based assays will be presented. In a cellfree system, production of H2O2 by CD3 antibodies was confirmed using our real time H2O2 monitoring method. Additionally we report the finding that splenocytes and Jurkat T cells also produce H2O2 when exposed to UV light.

MEK/ERK-mediated signals have recently been found to inhibit Fas-mediated cell death through inhibition of caspase-8 activity. It remains unknown whether MEK/ERK-mediated signals affect ionizing radiation (IR)-induced cell death. Here we demonstrate that MEK/ERK-mediated signals selectively inhibit IR-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and subsequent cell death. In Jurkat cells, TPA strongly activated ERK and inhibited the IR-induced caspase-8/Bid cleavage and the loss of DeltaPsi(m). The inhibitory effect of TPA was mostly abrogated by pretreatment of a specific MEK inhibitor PD98059, indicating that the effect depends upon MEK/ERK-mediated signals. Moreover, BAF-B03 transfectants expressing IL-2 receptor (IL-2R) beta(c) chain lacking the acidic region, which is responsible for MEK/ERK-mediated signals, revealed higher sensitivity to IR than the transfectants expressing wild-type IL-2R. Interestingly, the signals could neither protect the DeltaPsi(m) loss nor cell death in UV-irradiated cells. These data imply that the anti-apoptotic effect of MEK/ERK-mediated signals appears to selectively inhibit the IR-induced cell death through protection of the DeltaPsi(m) loss. Our data enlighten an anti-apoptotic function of MEK/ERK pathway against IR-induced apoptosis, thereby implying its contribution to radioresistance.

This study investigates the involvement of the c-cbl proto-oncogene during the first stages of the apoptotic process. We have already shown that a c-Cbl aptotosis-related protein of 90 kDa (CARP 90) is detected very rapidly in the cytoplasm as well as in the nucleus of murine thymocytes after hydrocortisone (HC) treatment. We report here that this protein appeared as well after in vivo treatment of mice by gamma-irradiation or injection of anti-CD3 monoclonal antibody, two potent thymic apoptosis inductors, providing a close relationship between the occurrence of apoptosis and the appearance of CARP 90. We showed that CARP 90 and p120(cbl) share numerous epitopes strikingly suggesting that CARP 90 is coded by c-cbl. In addition, KO mice do not sustain CARP 90 appearance. We finally showed that CARP 90 contains N- and C-terminal end epitopes of p120(cbl), which suggests that CARP 90 is an alternative spliced form of c-cbl. This protein was also observed under gamma-irradiation in tissues of different origin, which enlarges the physiological significance of this phenomenon. The very rapid CARP 90 appearance under apoptotic conditions in the nucleus of cells originating in different tissues makes this protein if not a possible new actor of the apoptotic process, at least an interesting marker of this process.

The effect of dipyrone (metamizol) on cell viability was evaluated in human leukocyte cell lines upon different apoptotic treatments: arachidonic acid (AA), cycloheximide (CHX), tumor necrosis factor (TNF) and ultraviolet (UV) irradiation. Dipyrone had a dual effect: at high concentrations (beyond 300 microM), it was cytotoxic, leading to apoptosis, whereas at lower concentrations (37.5-300 microM), it was cytoprotective, delaying the loss of membrane integrity triggered by arachidonic acid (100-200 microM) and UV irradiation and the cytotoxicity of cycloheximide (25-50 microM). No effect of dipyrone was found on TNF-induced cytotoxicity (250 ng/ml). The cytoprotective effect of dipyrone is associated with a decrease in DNA fragmentation, as assessed by electrophoresis of genomic DNA and by flow cytometry; a reduction in the percentage of condensed nuclei, as evaluated by DNA staining with Hoescht 33342 and a decrease in poly(ADP)-ribose polymerase (PARP) cleavage, as assessed by Western blotting. The cytoprotective effect of dipyrone on leukocyte apoptosis occurs at concentrations usually found for the main active metabolite of the drug and may have implications on the therapeutic and side effects caused by this agent.

Human leukemic T lymphocytes (Jurkat cells) were induced to undergo apoptosis by brief irradiation with ultraviolet C light (254 nm). This was accompanied by accumulation of lipid peroxidation products in the form of conjugated dienes, a decrease of total glutathione content, and a shift of its redox state towards the oxidized form. Preincubation of the cells with 1 mM pantothenate resulted in a significant elevation of total glutathione content of the cells, reaching its maximum level, 160% of the control, after 3 h. Similar increase was observed after preincubation with 5 mM N-acetylcysteine, a known precursor of glutathione. Both pantothenic acid and N-acetylcysteine alleviated the ultraviolet-induced decrease of glutathione content, diminished lipid peroxidation, and partly protected the cells against apoptosis produced by ultraviolet irradiation.

A combination of antitumor approaches acting on different death pathways seems ideal for increasing therapeutic responses, especially when defined resistance mechanisms interfere with individual cellular processes. Apoptosis pathways triggered by ionizing radiation (XRT) and the death ligand TRAIL were analysed in Jurkat lymphoma cells. Both induced the activation of caspase-8, caspase-3, BID and mitochondrial potential loss. TRAIL induced apoptosis required caspase-8, whereas it was not essential for radiation induced apoptosis. The inhibition of mitochondrial damage by Bcl-2 abrogated XRT induced apoptosis and caspase activation, but only marginally attenuated TRAIL induced cell death. The combined treatment with TRAIL and XRT exerted additive apoptotic effects in control cells, whereas highly synergistic effects occurred in cells overexpressing Bcl-2. In addition, a strong effect of TRAIL on radiation induced clonogenic cell death was found. In conclusion, TRAIL seems to be of high potential value for a combination with ionizing radiation in tumor therapy.

The human T cell line Jurkat registers a sinusoidal extremely low frequency (ELF), 0.10 mT magnetic fields (MFs) at the level of the plasma membrane. In this study, the protein tyrosine phosphorylation (PY) of two membrane-associated proteins in Jurkat cells were examined following a short-term MFs exposure, the zeta chains and the Src kinases p56lck. These proteins are interesting to study since the earliest biochemical event upon T cell receptor (TcR) activation is PY of the zeta chains. These signalling chains in the TcR complex was assessed using Western blotting and the activation of the p56lck kinase was analysed by in vitro kinase assay. The MFs exposure of Jurkat for 5 min activated p56lck and resulted in PY of zeta. These findings are in line with earlier reports on how MFs exposure affects signal transduction in Jurkat.

Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)-6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1 beta, and tumor necrosis factor-alpha, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking-induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. (Blood. 2000;96:4300-4306)

We recently demonstrated that linker histone H1, which is thought to have a fundamental role in higher-order chromatin structure, becomes transiently dephosphorylated after ionizing radiation (IR) in a mutated ataxia telangiectasia (ATM) dependent manner. To establish whether H1 dephosphorylation was a component of a damage-response pathway that included dephosphorylation of other histones, we asked whether H3 was dephosphorylated in response to IR in a manner similar to H1. H1 and H3 are maximally phosphorylated in metaphase and both are dephosphorylated after IR. However, the duration of IR-induced H3 dephosphorylation is significantly longer than that of IR-induced H1 dephosphorylation. Moreover, H1 dephosphorylation is ATM-dependent, whereas H3 dephosphorylation is ATM-independent. These observations suggest that the damage-sensing pathways regulating H3 and H1 dephosphorylation diverge upstream of ATM.

Ionizing radiation is a major tool for cancer treatment. The response of eukaryotic cells to ionizing radiation includes apoptosis, a process which requires activation of multiple genes. We sought to determine whether radiation-induced gene expression plays a role in radiation-induced apoptosis. We found Apo2 ligand (Apo2L, also called TRAIL) mRNA induction following gamma-irradiation of Jurkat, MOLT-4, CEM, and PBMC, all human T lineage-derived cells. Increased Apo2L protein levels were found in MOLT-4 and Jurkat cells. Radiation also activated the Apo2L death receptor (DR)5 (also called Apo2, TRAIL-R2, or KILLER) in MOLT-4 cells, which harbor a wild-type p53. We isolated 1152 bp of 5' flanking region of the Apo2L gene and a shorter fragment of 716 bp, both of which showed promoter activity driving the expression of a luciferase reporter gene; however, the response to radiation in MOLT-4 cells was lost when only 430 bp of 5' proximal flanking sequence was maintained. Exogenous Apo2L induced phosphatidylserine exposure on cell membranes, caspase 8 and caspase 3 activation, key markers of apoptosis, confirming that the Apo2L/DR5 pathway is functional in these cells. Bid, a Bcl-2 family protein also known to contribute to receptor-mediated apoptosis, was also activated. To determine whether Apo2L and DR5 were critical for radiation signaling to apoptosis, we stably expressed a dominant negative DR5delta-receptor in Jurkat cells. Cell survival was significantly augmented, indicating that increased Apo2L expression contributed to radiation-induced apoptosis. Clonogenic assays demonstrated that purified, recombinant soluble Apo2L enhanced the lethality of low, therapeutic doses (1-2 Gy) of gamma-irradiation. These data suggest that production of Apo2L may cooperate synergistically with the cytotoxic effect of radiation, and that combinations of Apo2L and radiation may become a powerful tool in clinical therapy.

An effect on intracellular calcium continues to be proposed as a biochemical pathway for the mediation of biologic effects of electrical-power-frequency magnetic fields (MF). However, reproducible results among laboratories are difficult to attain and the characteristics of magnetic field effects on intracellular free calcium ([Ca(2+)](i)) are not well understood. We attempted to repeat the studies of Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line by a Weak 50 Hz Magnetic Field. J Cell Physiol 156:395-398 (1993)] by investigating the effect of a 1.5-G 50-Hz MF on [Ca(2+)](i) in the Jurkat lymphocyte T-cell line. Changes in [Ca(2+)](i) were determined using microscopic imaging of fura-2 loaded Jurkat cells on poly-l-lysine-coated glass coverslips. The MF was generated by a single coil constructed with bifilar wire and located in the same plane as the cells. Cells were randomly exposed for 8 min to MF, sham field (SF), or no field (NF) conditions. The exposure condition remained coded until data analysis was complete. Each exposure period was preceded by an 8-min data collection to establish a baseline for [Ca(2+)](i). After each exposure condition, cells were exposed to anti-CD3 antibody that induced a rapid increase in [Ca(2+)](i) in responsive cells; this provided a positive control. [Ca(2+)](i) was analyzed for individual cells as spatially-averaged background-corrected 340/380 nm ratios, and a [Ca(2+)](i) transient was considered significant for positive deviations from baseline of 3 [multiple] an estimate of noise in the baseline. Typically, 25-50 cells/field were viewed and approximately 50% had no [Ca(2+)](i) transients in the baseline period and also responded to positive control. Only cells responding to positive control and lacking changes in [Ca(2+)](i) during the baseline period were considered qualified for assessment during the exposure period. The incidences of [Ca(2+)](i) transients during the exposure period for two experiments (40 [multiple] objective) were 16.5, 14.6, and 14.2% for MF, SF, and NF, respectively, and were not statistically significantly different. Previous studies by Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line after Exposure to Extremely-Low-Frequency Magnetic Fields with Variable Frequencies and Flux Densities. Bioelectromagnetics 16:41-47 (1995)] showed a high response rate (92%) for exposure to 1. 5-G 50-Hz MF when individual cells were preselected for investigation. We found no such effect when examining many cells simultaneously in a random and blind fashion. These results do not preclude an effect of MF on [Ca(2+)](i), but suggest that responsive cells, if they exist, were not identified using the approaches that we used in this study.

IgG binding to multiple protein constituents in lysates of Jurkat cells was detected by Western blot in sera of patients with multiple sclerosis (MS) and systemic lupus erythematosus (SLE). The distribution patterns of bands with sera tested against protein lysates from normal Jurkat cells or from Jurkat cells exposed to apoptosis or oxidative stress inducing conditions were similar in most patients, but with inter-individual differences. The number of bands with sera of both patient populations far exceeded those (0 or 2 bands) detected with sera of healthy controls. Proteinase K, RNase and DNase pre-treatment of cell lysates suggested a protein nature for all of the antigens and a ribonucleoprotein (RNP) nature for some of the antigens recognized by serum IgG of MS and SLE patients. Only two MS patients had positive anti-nuclear antibody (ANA) titers, while all of them had positive Western blots. In addition to similarities, dissimilarities were also recognized between the humoral immune responses in MS and SLE. No IgG molecules were detected against phosphorylated proteins in the sera of MS patients, while multiple phosphoproteins were recognized by IgG molecules of SLE patients in immunoprecipitation experiments. These data suggest that in addition to ANA, the sera of MS patients contain autoantibodies directed against multiple intracellular proteins. The protein recognition patterns of immunoglobulins in MS share similarities, but also have distinct features when compared to those in SLE. The biological significance of these autoantibodies in MS remains to be understood.

The purpose of this study was to explore the connection between radiation-induced apoptosis and progression of cells through the phases of the cell cycle. Cells of the human T-cell line Jurkat were separated by centrifugal elutriation into populations enriched in G(1)-, S- and G(2)/M-phase cells before irradiation. After a dose of 20 Gy, the onset of massive apoptosis occurred at about 6 h in all populations regardless of the phase of the cell cycle in which they were irradiated. In contrast, after 2 Gy, cells died at various times after a pronounced G(2)/M-phase arrest. These results indicate that radiation-induced apoptosis can occur independently of cell cycle arrest and that the time for onset of apoptosis may be dependent on the radiation dose.

In many cells, the process of apoptosis is accompanied by endonuclease-mediated double-strand cleavage of DNA between nucleosomes, resulting in the production of discrete fragments of 200 bp or multiples thereof. To address the question of whether this endonuclease attack occurs randomly or nonrandomly along chromosomes, we first constructed chromosome fluorescence in situ hybridization probes from the 200- and 400-bp fragments from gamma-irradiated apoptotic human T cells along with similar-sized probes from randomly sheared DNA of nonirradiated cells. These probes were compared for their binding along normal human metaphase chromosomes after fluorescence in situ hybridization with and without the presence of unlabeled total human blocking DNA. The addition of blocking DNA to the apoptotic probes revealed a nonrandom pattern of hybridization that was not observed for the nonirradiated control probes. The most obvious areas of selective binding occurred around the centromeric and other heterochromatic regions along the chromosome arms, such as the long (q arm) of the Y chromosome. The converse of this experiment was also carried out. DNA probes from heterochromatic and euchromatic regions of the human Y chromosome were hybridized onto slot blots of apoptotic ladder-sized and randomly sheared nonirradiated human T-lymphocyte DNA. The slot blot results showed that for an equal mass of ladder-sized apoptotic DNA and randomly sheared nonirradiated control DNA, the apoptotic DNA sample contains a relatively larger proportion of Y heterochromatin DNA sequences (approximately 2.5-fold). Together, these results indicate that apoptosis-mediated endonuclease attack does not occur randomly in the genome but occurs preferentially in heterochromatin.

Alkyl-lysophospholipids (ALPs) represent a new class of antitumor drugs that induce apoptotic cell death in a variety of tumor cell lines. Although their precise mechanism of action is unknown, ALPs primarily act on the cell membrane, where they inhibit signaling through the mitogen-activated protein kinase (MAPK) pathway. Because stimulation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway is essential for radiation-induced apoptosis in certain cell types, we tested the effect of ALPs in combination with ionizing radiation on MAPK/SAPK signaling and apoptosis induction. Here, we present data showing that three ALPs, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, hexadecylphosphocholine, and the novel compound octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate (D-21266) induce time- and dose-dependent apoptosis in the human leukemia cell lines U937 and Jurkat T but not in normal vascular endothelial cells. Moreover, in combination with radiation, ALPs strongly enhance the induction of apoptosis in both leukemic cell lines. All tested ALPs not only prevented MAPK activation, but, like radiation, stimulated the SAPK/JNK cascade within minutes. A dominant-negative mutant of c-Jun inhibited radiation- and ALP-induced apoptosis, indicating a requirement for the SAPK/JNK pathway. Our data support the view that ALPs and ionizing radiation cause an enhanced apoptotic effect by modulating the balance between the mitogenic, antiapoptotic MAPK, and the apoptotic SAPK/JNK pathways. This type of modulation of specific signal transduction pathways in tumor cells may lead to the development of new therapeutic strategies.

We report here that stress stimuli such as gamma-irradiation or the anticancer drug doxorubicin activate expression of the death-inducing ligands (DILs) CD95-L, TNF-alpha and TRAIL. Apoptosis induced by gamma-irradiation or doxorubicin engages a FADD- and caspase-dependent apoptosis pathway which is inhibited by dominant negative FADD or the caspase inhibitor zVAD. zVAD did not prevent activity of JNK/SAPKs in response to doxorubicin suggesting that JNK/SAPK activity is independent of death receptor triggering during cellular stress-induced apoptosis. In addition, JNK/SAPKs remained activated by doxorubicin in resistant cell lines in which cleavage of caspases and apoptosis was not observed. These data uncouple JNK/SAPK activation and apoptosis signaling and indicate that cellular stress-induced apoptosis involves signaling via DILs which is paralleled by activation of JNK/SAPKs. Activation of these kinases may contribute e.g., to the expression of molecules involved in apoptosis but is not sufficient for induction of the apoptosis program following cellular stress.

Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.

Kinectin has been characterized as the first known receptor for the molecular motor kinesin, which is critically involved in microtubule-based vesicle transport and membrane trafficking. Here we identify kinectin as a target for caspase-mediated proteolysis during apoptosis. Treatment of cells with diverse apoptotic stimuli including TNF, anti-Fas, anticancer drugs, gamma-radiation or ceramide leads to rapid proteolytic cleavage of the 160-kDa form of kinectin to a 120-kDa fragment. Evidence is provided that kinectin cleavage is mediated by caspase 7.

T lymphocytes undergo apoptosis in response to a variety of stimuli, including exposure to UV radiation and gamma-irradiation. While the mechanism by which stress stimuli induce apoptosis is not well understood, we have previously shown that the induction of Fas ligand (FasL) gene expression by environmental stress stimuli is dependent on c-Jun N-terminal kinase (JNK) activation. Using inducible dominant-active (DA) JNK kinase kinase (MEKK1) expression in Jurkat cells, we map a specific MEKK1-regulated response element to positions -338 to -316 of the Fas ligand (FasL) promoter. Mutation of that response element abrogated MEKK1-mediated FasL promoter activation and interfered in stress-induced activation of that promoter. Using electrophoretic mobility shift assays, we demonstrate that activator protein 1 (AP-1) binding proteins, namely, activating transcription factor 2 (ATF2) and c-Jun, bind to the MEKK1 response element. Transient transfection of interfering c-Jun and ATF2 mutants, which lack the consensus JNK phosphorylation sites, abrogated the transcriptional activation of the FasL promoter, demonstrating the involvement of these transcription factors in the regulation of the FasL promoter. Taken together, our data indicate that MEKK1 and transcription factors regulated by the JNK pathway play a role in committing lymphocytes to undergo apoptosis by inducing FasL expression via a novel response element in the promoter of that gene.

Because glutathione (GSH) plays a central part in the endogenous defense against UV radiation, an increase in GSH might provide photoprotection. Two agents that increase GSH levels were investigated. Cultured human cells and mouse skin were treated with N-acetylcysteine (NAC) and glutathione ethylester (GSH-Et). After 30 min, the GSH level was determined by HPLC. Photoprotection was assessed by testing the ability of the thiols to scavenge UV-induced reactive intermediates in the same models. As compared to control cells, NAC and GSH-Et increased intracellular GSH in vitro to maximally 144% and 174% respectively. In vitro protection (maximum 23% for NAC and 21% for GSH-Et) did not correlate to the intracellular GSH level but to the concentration of the thiols in the medium. In vivo, epidermal GSH was increased to maximally 163% of the control level by NAC and 1234% by GSH-Et. The maximum in vivo photoprotection provided by GSH-Et was 55%, similar to what was found previously for NAC. Again, the protection seemed more closely correlated to the thiol dose than to the GSH level. The study showed that the protection against UV-induced reactive intermediates depends on a general antioxidant action of these thiols, rather than only on their role as GSH precursors.

Objective: To study the intracellular signaling events associated with ligation of the surface receptor CD95., Methods: A mutant clone of Jurkat T cells, DD3, which fails to transmit apoptotic signals through CD95, was selected for study. Surface expression of CD95 and the primary nucleotide sequence of CD95, as well as the functional effects of a mutant CD95 molecule found in DD3, were examined., Results: DD3, while exhibiting impaired ability to undergo apoptosis after CD95 ligation, retained the ability to die after ultraviolet light stimulation. A CD95 complementary DNA (cDNA) cloned from DD3 encoded a mutant transmembrane protein lacking the carboxy-terminal "death domain." Western blotting confirmed the presence of both wild-type and mutant CD95 protein in DD3. Transfection of the mutant CD95 cDNA into parental Jurkat cells conferred protection from CD95-mediated apoptosis., Conclusion: A mutant CD95 receptor lacking the cytoplasmic "death domain" can interfere with wild-type receptor function in T cells.

Purpose: The purpose of this study was to characterize the concentration-dependent induction of apoptosis by anticancer drugs in vitro., Methods: The apoptosis- and necrosis-inducing potential of the anticancer drugs cladribine (CDA), cytarabine (ARA-C), cisplatin (CDDP), and 5-fluorouracil (5FU) were studied in vitro in the human leukemia cell lines HSB2 and Jurkat using a flow-cytometry assay that permits the simultaneous quantification of vital, apoptotic, and necrotic cells by double-staining with fluorescein isothiocyanate (FITC)-labeled Annexin-V and propidium iodide. The results were fit to different multicompartmental models and the sensitivity of the cell lines to apoptosis and necrosis was estimated., Results: A time- and dose-dependent decrease in vital cells as well as an increase in apoptotic and necrotic cells was observed in HSB2 cells upon continuous incubation with 10(-5)-10(-7) MCDA, 10(-5)-10(-8) MARA-C, 5 x10(-5)-5 x 10(-6) M CDDP, and 10(-4)-10(-5) M 5FU, whereas no effect was observed relative to controls upon incubation with 10(-8)-10(-9) M CDA, 10(-9) M ARA-C, 10(-7)-10(-8) M CDDP, or 10(-6)-10(-9) M 5FU. In Jurkat cells, apoptosis- and necrosis-inducing effects were observed at 10(-4)-5 x 10(-6) M CDA, 10(-5)-10(-7) M ARA-C, 5 x 10(-5)-5 x 10(-6) M CDDP, and 10(-4)-10(-5) M 5FU. In all experiments, apoptotic cells reached a peak after 6-48 h of drug exposure. These data were best fit by a model in which vital cells became irreversibly apoptotic by a direct pathway and necrotic by an irreversible indirect pathway following the apoptotic state (mean R = 0.9876; range 0.9510-0.9993; mean modified Akaike's information criterion 3.88; range 1.86-5.82) and the rate constants of either pathway (Kva and Kan, respectively) were assessed. The sensitivity of both cell lines to apoptosis and necrosis (expressed as EC50 and Emax values) induced by the anticancer drugs could be calculated from the sigmoidal concentration-effect curves. Furthermore, it was shown that drug treatment (10(-6) M CDA or 10(-6) M ARA-C) potentiated the apoptosis-inducing effects of irradiation (6 Gy) but not its necrosis-inducing potential., Conclusion: This study demonstrates that CDA, ARA-C, CDDP, and 5FU possess concentration-dependent apoptosis-inducing potential in the cell lines studied. The cytotoxic mechanism and cell-killing potential of these drugs is different, which is reflected by different EC50 and Emax values. Furthermore, a method for pharmacodynamic modeling is introduced that permits a quantitative approach for the assessment of the sensitivity of tumor cells to anticancer drugs and combined treatments.

To explore possible biochemical mechanisms whereby electromagnetic fields of around 0.1 mT might affect immune cells or developing cancer cells, we studied intracellular calcium signaling in the model system Jurkat E6-1 human T-leukemia cells during and following exposure to a 60 Hz magnetic field. Cells were labeled with the intracellular calcium-sensitive fluorescent dye Fluo-3, stimulated with a monoclonal antibody against the cell surface structure CD3 (associated with ligand-stimulated T-cell activation), and analyzed on a FACScan flow-cytometer for increases in intensity of emissions in the range of 515-545 nm. Cells were exposed during or before calcium signal-stimulation to 0.15 mTrms 60 Hz magnetic field. The total DC magnetic field of 78.2 microT was aligned 17.5 degrees off the vertical axis. Experiments used both cells cultured at optimal conditions at 37 degrees C and cells grown under suboptimal conditions of 24 degrees C, lowered external calcium, or lowered anti-CD3 concentration. These experiments demonstrate that intracellular signaling in Jurkat E6-1 was not affected by a 60 Hz magnetic field when culture and calcium signal-stimulation were optimal or suboptimal. These results do not exclude field-induced calcium-related effects further down the calcium signaling pathway, such as on calmodulin or other calcium-sensitive enzymes.