Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.     

Arachidonic Acid Oxidation by Brain and Placenta Preparations from Normal and Placental Insufficient Fetal Rabbit

Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1-14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12-HETE and 15-HETE by fetal brain is time-dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 mumol/h/g wet weight are presumably responsible for the excessive accumulation of 12-HETE and 15-HETE in comparison to PGE2 (Km = 0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12-HETE and 15-HETE there is a substantial formation of 12-L-hydroxyheptadecatrienic acid. Formation of 12-HETE and 15-HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.     

Ways of apoptosis development in human lymphocytes, induced by UV-irradiation

The level of DNA damage and cytochrome c content in human lymphocytes in the dynamics of apoptosis induced by UV light (240?C390 nm) at doses of 151, 1510 and 3020 J/m² is studied. DNA fragmentation is revealed in 20 h after UV irradiation of lymphocytes at doses mentioned above. It is shown that DNA damages (single strand breaks) appear immediately after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² (comets of C1 type) and reach their maximum 6 h after cell modification (comets of C2 and C3 types). It is concluded that p53-dependent and receptor caspase pathways are involved in apoptosis development in the human lymphocytes, modified after UV irradiation.     

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.     

UVB照射后小鼠脾淋巴细胞DNA损伤程度的检测

用强度为30 μW/cm2、60 μW/cm2、90 μW/cm2、120 μW/cm2、150 μW/cm2的中波红斑效应紫外线(UVB)分别照射小鼠脾淋巴细胞5 min、15 min、30 min,采用单细胞凝胶电泳法(SCGE)检测UVB照射对细胞DNA的损伤,结果照射5 min和15 min时,DNA损伤程度与照射强度呈正相关;而照射30 min时,30 μW/cm2组DNA损伤程度最高,60 μW/cm2、90 μW/cm2、120 μW/cm2组损伤程度有所降低,而150 μW/cm2组又出现了高DNA损伤.认为通过SCGE来检测UVB照射对小鼠脾淋巴细胞的损伤程度,可广泛应用于环境毒理学中对DNA具有损伤作用的分析,为环境生物学研究辐射对生物体的影响提供借鉴.     

Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase Calpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis

Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). We expressed PKCalpha fused to green fluorescent protein and examined its real time translocation to the plasma membrane in living human corneal epithelial cells. Upon 10 min of stimulation with epidermal and hepatocyte growth factors (EGF and HGF), PKCalpha translocated to the plasma membrane. Keratinocyte growth factor did not stimulate PKCalpha translocation up to 1 h after stimulation. Pretreatment with the 15-lipoxygenase metabolite, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), followed by EGF or HGF, produced faster translocation of PKCalpha detectable at 2 min. However, the same concentration of 15(S)-HETE alone did not stimulate translocation. 15(S)-Hydroperoxyeicosatetraenoic acid and 5(S)-HETE did not affect growth factor-induced translocation of PKCalpha. PD153035, a specific inhibitor of tyrosine kinase activity of the EGF receptor, completely blocked PKCalpha translocation induced by EGF. PD98059, a specific MEK inhibitor, significantly inhibited EGF- and HGF-mediated PKCalpha translocation, which was reversed by addition of 15(S)-HETE. Phosphorylation of ERK1/2 by EGF was followed by phosphorylation of cytosolic phospholipase A(2) (cPLA(2)), and blocking ERK1/2 inhibited cPLA(2) activation. Immunofluorescence demonstrated translocation of p-cPLA(2) to plasma and nuclear membranes as early as 2 min. This may further increase arachidonic acid release from membrane phospholipid pools and increase the intracellular pool of HETEs. In fact, in cells prelabeled with [(3)H]arachidonic acid, EGF stimulated synthesis of 15(S)-HETE in the cytosolic fraction. 15(S)-HETE also reversed the effect of LOX inhibitor on EGF-mediated cell proliferation. Our results indicate that 15(S)-HETE is an intracellular second messenger that facilitates translocation of PKCalpha to the membrane and elucidate a mechanism that plays a regulatory role in cell proliferation crucial to corneal wound healing.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Characterization of 11-HETE and 15-HETE, together with prostacyclin, as major products of the cyclooxygenase pathway in cultured rat aorta smooth muscle cells

Arachidonic acid is the precursor of several potent derivatives that regulate physiological functions in the cardiovascular system. Thromboxane (TXA2) and prostacyclin (PGI2) are synthesized by the cyclooxygenase enzyme. The proaggregatory and vasoconstrictive TXA2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of PGI2 synthesized by blood vessels. Arachidonic acid is also converted via a 5-lipoxygenase to leukotrienes, the vasoconstrictive components of SRSA. We have shown that this latter pathway is regulated by 15-HETE, a product of the 15-lipoxygenase present in lymphocytes. Confluent cultures of rat aorta smooth muscle cells (RSM) were superfused briefly with [14C]arachidonic acid. The products were isolated and analyzed by thin-layer chromatography-radioautography, high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. Prostacyclin (PGI2) was identified as the major product both by its biological properties in a platelet aggregation assay and by the mass spectrum of its tetra-trimethylsilylether-methyl ester derivative. Minor quantities of PGE2, PGD2, and PGF2 alpha were also synthesized. Three other compounds with chromatographic properties of mono-hydroxy eicosanoic acids were also formed in major amounts. These were shown to be cyclooxygenase products since their synthesis, together with that of prostacyclin, was blocked by the cyclooxygenase inhibitors aspirin (0.2 mM) and indomethacin (10 microM). Quantities of the hydroxy-eicosanoids were isolated from large scale incubations by silicic acid chromatography. Following methylation and reduction with platinum oxide/H2, the compounds were converted to their trimethylsilylether derivatives and analyzed by gas-liquid chromatography-mass spectrometry. The compounds were identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and hydroxy-5,8,10-heptadeca-trienoic acid (HHT) by simultaneous ion monitoring of characteristic ions at M/e ratios of 287, 258, 229 for 11-HETE and 343, 314, 173 for 15-HETE, and by comparison with the mass spectra of authentic samples. Rat smooth muscle cells, prelabeled by 24-hour incubation with [14C]arachidonic acid, released large amounts of prostacyclin together with enhanced amounts of 11- and 15-HETE in response to physiological levels of thrombin (0.5-5 units/ml). These experiments demonstrate that, in addition to the thromboxane antagonist prostacyclin, vascular smooth muscle cells produce significant quantities of the leukotriene inhibitor 15-HETE via the cyclooxygenase pathway in response to physiological stimuli such as thrombin. The release of both prostacyclin and 15-HETE by vascular smooth muscle cells may thus play an important role in vascular homeostasis.     

Changes in arachidonic acid content of X-irradiated human lymphocytes and plasma membrane fractions

The 14C-arachidonic acid content of pre-labelled human lymphocytes was followed during in vitro incubation in untreated and X-irradiated cells. Loss of about 35-40 per cent of the incorporated radioactivity was found already at 10 minutes after irradiation as detected in whole cells, phospholipid, free fatty acid and phosphatidylcholine fractions of plasma membranes. When pulse labeling with 14C-arachidonic acid was performed at various intervals after in vitro irradiation, the uptake of precursor by irradiated cells did not differ from untreated ones. At the same time the activity of phospholipid, free fatty acid and phosphatidylcholine fractions was approximately 50 per cent lower compared to the control, then it increased gradually reaching the control level.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

The content of cyclic nucleotides, free cytoplasmic Ca2+ and malondialdehyde in the splenic lymphocytes and thymocytes of rats under the action of moderate doses of radiation]

It has been shown that the malonic dialdehyde (MDA) content in spleen lymphocytes of rats increases after whole-body X irradiation with a dose of 0.5 Gy to reach the maximum level in 24 h. Simultaneously, the concentration of cGMP and free cytosol Ca2+ increases. With a dose of 1 Gy MDA content of cells increases 6 h following irradiation. The maximum drop of the release of viable lymphocytes from the spleen and thymus, observed 24 h and 3 days after irradiation respectively, coincides with the appearance of the second peak of the MDA content. The level of cGMP remains decreased throughout the period of about 6 days. The onset of lymphocyte repopulation in the spleen on day 6 coincides with the decrease in the MDA level and increase in the cytosol Ca2+ concentration.     

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.     

PID15, a novel 6?kDa secreted peptide,mediates Naja naja venom phospholipase A2 induced apoptosis in isolated human peripheral lymphocytes

Background

Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A₂ (NV-PLA₂) using isolated human peripheral lymphocytes.

Results

Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A₂ (NV-PLA₂) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA₂ at 15^th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A ₂ Induced DNA fragmentation factor secreted at 15 ^th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF).

Conclusion

Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA₂ treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-14C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF1 alpha, TxB2 and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF2 alpha, PGE2, PGD2, LTB4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB4, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB4 is considerably increased after adrenalectomy. In the spleen, PGD2 and 12-HETE are decreased after adrenalectomy. The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA2 inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).     

Immunospecific protein of 34.5 kDa from DNA-protein cross-links induced by cis- and trans-diamminedichloroplatinum

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most often used anticancer drugs. It is generally accepted that the antitumor activity of the drug results from its interactions with DNA. Trans-diamminedichloroplatinum(II) (trans-DDP) also binds to DNA effectively, but is clinically ineffective. In the present work the lymphocyte nuclear proteins that participate in DNA-protein cross-links induced by cis- and trans-DDP are investigated. In lymphocytes which are incubated without platinum compounds there are DNA-binding proteins in the range of 45-71 kDa. It is shown that additional proteins of 28, 30, 34.5, 45 and 120 kDa are cross-linked with DNA in lymphocytes after 2-h incubation with cis-DDP at concentrations of 0.1 and 0.5 mM. Trans-DDP does not bind additional proteins to DNA after the same incubation time. Electrophoretic analysis shows that trans-DDP binds much more of the same nuclear proteins to DNA than cis-DDP after 12-h incubation. In this study a test for the identification of 34.5 kDa protein is also undertaken. This protein appears in the samples obtained after 12-h incubation of lymphocytes with cis- and trans-DDP at 0.5 and 1 mM, especially. The protein of 34.5 kDa from cross-links induced by 1 mM trans-DDP is recognized by antibodies against the protein of the same molecular weight from the nuclear matrix of the lymphocytes. The results obtained here are discussed in relation to the biological activity of diamminedichloroplatinum isomers.     

DNA loop organization and DNA fragmentation during radiation-induced apoptosis in human lymphocytes

Apoptotic DNA fragmentation induced by gamma-rays has been compared with the DNA loop sizes in G0-human lymphocytes using pulsed field gel electrophoresis (PFGE). Genomic DNA was cleaved into the DNA loops at the topoisomerase II mediated attachment points using short treatment of cells with etoposide. The apoptotic fragmentation, with a distinct cut-off around 50 kb for a maximum length of fragments, appeared 5 h after irradiation when the most part of radiation-induced DNA double strand breaks (DSBs) have been repaired. The data indicate that apoptotic fragmentation of DNA in the G0-human lymphocytes begins when repair of radiation-induced DSBs has been completed. Similar apoptotic DNA fragmentation was also observed following the treatment of cells with etoposide. All genomic DNA was fragmented into 50-kb fragments during the final stages of apoptosis. Most of the DNA in resting lymphocytes is organized into Mb-size loops but loops of sizes down to 50 kb were also observed. A sharp border between the size distributions of DNA loops and apoptotic fragments was found. The data suggest that 50 kb apoptotic fragmentation is not based on excision of the DNA loops. No apoptotic fragments with the sizes more than 5.7 Mb were seen during the whole course of apoptosis. This observation indicates that despite intensive apoptotic fragmentation into the 50-kb fragments the chromosomes maintain integrity during radiation-induced apoptosis in human lymphocytes. We propose a model for radiation-induced apoptotic fragmentation in human lymphocytes that involves four stages: induction of DNA breaks and relaxation of DNA loops; DNA repair followed by reorganization of the DNA loops into the 50-kb units of condensed chromatin; co-operative fragmentation of the reorganized DNA loops into the distinct 50-kb fragments and resealing of the chromosome ends at the sites of this fragmentation; cleavage of the 50-kb fragments at the internucleosomal spacers.