Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation.

Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.

[Chromatin degradation in the death of thymic lymphocytes induced by radiation or dexamethasone: the necessity for a stage of preliminary proteolysis]. / Degradatsiia khromatina pri gibeli limfotsitov timusa, indutsirovannoi radiatsiei ili deksametazonom: neobkhodimost' étapa predvaritel'nogo proteoliza.

Proteolytic activity in a protein fraction of a rat thymocyte nuclear matrix was found to increase 1-2 h after gamma-irradiation or administration of dexamethazone. Cycloheximide did not prevent the observed protease activation. Neither histons nor thymocyte nuclear matrix proteins were subjected to proteolysis after exposure to radiation or the hormone. Such proteolysis inhibitors as phenylmethylsulfonyl fluorine, trasilol, and partly leupeptine inhibited nuclear DNA degradation in irradiated and dexamethazone treated thymus lymphocytes. In all appearance, this effect was not due to Ca/Mg-dependent endonuclease inactivation. The same was observed in the system of autolytic chromatin degradation in isolated thymocyte nuclei.

Is the NAD-poly (ADP-ribose) polymerase system the trigger in radiation-induced death of mouse thymocytes?

Agents that induce DNA strand breaks evoke a drop in the NAD content in mouse thymocytes. A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymocytes is entirely attributed to the activation of poly(ADP-ribosylation). The addition of 5 mM benzamide before irradiation prevents the postirradiation drop of the NAD level but has no effect on chromatin degradation and cell death. In contrast to liver nuclei, pre-incubation of mouse thymic nuclei with NAD had no effect on the subsequent chromatin endonucleolysis by Ca2+/Mg2+-dependent endonuclease. It is suggested that the NAD-poly(ADP-ribose) polymerase system is probably not the trigger in the radiation-induced programmed death of mouse thymocytes, but may merely be indicative of the radiation response of these cells.

Early postirradiation chromatin degradation in thymocytes.

The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.

[Spontaneous DNA damage and DNA polynucleotide ligase activity in thymocyte populations differing in radiosensitivity]. / Spontannye povrezhdeniia DNK i aktivnost' DNK-polinukleotidligaz v populiatsiiakh timotsitov, razlichaiushchikhsia po radiochuvstvitel'nosti.

Single-strand breaks (SB) in DNA of irradiated thymocytes are first repaired, at a half-recovery period of a few minutes, then the number of breaks rapidly increases indicating that in addition to chromatin endonucleolysis there is an imbalance between the repair enzymes that generate DNA breaks and eliminate them. The rate of SB accumulation in thymocyte fractions, upon incubation of the cells with 1-beta-D-arabinofuranosyl cytosine (AraC) and hydroxyurea (HU), is directly proportional to their radiosensitivity, whereas DNA ligase activity is higher in a fraction of highly radioresistant thymocytes. Chemical compounds that increase the survival rate of irradiated thymocytes decelerate SB accumulation in DNA and sensitivity of cells to AraC and HU. Ineffective substances have no such an effect.

[Participation of the NAD-poly(ADP ribose) system in the degradation of chromatin in irradiated thymocytes]. / K voprosu ob uchastii sistemy NAD-poli(ADF-riboza) v degradatsii khromatina v obluchennykh timotsitakh.

The diminution of NAD level in mouse thymus lymphocytes precedes their death under the effect of various genotoxic agents and manifests itself by the time of the onset of chromatin degradation. At the same time, in vitro, NAD does not influence the activity of micrococcus nuclease of Ca2+,Mg2+-dependent endonuclease from human spleen. Stimulation of protein poly(ADP-ribosylation) by exogenous NAD does not change the sensitivity of chromatin to micrococcus nuclease. In contrast to hepatocytes, in the thymus, no inhibition of Ca2+,Mg2+-endonuclease, resulting from ADP-ribosylation, occurs which may be due to low activity of ADP-ribosyl transferase in thymocytes. Incubation of thymus lymphocytes with benzamide prior to irradiation does not inhibit chromatin degradation. It is suggested that the decrease in the NAD level is one of the indications of the injury to thymocytes which is not related to the induction of their death. In contrast to thymocytes, the pretreatment of Ehrlich ascites tumor cells with benzamide produces a radiosensitizing effect.

[Poly(ADP-ribosylation) of proteins determines the pool of endogenous NAD and the radiosensitivity of thymus lymphocytes]. / Poli(ADF-ribozilirovanie) belkov opredeliaet fond éndogennogo NAD i radiochuvstvitel'nost' limfotsitov timusa.

A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymus lymphocytes is attributed to activation of poly (ADP-ribosylation) and not to changes in the activity of NAD-glycohydrolase and/or to the release of NAD from cells. The addition of benzamide 60 min before irradiation prevents the postirradiation drop of the NAD level and produces a radioprotective effect. At the same time, benzamide inhibits nuclear superhelix DNA repair and causes an average of 40 per cent decrease in the activity of DNA ligases I and II. The authors discuss the idea that the content of intracellular NAD in thymocytes is a critical factor responsible for the vitality of these cells.

[Role of protein poly(ADP-ribosylation) in the radiosensitivity of thymus lymphocytes]. / Rol' poli(ADF-ribozilirovania) belkov v radiochuvstvitel'nosti limfotsitov timusa.

The inhibitors of poly(ADP-ribose) polymerase did not exert a radiomodifying effect on thymocytes. The inhibitors did not also influence single-strand breaks repair in DNA of nucleoids of irradiated cells. The participation of poly(ADP-ribosylation) system in increasing the availability of thymocyte DNA damages for repair enzymes was hardly probable since benzamide in these cells did not influence the level of spontaneous genome lesions recognized by endonucleases. A possible role of protein poly(ADP-ribosylation) in thymocyte DNA repair is discussed.

[Autolytic degradation of RNA; the effect of various factors on the dynamics of the process]. / Avtoliticheskaia degradatsiia RNK: vliianie razlichnykh faktorov na dinamiku protsessa.

As found earlier RNA preparations from different eukaryotic tissues followed by thorough deproteinization contain "masked" RNases, that cause autolytic degradation. The present paper indicates that the autolytic degradation of a deproteinized preparation of RNA from Ehrlich's cells by gel electrophoresis under denaturated conditions and ultracentrifugation in sucrose gradient is submitted by definite regularities and is essentially dependent on the concentration change of Mg2+, Na+, K+, NH4+ ions and also GTP and guanosine. It is proposed the activity of "masked" RNases is the manifestation of ribozyme properties of RNA molecules. Similar processes may take place in processing and catabolism of RNA in vivo.