Calibration of CR-39-based thoron progeny device.

Radon isotopes and their progenies have proven significant role in respiratory tumour formation. In most cases, the radiological effect of one of the radon isotopes (thoron) and its progenies has been neglected together with its measurement technique; however, latest surveys proved that thoron's existence is expectable in flats and in workplace in Europe. Detectors based on different track detector measurement technologies have recently spread for measuring thoron progenies; however, the calibration is not yet completely elaborated. This study deals with the calibration of the track detector measurement method suitable for measuring thoron progenies using different devices with measurement techniques capable of measuring several progenies (Pylon AB5 and WLx, Sarad EQF 3220). The calibration factor values related to the thoron progeny monitors, the measurement uncertainty, reproducibility and other parameters were found using the calibration chamber. In the future, the effects of the different parameters (aerosol distribution, etc.) will be determined.

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts.

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF-CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIP(S+L), FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8.

Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), in the concentration range of 20 microM and above, induced arrest in the S-phase and apoptosis in the T cell-derived T-ALL lymphocytic leukemia cell line CEM-C7H2 which is deficient in functional p53 and p16. Expression of transgenic p16/INK4A, which causes arrest in G0/G1, markedly reduced the percentage of apoptotic cells. Antagonist antibodies to Fas or FasL, or constitutive expression of crmA did not diminish the extent of resveratrol-induced apoptosis. Furthermore, a caspase-8-negative, Fas-resistant Jurkat cell line was sensitive to resveratrol-induced apoptosis which could be strongly inhibited in the Jurkat as well as in the CEM cell line by z-VAD-fmk and z-IETD-fmk. The almost complete inhibition by z-IETD-fmk and the lack of inhibition by crmA suggested caspase-6 to be the essential initiator caspase. Western blots revealed the massive conversion of procaspase-6 to its active form, while caspase-3 and caspase-2 were proteolytically activated to a much lesser extent.

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts.

The histone deacetylase inhibitor and potential anti-cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF-CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline-regulated expression of transgenic p16/INK4A protected the cells from butyrate-induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide 'effector caspase' inhibitors benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Glu-Ile-Asp.fluoromethyl-ketone (VEID); however, the viral protein inhibitor of 'inducer caspases', crmA, had no effect. Bcl-2 overexpression partially protected stably transfected CCRF-CEM sublines from butyrate-induced apoptosis, but showed no effect on butyrate-induced growth inhibition, further distinguishing these two butyrate effects. c-myc, constitutively expressed in CCRF-CEM cells, was down-regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline-induced transgenic c-myc sensitized stably transfected CCRF-CEM derivatives to butyrate-induced cell death.

Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.

Butyrate, aspirin and colorectal cancer.

In vitro, for animal cells generally, butyrate at millimolar concentrations is an inhibitor of growth. In vivo, however, colonocytes are able to grow in the environment of about 20 mM butyrate produced by bacterial fermentation on the luminal side of the colonic epithelium. An in vivo increase of the butyrate supply results in growth stimulation of cells in the colonic crypts. This discrepancy, namely, that in cell cultures butyrate is an inhibitor of growth, whereas in vivo it has a trophic effect, is the so called in vivo paradox of butyrate. In the present review it is pointed out that butyrate is an inhibitor of histone deacetylases and there is sufficient evidence for hyperacetylation being the mechanism of the in vitro growth-inhibiting effect of butyrate. As within animal cells hyperacetylation has to occur at a certain butyrate concentration (1-10 mM), it is postulated that the in vivo lack of inhibition and 'paradoxical' stimulation of growth is a result of a low intracellular steady state concentration of butyrate in the lower layers of the crypt in spite of the much higher butyrate concentration on the luminal side. As butyrate is the preferential source of energy for colonocytes, the in vivo trophic effect is not paradoxical, when in spite of an increase of the butyrate concentration in stool, the intracellular butyrate concentration of intestinal epithelial cells still remains below the inhibiting level. For mature non-dividing colonocytes which are programmed for apoptosis, there is no difference between the observations made in vitro or in vivo. Furthermore, recent developments are discussed which suggest that cyclo-oxygenase-2 may play an essential role in colonic carcinogenesis. Cyclo-oxygenase-2 is found to be expressed in most colorectal carcinomas, but not in normal non-transformed intestinal epithelial cells (DeWitt and Smith, 1995). Cyclo-oxygenase-2 overexpression makes intestinal epithelial cells resistant to butyrate-induced apoptosis (Tsujii and DuBois, 1995). This escape from butyrate-induced apoptosis appears to be an essential prerequisite for the development of colorectal cancer and suggests a functional role of butyrate in growth, differentiation and programmed cell death of colonic epithelial cells.

In-vivo treatment with 5-azacytidine causes degeneration of central lymphatic organs and induces autoimmune disease in the chicken.

In-vitro evidence suggests that DNA methylation may be involved in the development of forbidden immune responses that can result in autoimmune disease. In the present study we examined in-vivo effects of 5-azacytidine (5-azaC), a substance that inhibits DNA methylation, on the immune system and the occurrence of a spontaneous autoimmune disease in the chicken model. We found that (1) treatment of young normal chickens with 1.0 mg/kg 5-azaC on 7 consecutive days caused a rapid degeneration of the central lymphoid organs thymus and bursa; (2) this regimen with 5-azaC apparently inhibited B cell maturation, as the frequency of cytoplasmic Ig+ plasma cells in the bone marrow was found to be significantly reduced, whereas the total number of bone marrow cells was unchanged; and (3) a chronic low-dose (0.5 and 1.0 mg/kg) application of 5-azaC through 6 weeks was found to significantly enhance the spontaneous autoimmune thyroiditis in newly hatched chickens of the Cornell C strain, as determined by anti-thyroglobulin autoantibody titres and histological analysis of thyroid gland infiltration. The possible implications of these data for the generation of pathogenic autoimmune responses are discussed.

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation.

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (10:0), lauric (12:0), myristic (14:0), oleic (cis-18:1) and elaidic (trans-18:1) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37 degrees, but also at 42 degrees and 47 degrees C lauric acid (12:0) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.

Increased acetylation of histones at an early stage of oestradiol-mediated gene activation in the liver of immature chicks.

The vitellogenin system of chicken was used to examine alterations in the microheterogeneity of chromosomal proteins in the course of steroid hormone-mediated gene expression. After administration of oestradiol-17 beta there is a dramatic increase in the number of copies of vitellogenin m-RNA in the liver of male oviparous animals, like Xenopus and chicken. According to earlier reports the rapid increase in transcriptional activity starts after a lag of 4 h. The system has also been examined as to the number of DNase I hypersensitive sites which appear to correlate with the degree of differentiation and hormonal activation. The time course of [3H]acetate incorporation into the histone fraction was monitored and the microheterogeneity of histones analysed by acid-Triton-urea gel electrophoresis. The results show that there is an increased degree of acetylation of histones in the liver of immature chicks as a result of oestradiol-17 beta administration. The change in the modification pattern of histones was found to be an early event, correlated with the time course of appearance of new DNase I hypersensitive sites and the onset of vitellogenin m-RNA synthesis. These observations are in agreement with an earlier report about the increase in the acetylation of histones in fetal guinea pig uterus after oestradiol treatment [1]. The results suggest that the acetylation of histones might be a prerequisite for the removal of structures for efficient gene repression and the establishment of DNase I hypersensitive sites.

Depression of histone acetylation by alkylating antitumor agents: significance for antitumor activity and possible biological consequences.

Treatment of Ehrlich ascites tumor cells with the alkylating antitumor agents triaziquonum, N-mustard and cyclophosphamide leads to a reduction in the posttranslational incorporation of 3H-acetate into histones and the extent of histone acetylation in Ehrlich ascites tumor cells. All core histones are affected. The depression of histone acetylation is not the result of a decrease in acetyl-CoA. Evidence is presented for an activation of histone deacetylase by alkylating agents. A reduction of histone deacetylation is observed after exposure to all concentrations of alkylating agents which inhibit cell proliferation. In order to evaluate the biological consequences of a reduction of histone acetylation, the extent of acetylation was modulated by either chemical acetylation or treatment with butyrate. In all cases an increase in histone acetylation leads to an enhancement of the rate of transcription. In accord with previous reports from our laboratory (1), it is concluded that the reduction of histone acetylation affects RNA synthesis. It is emphasized, however, that besides a regulation of transcription, histone acetylation may be involved in other cell functions. Thus, the complete biological consequences of the reduction of histone acetylation remain to be elucidated. In view of the antitumor activity of the alkylating agents it seems noteworthy that hepatoma AS30D cells are characterized by a remarkably higher extent of histone H4-acetylation compared to normal, adult, fetal, or regenerating liver.

Conservation of the acetylation pattern of histones and the transcriptional activity in Ehrlich ascites tumor cells by sodium butyrate.

Histone deacetylases of Ehrlich ascites tumor cells are active at low temperatures (0-4 degrees C). The so-called hyperacetylated state of histones is the physiological state of histones in intact Ehrlich ascites tumor cells which is conserved by the continuous presence of 10 mM sodium butyrate during the preparation of nuclei and histones. Isolation of histones in the absence of butyrate causes an artificial decrease in histone acetylation. This artificial loss of histone acetylation produces a decrease of the elongation reaction in the RNA synthesis. The initiation of RNA synthesis is not affected.