Chromatin degradation during the exposure of thymus lymphocytes in vitro to differentiation inducers and gamma irradiation

Chemical inductors of differentiation were shown to cause chromatin degradation in thymus lymphocytes. This process was prevented by the protein synthesis inhibitors. The fragments formed after the effect of chemical differentiation inductors on thymocytes were fully identical to chromatin internucleosome degradation products formed in the exposed cells. Chromatin degradation under the effect of chemical differentiation inductors was most pronounced in a more radiosensitive thymocyte fraction.     

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.     

Effects of polysaccharide from Thuja occidentale L. on stromal precursor cells of hematopoietic microenvironment in mice]

The effect of high molecular polysaccharide subfraction from Thuja occidentale L. (TPS) on stromal precursor cells of hematopoietic microenvironment under the "steady-state" conditions and after sublethal irradiation was investigated. The stromal precursor cells of different stages of differentiation were detected by the implantation of mouse bone marrow under the renal capsule of syngeneic intact recipients and chimeras. It was shown that TPS did not occur the toxic influence on the stromal precursor cells and provided the defense effect on them under the strong (6 Gy) radiation damages.     

Radiobiology of differentiating mammalian cells

The data are reviewed in a comparative aspect concerning the influence of ionizing radiation and chemical inductors on mammalian cell differentiation. Molecular bases for the effects observed are discussed. Such an approach is shown to be adequate in studying the mechanism of radiation death of thymus lymphocytes.     

On the mechanism of a radiation-induced change in enzymic differentiation during development

In experiments on glucose-6-phosphatase and tyrosine aminotransferase it was shown that radiation induces changes in enzymic differentiation in perinatal rat liver. A study was made of the probable reasons for the observed changes. It was shown that the macromolecular system of the protein enzyme synthesis was not damaged by the radiation doses used. The observed decrease in glucose-6-phosphatase activity during late embryogenesis, after pre-irradiation at early organogenesis, is eliminated by administration of exogenous thyroxine. A radiation-induced rise in the tyrosine aminotransferase activity during the perinatal period correlated with the cyclic AMP system status. It is proposed that modification of enzymic differentiation after irradiation results from the change in the amount of inductors.     

Regulation of the 26S proteasomes' endoribonuclease activity specificity in k562 cells under effect of differentiation and apoptosis inductors

The specificity of 26S proteasomes' endoribonuclease activity has been shown to be changed under effect of erythroid differentiation (hemin) and programmed cell death (diethylmaleate) inductors in proerythroleukemic K562 cells. Treatment of K562 cells with apoptosis and differentiation inductors leads to the specific stimulation of RNase activity towards certain mRNA and to reduction of proteasome RNase activity towards other mRNA. The enzymatic activity under study has been demonstrated to be specifically and selectively dependent on phosphorylation of 26S proteasome subunits as well as on Mg and Ca ions. The conclusion is drawn that the specificity of the proteasomes' RNAse activity is regulated during differentiation and apoptosis, and selective regulation of the activity of different nuclease centers is suggested, the mechanism involving changes in phosphorylation of proteasome subunits and cation homeostasis.     

Combined effect of interferon inducers, radiosensitizing and antineoplastic agents on solid tumors

In experiments with mice bearing solid sarcoma 37 a study was conducted on the combined effect of radiation and inductors of endogenous interferon synthesis (IEIS), together with hyperthermia or together with an alkylating and carbomoilating agent, dimethinur. The effect was estimated by the tumor growth coefficient and by the number of animals with the regressed tumors. Po I. polyC was not shown to influence the efficiency of hyperthermia combined with radiation; dextransulphate and tiloron increased the radiosensitizing effect of hyperthermia. Dimethinur aggravated the effect of radiation, but with IEIS used together with dimethynur and radiation, the response of the tumor increased insignificantly as compared to the effect of IEIS together with radiation.     

Radiation disorder of enzyme synthesis in the perinatal period of ontogeny

A change of enzymatic differentiation in the rat liver during the perinatal developmental period after gamma-irradiation on the 7-9th and 19th days of embryogenesis in doses 0.5, 2 and 6 Gr has been shown on the example of glucose-6-phosphatase (G-6-P-ase) and tyrosine aminotransferase (TAT). The protein-synthesizing machinery was not damaged at these doses. The radiation inhibition of G-6-P-ase synthesis was relieved by the injection of thyroxine. A dependence was shown between the radiation increase of TAT activity and changes in cAMP system (increase of cAMP level, decrease of phosphodiesterase activity, intensification of response of adenylate cyclase complex to biogenic amines). A suggestion is put forward that the radiation damage of the enzymes under study is mediated by a change in the number of hormonal inductors.     

The diethyldithiocarbamate concentration effects and interactions with other cytotoxic agents on Chinese Hamster cells (V79)

A metal chelator, diethyldithiocarbamate (DDC) perturbs the chromosome condensation processes in dividing cells. The length of the metaphase chromosomes in Chinese hamster cells (V79) treated with 17.2 micrograms/ml of DDC for 2 hr is about half of that in untreated cells. However, concentrations of 1.7 microgram or 172 micrograms/ml DDC apparently do not produce this effect. DDC at 17.2 micrograms/ml also disrupts spindle fibers. Penicillamine, EDTA, EGTA, and diamide show no effect on chromosome condensation. Bleomycin, but not mitomycin and cisplatin, added simultaneously with DDC can prevent the DDC effect on chromosomes. The cytotoxic effect of increasing concentrations of DDC to V79 cells incubated at 37 degrees C exhibits a similar biphasic response. This concentration biphasic toxic effect is not altered when the cells are treated with DDC in combination with radiation, heat, or other cytotoxic drugs. These observations suggest that the different effects of DDC concentrations on chromosome condensation should be considered as one important modification factor for DDC related toxicity.     

Interaction of platinum(II) tetrachlorodianion (Fast Black)2 with superhelical DNA and with radiation in vitro and in vivo

A new complex of tetrachloroplatinum(II) and the azoic diazo dye, Fast Black K, Pt(Fast Black)2, was made in an attempt to produce an uncharged molecule which could readily gain access into cells and could bring a high concentration of tetrachloroplatinum into the vicinity of the DNA. Even the lowest concentration of Pt(Fast Black)2 tested in the superhelical pBR322 plasmid DNA assay in vitro completely converted the superhelical DNA to the circular and linear forms by 24 h. When the cytotoxicity of the Pt(Fast Black)2 and Fast Black were tested in exponentially growing EMT6 cells. Pt(Fast Black)2 was slightly more toxic to normally oxygenated than to hypoxic cells at pH 7.40, but was far more toxic to cells at pH 6.45 with no difference based on cellular oxygenation. Fast Black was much less toxic than Pt(Fast Black)2 and its cytotoxicity was unaffected by pH. Pt(Fast Black)2 had a small radiosensitizing effect on hypoxic EMT6 cells with a dose-modifying factor of 1.3, but exposure to the drug entirely removed the shoulder region on the radiation survival curves for both the oxygenated and hypoxic cells. In contrast, Fast Black reduced the shoulder in hypoxic but not in oxygenated cells. When Pt(Fast Black)2 (500 mg/kg), Fast Black (300 mg/kg) (the maximally tolerated dose), or misonidazole (1 g/kg) were given intraperitoneally 15 min prior to irradiation of FSaIIC tumors with 0, 10, 20, or 30 Gy, Pt(Fast Black)2 alone caused a tumor growth delay of 6 days versus 3 days for Fast Black. With radiation, Pt(Fast Black)2 produced the greatest enhancement in tumor growth delay of the drugs tested, especially at the lowest (10 Gy) radiation dose (i.e., in the in vivo "shoulder region"). These results indicate that Pt(Fast Black)2 may be suitable for clinical development because it causes both significant direct cytotoxicity and enhancement of radiation killing. The fact that its cytotoxicity is markedly increased at an acidic pH and its radiation enhancing effects are greatest in combination with relatively low single-fraction radiation doses make it especially interesting. The cytotoxicity of Pt(Fast Black)2 may be influenced by the tumor environment, and the radiosensitizing properties appear well suited for use with radiation fraction sizes that are employed in the clinic.     

Combined effect of metronidazole, interferon synthesis inducers and DNA repair inhibitors

In experiments on mice with solid sarcoma 37, a study was made of modification of the radiosensitizing effect of metronidazole. The increase in the leucotic interferon titre, induced by the interferon synthesis inductors, did not influence the radiosensitizing efficiency of metronidazole: after the administration of the repair inhibitor, 8-bromocaffeine, the coefficient of suppression of tumor growth was higher and the number of mice with the regressed tumors greater as compared to animals affected by metronidazole and radiation.     

Antiviral activity of interferon and its inducers in human lymphoblastoid and somatic cells]

The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells.     

Effect of canatoxin on cell cultures

Canatoxin, a toxic protein isolated from Canavalia ensiformis, was shown to inhibit DNA synthesis and to produce a cytolytic effect when added in vitro to various cells, in doses ranging from 50 to 500 nM. In this case no selectivity was found for a certain cell type, as both normal and transformed cells could be affected by the toxic protein. The cytostatic effect was irreversible upon removal of the toxic protein from the medium and could be fully attained after exposing the cells to canatoxin for only 30 min. The use of an in vitro cell culture system may allow for a better insight on the mode of action of canatoxin.     

4-Thiothymidine sensitization of DNA to UVA offers potential for a novel photochemotherapy

Photochemotherapy, in which ultraviolet radiation (UVR: 280-400 nm) or visible light is combined with a photosensitizing drug to produce a therapeutic effect that neither drug or radiation can achieve alone, is a proven therapeutic strategy for a number of non-malignant hyperproliferative skin conditions and various cancers. Examples are psoralen plus UVA (320-400 nm) radiation (PUVA) and photodynamic therapy (PDT). All existing photochemotherapies have drawbacks - for example the association of PUVA with the development of skin cancer, and pain that is often associated with PDT treatment of skin lesions. There is a clear need to develop alternative approaches that involve lower radiation doses and/or improved selectivity for target cells. In this review, we explore the possibility to address this need by exploiting thionucleoside-mediated DNA photosensitisation to low, non toxic doses of UVA radiation.     

Effect of melanin on radiation response of CHO cells

The effect of the presence of melanin on the response of mammalian cells to ionizing radiation was investigated in a model system utilizing the ability of Chinese hamster ovary cells to incorporate melanin by endocytosis. Cells were incubated in monolayer cultures from 2 to 20 hours with melanin prepared from 'beef eye' or synthesized by air oxidation of 3,4-dihydroxyphenylalanine. For asynchronous cultures, the survival curve parameters for cells incubated with both types of melanin were indistinguishable from those of the same cells without added melanin. The radiation response to fractionated doses of 6 Gy separated by various periods did not indicate any effect of melanin on the extent or kinetics of repair of sublethal damage. Likewise, the repair of potentially lethal damage in plateau phase cultures was unaffected by the presence of melanin. Thus the explanation for the clinical radiation resistance of melanomas in the absence of a direct radiation effect might more likely be found in consideration of other factors such as the role of melanin in oxygen consumption or in differentiation.     

Relative responses of an X-ray-resistant hybrid cell-line and its parent line to X-irradiation, ultraviolet light, actinomycin D and cordycepin.

Using colony formation as an assay, a rat-mouse hybrid cell-line (HD1) and one of its parent lines (H4) have been studied as to their abilities to survive exposure to ionizing radiation, ultraviolet light, and the drugs actinomycin D and cordycepin. HD1 cells are more resistant than H4 to ionizing radiation, actinomycin D and cordycepin. Both cell lines respond similarly to ultraviolet light. When both cell-lines were co-treated with actinomycin D or cordycepin, the toxic effect of ionizing radiation was enhanced, whereas that of ultraviolet light (U.V.L.) was unchanged. The data suggest that RNA synthesis is more important immediately after irradiation with X-rays than with U.V.L. and that cells resistant to the toxic effect of ionizing radiation are also resistant to the toxicity induced by inhibitors of RNA synthesis.     

Experimental and biochemical aspects of crystalline lens induction during embryogenesis]

The lens induction is a two-step process and involves morphogenetic influences from the archencepalic endoderm and optic vesicle. One can suggest that the lens induction is primed by specific proteins which are synthesized and secreted by the optic vesicle cells. The proteins-inductors appear to penetrate in the cells and, while interacting (directly or via the cytoplasm) with the nuclei, "programme" the ectodermal cells towards the lens differentiation. The contact interactions and extracellular matrix are of substantial, but not crucial value for the lens induction. The synthesis of specific proteins (crystallins) is to be considered as the most objective criterion of lens differentiation. In vertebrates, there is a lag-period between the moment of lens induction and synthesis of crystallins which is the most long-term in amphibians. The chick embryos constitute an exception and the synthesis of crystallin mRNA occurs in them a few hours after the lens induction. The developing retina loses its capacity to induce lens but stimulates the processes of fiber formation and synthesis of crystallins. A factor was found in the definitive lens epithelium which may be considered as a possible regulator of lens differentiation. On the basis of experiments with heterogenous and native lens inductors, a suggestion is put forward to the effect that the activity of inducing substances is determined by a definite determinant group of the molecule, rather than by the whole molecule.     

The effect of gamma-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes.

There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of gamma-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cells, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.     

RESULTS OF IRRADIATING SACCHAROMYCES WITH MONOCHROMATIC ULTRA-VIOLET LIGHT : II. THE INFLUENCE OF MODIFYING FACTORS

Possible variation in the probability that absorbed quanta of ultra-violet energy will produce observable inhibitory and lethal effects in the yeast cell, due to non-uniformity in sensitivity of the different regions of the cell, may be further modified by the reproductive stage of the cell at the time of irradiation. Tests of the survival of yeast cells of 15 day and 24 hour cultures indicate that the older resting cells are more resistant to ultra-violet irradiation effects than cells undergoing rapid cell division. The effects of temperature changes within the range of normal growth are evidently small, as judged from the temperature coefficient (1.10). Possible inhibitory effects due to the action of ultra-violet radiation on the malt agar medium and to toxic substances diffused from cells killed by irradiation were not found under the conditions of the experiments. Tests of the validity of the Bunsen-Roscoe reciprocity law for variation in the intensity of the incident ultra-violet radiation up to 30 per cent indicate that for this range the rate of absorption of quanta by the cell does not produce any marked change in the lethal effects observed.     

The role of spontaneous DNA damage in the radiosensitivity of thymus lymphocytes

The most radiosensitive thymus cortical lymphocytes are highly sensitive to inhibitors of repair of spontaneous DNA lesions known by endonucleases: this is indicative of different level of injury to cells. The preincubation of cells with chemical inductors of differentiation increases the number of spontaneous lesions in them. The preincubation of thymocytes with concanavalin A removes almost completely the differences in the level of injury.