The effect of irradiation and alkylating agents on chromatin breakdown in normal and malignant lymphoid cells

Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chromatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/BL cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes.     

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.     

Pre-TCR signaling and CD8 gene bivalent chromatin resolution during thymocyte development

The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.     

Surface immunoglobulin of mouse thymus cells and its in vitro biosynthesis

Surface immunoglobulin (Ig) was demonstrated on thymocytes from BALB/c and CS7BL mice by lactoperoxidase radioiodination of the cells. Active synthesis of Ig in these cells was demonstrated in short-term tissue culture using ¹⁴C-labeled amino acids. The demonstration of intracellular and surface Ig required procedures that minimize proteolytic degradation.Monomeric α chains and light chains were found in the cytoplasm and on the surface of BALB/c thymocytes, whereas monomeric μ chains and light chains in the cytoplasm and on the surface of CS7BL thymocytes. Cytotoxic tests with an alloantiserum revealed that in the thymus of BALB/c mice the IgA monomeric subunits are synthesized by the θ⁺ cells.     

Effect of the cytoplasm of irradiated cells on chromatin degradation in mouse thymocytes and hepatocytes

The addition of a cytoplasmic fraction, isolated from cells 3h after irradiation of mice, to exposed or intact thymocyte nuclei causes a 2- or 3-fold acceleration of chromatin degradation in the nuclei incubated in conditions optimum for activity of Ca2+,Mg2+-dependent endonuclease to be manifest. In contrast to thymocytes, no chromatin degradation products are found in liver cells of irradiated mice. The cytoplasmic fraction isolated from hepatocytes of irradiated animals fails to activate chromatin degradation in thymocyte nuclei.     

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.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

Effect of cycloheximide on the morphological and biochemical changes in chromatin in non-irradiated and irradiated rat thymocytes

The level of chromatin degradation was studied and the method of electron-microscopy was used to estimate the changes in the ultrastructure of irradiated and nonirradiated thymocytes of rats treated with cycloheximide. The latter was found to decrease the degree of derangement of nuclear ultrastructure and the level of chromatin degradation in exposed animals and to increase the yield of these damages in thymocytes of nonirradiated animals. The electronmicroscopic determinations showed that the percentage of thymocytes with the impaired nucleus structure is twice as high as that of degraded chromatin. The causes of the quantitative disagreement between the morphological and biochemical indices of the interphase thymocyte death are discussed.     

Endonucleolysis of chromatin in thymocytes of irradiated and non-irradiated rats

It was shown that in conditions optimal for Ca/Mg endonuclease, chromatin endonucleolysis in the nuclei and thymocytes occurs due to internucleosome fragmentation of DNA. Irradiation activates chromatin degradation in thymocytes washed by a buffer containing 0.25 M sucrose, 10 mM tris-HCl, pH 7.2, 3 mM MgCl2, and does not influence this process in thymocytes washed by 10 mM tris-HCl, pH 7.2, 3 mM MgCl2.     

Structural organization of chromatin as a factor determining the rate of its endonucleolysis in irradiated and nonirradiated thymocytes

A study was made of chromatin endonucleolysis in hypotonic thymocytes incubated in digestive buffers containing different concentrations of potassium, magnesium, calcium, and mercaptoethanol. Inhibition of endonucleolysis by univalent cation during the first 20 min of incubation was followed by intensive chromatin degradation. A decrease in free potassium content retarded chromatin degradation and enhanced the inhibiting effect of the univalent cations. The regularities of changes in the rate of chromatin endonucleolysis in different digestive buffers were similar with both exposed and intact thymocytes.     

Degree of chromatin fragmentation and frequency of nuclear pyknosis in Percoll-fractionated thymocytes of irradiated rats

The relationship between nuclear chromatin degradation to polydeoxyribonucleotides (PDN) and other features of interphase death were studied using thymocytes of normal and X-irradiated rats. Fractionation of the thymic cells in Percoll gradients was performed in order to separate dead from intact cells. The degree of radiation-induced chromatin fragmentation, as assessed by electrophoresis, was similar for PDN from all Percoll bands. Following irradiation 87-98 per cent of 'heavy' thymocytes were pyknotic and almost devoid of receptors to autologous erythrocytes thus comprising a dead cell population. A direct relationship between PDN content and nuclear pyknosis was noted throughout the gradient. The loss of autologous rosette-forming ability was directly related to other indices of interphase death. The possibility of PDN originating from pyknosis-prone cells and the capacity of radiosensitive thymocytes to form autologous rosettes are discussed.     

The radiosensitivity of lymphosarcoma cells as determined by the liver colony method

The liver colony method is based on the observation that intravenous injection of an appropriate number of LS/BL cells into isologous non-irradiated hosts leads to the formation of colonies of proliferating cells in the livers of these animals. The relationship between the number of cells injected and the number of colonies appearing in the livers was determined. This technique was used to measure the radiation sensitivity of LS/BL cells and it yielded a Do of 1.05 Gy. The results show that LS/BL cells have a similar radiation sensitivity as other mammalian cells. The liver colony assay used to determine the radiation sensitivity of the lymphosarcoma LS/BL cells can also be used whenever the number of viable tumour cells in a suspension is to be estimated.     

Participation of the NAD-poly(ADP ribose) system in the degradation of chromatin in irradiated thymocytes

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.     

On the role of Ca, Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues

The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.     

Biosynthesis of chromatin proteins in the thymocytes of irradiated rats

Biosynthesis of chromatin proteins in thymus cells of irradiated rats has been studied. During the period immediately preceding DNA degradation, new proteins, which are not found in the chromatin of the control cells, are synthesized in the exposed thymocytes and enter the chromatin. Besides, the rate of biosynthesis of a large number of other chromatin proteins decreases. The data obtained indicate that interphase death of lymphoid cells is an active, genetically programmed process.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

Reasons for the quantitative differences in the formation of chromatin breakdown products following exposure to neutrons and gamma radiation

Relative content of salt-soluble chromatin fragments in rat thymus increased after the effect of neutrons, as compared to gamma-radiation, while cell depletion of the organ was similar with both types of radiation. This was not associated with the rate and extent of chromatin degradation in thymocytes and was an indication of death of a larger number of thymocytes after the effect of neutrons.     

Thymidine excretion from thymocytes in irradiated and nonirradiated rats during endonucleolysis

The concentration of free thymidine (dT), excreted from thymocytes of irradiated and nonirradiated rats, was determined after incubation of cells in various digestive buffers. The release of dT from thymocytes depended upon the rate of DNA fragmentation in conditions of chromatin endonucleolysis. The increase in the thymidine content, in conditions of chromatin endonucleolysis in buffers containing no calcium ions, was only noted in thymocytes of exposed rats: this was the consequence of chromatin DNA damages already available in these cells.     

Interphase death of thymus cells in combined radiation-thermal injury against a background of prophylactic administration of alpha-tocopherol and indomethacin

The administration of alpha-tocopherol prior to radiation and thermal affection enhances chromatin degradation in thymocytes, increases the number of pyknomorphic cells in the critical zone of thymus, and enhances injury to erythrocyte membranes. The preventive administration of indomethacin does not influence the interphase cell death.