Effect of subtherapeutic doses of docetaxel (taxotere) on the efficacy of radiotherapy and pro-oxidant-antioxidant balance in rats with Guerin's carcinoma

In the experiments at Wistar male rats the effect of subtherapeutic doses of docetaxel (5 and 10 mg/kg) on the radiotherapy efficacy (20 Gy of single-dose X-rays) namely growth rate of Guerin's tumor and prooxidant-antioxidant balance in liver and blood of animals bearing tumors was investigated. It has been demonstrated that docetaxel at dose 5 mg/kg given 18 hours before irradiation resulted in significant tumor growth delay (2.3-2.7-fold) in comparison with group of rats that received only irradiation. After application of higher dose of docetaxel there was no statistically significant change of tumor size along the whole experiment (14 and 21 days after tumor implantation). Content of lipid peroxidation products was revealed to be considerably increased after chemotherapy and concurrent irradiation when docetaxel was used in a dose of 10 mg/kg. At the same time glutatione peroxidase activity and antioxidative activity of blood plasma were reduced. In the rat liver chemoradiotherapy led to decrease of glutathion peroxidase and glutathione-S-transferase activity to greater degree at docetaxel dose of 10 mg/kg. The obtained results allow to conclude that higher dose of docetaxel and concurrent irradiation resulted in the most effective Guerin's carcinoma growth delay and considerable deviation of antioxidant-prooxidant balance of tissues in the direction of the last.     

Influence of small doses of fractionated X-ray radiation on the activity of histone-specific proteases and the content of malonic dialdehyde in chromatin of the cells of rat liver

The fractionated X-ray irradiation resulted in accumulation of malonic dialdehyde (MDA) in chromatin of the cells of rat liver and activation of nuclear endogenous histon-specific proteases. Seven days after the termination of irradiation, the activity of the nuclear proteases returned to the control level and the MDA level remained 10% above the control parameter. The correlation (r = 0.71) of the processes of MDA accumulation in chromatin and activation of histon-specific proteases in the cells of rat liver after the termination of irradiation showed that these processes were coupled. Transplantation of Guerin's carcinoma to the irradiated animals caused the changes in nuclear proteolysis of the histones and MDA level in chromatin of the tumor-carrier liver cells, which lessened the influence of preliminary X-ray irradiation on rats.     

Inhibition of the repair of neutron-induced DNA single-strand breaks in experimental tumor cells

Nicotinamide and arabinoside cytosine mixed with hydroxyurea were shown to influence the relative amount of double-stranded DNA in Ehrlich ascites tumor cells in vitro subjected to single irradiation (10-30-52 Gy) and in Guerin's carcinoma in rat lungs exposed to fractionated 6 MeV neutron-radiation (1.25 Gy X 4). The DMF values for Ehrlich ascites tumor were a function of a dose range and the duration of the drugs' effect. Guerin's carcinoma DNA was found to be affected more readily when treated with radiation and drugs than when exposed to neutron radiation alone.     

Influence of tumor drug resistance phenotype on the dynamics of cisplatin-induced changes of rat peripheral lymphocyte chromatin structure in Guerin's carcinoma

The correlation between the tumor drug sensitivity and the degree of lymphocyte interphase nuclei chromatin damages induced by cisplatin in rats was found using sensitive and resistant to cisplatin variants of Guerin's carcinoma. Increased optical density of lymphocyte chromatin in first minutes after cisplatin injection both in rats without Guerin's carcinoma and with sensitive to cisplatin variants of this tumor was observed. Lymphocyte chromatin structure remains unchanged in rats with cisplatin resistant carcinoma. Normal blood cells are suppose to change their sensitiveness to cisplatin under the humoral influence of the growing tumor in according with its phenotype.     

Histone specific proteinase and DNase activity of spleen lymphocyte nuclei during oncogenesis and under the effect of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil

The features of histone proteolysis, their carbonylation level and endonucleolysis intensity were studied in the spleen lymphocytes of rats with transplanted Guerin's carcinoma and under the conditions of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil (BCU) action. The intensification of oxidizing histone destruction and histone-specific protease activity during oncogenesis and under conditions of BCU action was shown. DNase I and DNase II enzymatic activities of spleen lymphocytes of rats with tumour were decreased on different stages of tumour development, however, they were increased under conditions of chemical compound administration.     

Regulation of lipid oxidation and structural state of the nuclear membrane after the irradiation of tumor-bearing animals

Changes of antioxidative activity (AOA), lipid composition and microviscosity of different membrane regions in tumor cell nuclei and in the liver of tumor-host with Ehrlich ascite carcinoma (EAC) after irradiation were studied. On the basis of the obtained data the analysis of the control system of lipid oxidation in the membrane was carried out. This control system involves a relationship between AOA changes, lipid composition, their oxidative ability and the nuclear membrane structure. It was shown that after irradiation the control system in the nuclei of tumor cells had the same state as before irradiation and was different from the normal one. The control system in the nuclei of tumor-host liver after irradiation starts to work in a regime which is characteristic of irradiated cells. It was shown that the principle difference in the control system functioning in tumor and tumor-host nuclei disappeared after irradiation.     

Biochemical features of cancer cachexia under conditions of different provision with vitamin A

On the model of tumor growth of Guerin's carcinoma and experimental modeling of vitamin A provision the biochemical characteristics of tumor cachexia in animals with malignant tumors and different vitamin A provision status were studied. It is determined that tumor growth in the body, deprived of vitamin A, is characterized by negative nitrogen balance, decrease of glucose, free fatty acids and ketone bodies level in the blood serum, which indicates the increase of cancer cachexia.     

Effects of ubiquinone-9 on lipids of nuclei and chromatin of the rat liver under continuous irradiation]

The influence of continuous gamma irradiation on the lipids of nuclei and chromatin of rat liver at a dose-rate of 0,129 Gy/day for 155 days (a total dose of 20 Gy) and by feeding of ubiquinone-9 has been studied. The amount of phosphatidylcholine with phosphatidylserine and phosphatidyl-ethanolamine in liver nuclei of irradiated rats was found to increase. Ubiquinone-9 had a normalizing effect. A decrease of cardiolipin was observed in the liver chromatin of irradiated rats. The amount of free fatty acids had a tendency to decrease in homogenate, nuclei and liver chromatin of irradiated rats. Ubiquinone was found to increase the amount of free fatty acids up to the control level. The amount of cholesterol in nuclei was increased after irradiation and that in chromatin tended to rise. Ubiquinone-9 significantly decreased the amount of cholesterol in nuclei and chromatin of irradiated rats.     

Effect of liposomal antitumor preparation 5,6-benzocoumarin-5-uracil on intensity of free-radical processes in the liver microsomes and tumor cells of rats with transplanted Guerin's carcinoma

The contents of primary and secondary (TBA-active) products of lipid peroxidation were investigated in microsomal fraction of the liver and tumor cells of rats with transplanted Guerin's carcinoma and under the condition of antitumor liposomal preparation 5,6-benzcumarine-5-uracil (BCU) action. High level of lipid peroxidation process in the microsomal fraction is shown in the rat liver and tumor cells under the condition of BCU action in the period of intensive carcinoma growth. It remains till the period of tumor growth braking. This fact testifies to the prooxidation action of the preparation. Liposomal antitumor preparation BCU raises the process of lipid peroxidation in microsomal fraction of tumor cells and its action increases according to the malignant growth. The processes of lipid peroxidation in microsomal rat liver fraction approach the control data under the condition of the mentioned preparation. The investigated liposomal form of BCU possesses directed prooxidation action on the malignant tissue.     

Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.     

Effect of irradiation and endogenous nucleases on rat liver chromatin

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.     

Thermo- and radioinduced enzymatic relaxation of superhelical DNA from mud loach Misgurnus fossilis L. spermatozoa

Actions of environmental impacts on mud loach spermatozoa were studied using various model systems: a) temperature stress, b) X-ray irradiation in vivo only of the animal head (a condition to trigger stress reaction), c) X-ray irradiation in vivo only of the animal body (a condition to exclude a direct activation of principal stress-realizing organism systems), d) gamma-irradiation in vitro of the cell suspension. It has been demonstrated that the temperature stress or X-ray irradiation of the mud loach head induced three lines of effects: 1) significant decrease in DNA superhelical density, 2) activity redistribution (functional activation) of DNase II between chromatin subfractions (with the increase of its association to chromatin), and 3) intracellular acidification up to pH value to satisfy the DNase II initiation. The obtained facts allow to suggest that, first, DNase II participates in the presented temperature- and radio-induced supercoiled DNA relaxation in spermatozoa, and, second, DNase II is involved in physiological (season elimination of spermatozoa that remained within male gonads after fertilization) or environmentally-induced DNA degradation.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model

We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.     

Changes of chromatin structure state in thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation

The paper deals with changes in the structural state of chromatin in isolated thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation. Content of necrosis and apoptosis cells in the suspension of the isolated rat thymocites, during 3-hour incubation after X-ray irradiation in a dose of 4.5 Gy or with the presence of 0.1 microM of H2O2 by the method of double lifetime staining by fluorescent dye Hehst 33342 and propydium iodide has been estimated. Apoptogenic effect of the studied effects has been found out, the dynamics of condensation and internucleosomic chromatin fragmentation has been established. It has been shown that 100 microM alpha-tocopherol inhibited completely DNA fragmentation in the cells incubated with H2O2 and only partially in irradiated cells. Introduction of postmitochondrial supernatant, isolated from the incubated control or irradiated cells, into the cell-free system which included the ATP-regenerating system and nuclei of control thymocites did not affect the level of DNA fragmentation, while the increase of the level of fragmented DNA in nuclei was observed in the presence of the supernatant obtained by centrifugation of the cells treated by H2O2. Differences of mechanisms of thymocite apoptosis initiation, as affected by hydrogen peroxide and ionizing radiation, is discussed.     

Inhibition of poly(ADP-ribose) polymerase as a possible reason for activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats

The molecular mechanism of activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats was studied. Thymocyte nuclei of control and irradiated rats were pre-incubated with NAD under conditions favourable for poly ADP-ribosylation. Pre-incubation results in a decrease in the rate of autolytic DNA digestion by Ca2+/Mg2+-dependent endonuclease of 6-7- and 2-3-fold for control and irradiated animals, respectively. The activity of Ca2+/Mg2+-nuclease extracted from the nuclei pre-incubated with NAD is also considerably decreased. The presence of nicotinamide and thymidine in the preincubation medium prevents the suppression of Ca2+/Mg2+-nuclease activity. In the experiments performed with isolated nuclei and permeabilized thymocytes the synthesis of poly(ADP-ribose) does not significantly change within 1 h after irradiation at a dose of 10 Gy, whereas 2 and 3 h after the exposure it decreases by 35-40 and 45-55 per cent, respectively. The activity of poly(ADP-ribose) glycohydrolase in this period is similar to that in the controls. The average size of the de novo synthesized chains of poly(ADP-ribose) increases from 11 to 17 ADP-ribose units by the second hour after irradiation. Inhibition of poly(ADP-ribose) polymerase in the postirradiation period preceded the internucleosomal fragmentation of chromatin. The results suggest that activation of Ca2+/Mg2+-nuclease in irradiated thymocytes is accounted for by the disturbance of its poly ADP-ribosylation.     

Radiation-induced formation of apoptotic bodies in rat thymus

The process of interphase death of thymocytes in whole-body X-irradiated rats were studied. Cell size distribution analysis indicates that cell fragments (= apoptotic bodies) appeared in the thymus and increased in number depending on dose (200-1000 R) and time (2-6 hr) after irradiation with corresponding decrease in normal-size thymocytes. Occurrence of nuclear fragmentation in association with the cellular fragmentation was proved with cytofluorometric determination of DNA content in individual cells. Scanning electron microscopic observations also revealed extensive fragmentation of cells in the irradiated rat thymus. The results show clearly that cells as well as nuclei fragment rapidly into smaller pieces of various sizes in the irradiated rat thymus as commonly observed with apoptosis.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage

 We have tested the specific hypothesis that the pathway of nuclear collapse in apoptosis is governed by the early attack on active chromatin at spatially restricted nuclear sites. Cell death in PC12 pheochromocytoma cells deprived of serum growth factors, in HL-60 leukemic cells treated with inhibitors of protein or RNA biosynthesis, and in U937 histiocytic lymphoma cells exposed to the cytokine tumor necrosis factor alpha showed a common mechanism in the targeting of DNA for degradation. An incorporation assay with labeled nucleotide revealed an early selective nicking in peripheral nuclear chromatin with concomitant diminution in the amount of immunoreactive lamin B protein. This was followed by a phase of more extensive cleavages, continued nuclear protein loss, chromatin collapse, and fragmentation of nuclei. The spatial restriction of early cleavages is similar to the nicking obtained by the application of exogenous DNase I to fixed nuclei of normal cells and to that obtained in the activation of the endogenous endonuclease of liver nuclei by Ca²⁺. These similarities suggest that, in apoptosis, activation of an endonuclease preferentially recognizing a specific chromatin configuration, such as that of active (DNase I-sensitive) genes, underlies the early spatial demarcation of cleavages. Accepted: 12 October 1998