The role of H3K4me3 and H3K9/14ac in the induction by dexamethasone of Per1 and Sgk1, two glucocorticoid [corrected] early response genes that mediate the effects of acute stress in mammals.

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties.

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response.

Circadian rhythms, which measure time on a scale of 24h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD(+)-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.

Ursolic acid triggers apoptosis and Bcl-2 downregulation in MCF-7 breast cancer cells.

In this report we determine the ability of ursolic acid (UA) to induce apoptosis and to modulate glucocorticoid receptor (GR) and Activator Protein-1 (AP-1) in MCF-7 cells. The UA-induced apoptosis (53 microM), the PARP cleavage, and the decrease in Bcl-2 protein (53 microM) support the notion that UA induces apoptosis through the intrinsic mitochondrial pathway. UA binds GR (relative binding affinity: 2.57) and translocates GR into nucleus, suggesting its potential as a GR modulator. UA had no effect on GRE- or TRE-driven gene expression. In summary, UA is a GR modulator and may be considered as a potential anticancer agent in breast cancer.

Effect of the histone deacetylase inhibitor trichostatin a in human peripheral blood lymphocytes as a function of donor age.

The histone deacetylase inhibitor trichostatin A (TSA) is a promising agent for the treatment of certain types of cancers alone or in synergistic combination with other anticancer agents. One of the advantages of the use of histone deacetylase inhibitors, such as TSA, is that its effects have been found to be more potent toward cancer cells compared to normal cells. The effect of anticancer agents on the immune system, and on lymphocytes in particular, is of major importance to the success of anticancer regimens. In this respect, information documenting the effect of such agents on normal lymphocytes compared to malignant cells may be of significant value for the successful designing of clinical protocols. Moreover, the parameter of age may be a factor in the differential effects of such protocols. Histone deacetylase inhibitors lead to the accumulation of acetylated histones and, depending on the cell type, may induce either apoptosis, cell cycle arrest, or differentiation. Previous work from our lab has shown that TSA induces the accumulation of histone H4 acetylation and apoptosis in human peripheral blood lymphocytes. In light of the above, we have extended our investigation of the effects of TSA on human lymphocytes to include the parameter of age, which has not been previously studied. Our results show that TSA induces apoptosis of lymphocytes from donors of all age groups, but no age-related changes in the levels of apoptosis are observed.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Synthesis, chemical, radiochemical and radiobiological evaluation of a new 99mTc-labelled bombesin-like peptide.

A new pentadecapeptide bombesin analogue was prepared by Fmoc synthesis, purified by HPLC and identified by electron ionization mass spectrometry. The biological activity of the new peptide was tested on isolated human colonic muscle cells and compared to native bombesin. Labelling of the new biomolecule with Tc-99m yielded a single radioactive species which remained stable at room temperature for eight hours. In a binding assay, the radiolabelled peptide showed high affinity for oat-cell carcinoma (Kd = 9.8 nM) and colorectal adenocarcinoma (Kd = 27.2 nM). Biodistribution studies, performed in normal rodents, indicated uptake by organs that normally express bombesin receptors, such as liver, intestines and kidneys. Scintigraphic studies, performed in nude mice transplanted with small cell lung carcinoma and colon cancer cells, showed significant tumor uptake two hours p.i. The new synthetic pentadecapeptide appears to have promise for several malignancies, including oat-cell lung carcinoma, colorectal cancer and gastroenteropancreatic (GEP) tumors.

Technetium labeled bombesin-like peptide: preliminary report on breast cancer uptake in patients.

Bombesin-like peptides are neurotransmitters and cancer growth factors. Several tumors, breast cancer among them, show one or more than one of the three known bombesin receptors. We have synthesized and labeled with technetium 99m a new pentadecapeptide, analogue to the leu13 amphibian bombesin (99mTc BN). Labeling yield was 83 +/- 4%. Prone Scintimammography was performed on five patients affected by breast cancers (T categorization: two T1b and three T1c), after injecting 0.7 mg, 185 to 296 MBq (5 to 8 mCi) of the peptide. Total body scan did not show free technetium biodistribution. No adverse reaction was observed. Prone Scintimammography with 99mTc Sestamibi (99mTc SM) was also performed few days later. 99mTc BN detected all 5 cancers, whereas 99mTc SM only four: all the T1c and one T1b cancer. Two of them showed axillary node invasion that was detected by both the radiotracers. A fibroadenoma present on contralateral breast to the one with cancer, was not detected neither by 99mTc SM nor by 99mTc BN. Tumor/breast normal tissue ratio (T/B) was constantly higher with 99mTc BN than with 99mTc SM. Maximal T/B was measured as 1.79 with 99mTc SM and 2.25 with 99mTc BN 5 min after fast i.v. administration. In conclusion our 99mTc BN is taken up by primary breast cancer showing higher T/B than 99mTc SM (p < 0.01). In our limited scale, 99mTc BN appears to be safe and, in our limited scale, even more accurate than 99mTc SM for detecting breast cancer.

mRNA levels of the differentiation-associated linker histone variant H1 zero in mitotically active and postmitotic senescent human diploid fibroblast cell populations.

The mRNA levels of the linker histone variant H1o, which is tightly associated with differentiation, have been studied in the present investigation in an in vitro model ageing human diploid fibroblast (HDF) cell system as a function of cumulative population doublings (CPDs) in mitotically active and senescent cell populations. According to our previous findings the synthesis rate of the H1o protein does not change as a function of CPDs as long as the cells are proliferating. However, when cells reach senescence, the synthesis rate of H1o increases in both naturally aged as well as in cell populations artificially aged by treatment with sodium butyrate. In the present investigation, it is shown that the H1o mRNA levels remain relatively constant in mitotic cells with a slight decrease in cell cultures of late CPDs, i.e. in populations which still retain a mitotic potential, but are toward the end of their proliferative lifespan. However, when cells senesce and are no longer capable of synthesizing DNA, the H1o mRNA levels increase in naturally aged cells while artificially aged cells still maintain mRNA levels comparable to those of mitotic cells.

Histone deacetylase inhibitors induce apoptosis in peripheral blood lymphocytes along with histone H4 acetylation and the expression of the linker histone variant, H1 degrees.

The results of this study show that H1 degrees can be induced by sodium butyrate and trichostatin A in peripheral blood lymphocytes, a cell system which does not normally express this linker histone variant. Moreover, this induced expression was found to be correlated in a dose-dependent manner with the concomitant induction of apoptosis and increased levels of histone H4 acetylation. Sodium butyrate and trichostatin A, both inhibitors of histone deacetylases, are known to induce terminal differentiation and at the same time the induction of the linker histone variant, H1 degrees, in a number of tissue/cell systems. Moreover, aside from induced expression by histone deacetylase inhibitors, H1 degrees gene expression has also been tightly associated with the process of terminal differentiation in many physiological tissue/cell systems. The concomitant induction of H1 degrees expression along with apoptosis and histone acetylation in the same cell system has not been previously reported. Histone acetylation is known to be involved in chromatin remodelling events. Such events also occur during apoptosis. The association of H1 degrees gene expression with apoptosis, and not with differentiation in these cells, leads to more general implications as to a potential functional role of H1 degrees during chromatin remodelling.

A morphological study of the effect of chlorambucil during the S and G2 phases of the cell cycle of synchronized HEp-2 cancer cell populations using computerized morphometry.

Chlorambucil, a bisalkylating agent, used extensively in the treatment of autoimmune and neoplastic diseases, is known to affect DNA synthesis. However recent studies have revealed that it also affects the synthesis of other nuclear protein constituents, especially histones. Since histones play a major role in both the structural and functional integrity of chromatin, we have analyzed the morphological effects of this agent, using low dose conditions and synchronized populations of HEp-2 cancer cells in the S and G2 phases of the cell cycle. Analyses at the light and electron microscopy levels were undertaken using synchronous image analysis techniques. Computerized morphometry was used so as to evaluate various nuclear and cytological morphological parameters. It was found that chlorambucil affects the organization of chromatin, as well as other cellular parameters in a manner characteristic of decreased tumor aggressiveness. A finding of significance in this study was that chlorambucil exerted its influence on all these morphological parameters only when treatment was initiated at the beginning of the S phase and not during the second half of the S phase or the G2 phase.

mRNA levels of the linker histone variant, H1o, in mitotically active human diploid fibroblasts as a function of the phases of the cell cycle and cumulative population doublings.

Senescence and differentiation have many similarities with respect to certain aspects of gene expression and cell cycle related events. One linker histone variant tightly associated with differentiation is the H1 variant, H1o. The work of this investigation has focused on the expression of H1o during the phases of the cell cycle and as a function of increasing cumulative population doublings (CPD) in an in vitro model ageing cell system, namely, human diploid fibroblasts. Increased H1o mRNA levels were found during the S phase of the cell cycle contrary to H1o protein relative synthesis rates, which were found to be increased during the Go phase of the cell cycle. These results were obtained in actively proliferating cell populations. However when the proliferative rate of the overall population begins to drop (CPD 50), H1o mRNA levels tend to remain stable throughout the Go, G1 and S phases. On the other hand, no changes in the H1o relative synthesis rates were found as a function of increasing CPD. Uncoupling of H1o protein and mRNA levels has been observed in numerous differentiating systems. The analogous mode in which H1o gene expression is regulated in both these two systems reinforces the opinion that senescence and differentiation may have similarities at the level of chromatin remodelling.

T-lymphocyte long-term cultures have a constant histone variant pattern during aging.

Human peripheral long-term T-lymphocyte cell cultures show some characteristics similar to those of fibroblast cell lines, the latter of which have been used as in vitro systems for cellular aging studies for many years. Both show a limited in vitro life span, as well as a progressive prolongation of their cell cycle with increasing age. However, whereas T-cell cultures die from apoptosis at the end of their proliferative capacity, fibroblasts can be maintained for long periods of time in stationary cultures as postmitotic senescent cells. Previous studies analyzing the histone variant pattern of a human lung embryonic fibroblast cell line have shown that this pattern changes as a function of cumulative population doublings in a manner not unlike that found in terminally differentiating systems. In the present study the histone variant composition of long-term T-cell cultures was analyzed as a function of population doublings and compared to a human diploid fibroblast system. The results from this study provide a distinction at the molecular level among these two in vitro aging model systems, because it was found that long-term T-cell cultures show a constant histone variant constitution throughout their in vitro life, dissimilar to previous findings using the fibroblast cell system.

Peripheral blood lymphocytes of bipolar affective patients have a histone synthetic profile indicative of an active cell state.

1. Although abnormalities of the immune system have been described in depression, no information exists regarding the biochemical parameters which could characterize the physiological state of lymphocytes from patients with bipolar affective disorder. 2. Lymphocytes of normal control subjects are known to be in the Go resting phase of the cell cycle. Histone synthesis is characteristically different during the Go, G1/G2 and the S phases of the cell cycle. As such, it can be used as a biochemical marker with which to distinguish between cycling and noncycling cells. 3. In order to investigate the possibility of whether or not the lymphocytes of patients with bipolar affective disorder are in an activated state, typical of cycling cells, total histone and histone variant synthesis were analysed in peripheral blood lymphocytes of a group of 12 patients with bipolar affective disorder and 7 normal controls. 4. According to the histone variant synthesis pattern, lymphocytes of patients in normothymia have values similar to those of controls, i.e., of noncycling cells, while patients in either the depressed or the manic phase have values intermediate to those of resting and cycling cells. 5. This study shows that histone synthesis can perhaps be used as a biochemical parameter of possible significance in differentiating amongst the three phases of the illness.

S and G2 phase histone biosynthesis of HEp-2 cells after the influence of the bisalkylating agent, chlorambucil.

Chlorambucil was previously found to be a specific inhibitor of total histone synthesis without affecting total cellular protein synthesis. In this study, we used S and G2 phase HEp-2 cancer cells to further analyze and specifically localize this effect. One hour (S phase), 4 hours (S phase) and 9 hours (G2 phase) after release from an aphidicolin double block synchronization procedure, cells were preincubated for 60 min with 30 microM chlorambucil and then radiolabeled for another 60 min in the continued presence of the agent. At the end of each of these time intervals, cells in almost mid-S phase, late S phase and toward the end of the G2 phase were obtained for analysis. It was found that chlorambucil partially inhibits total histone synthesis nonspecifically as to the variants being synthesized (S phase and basal variants) but only during the first half of the S phase. DNA synthesis is also inhibited partially, but during the second half of the S phase. The position during the S phase where chlorambucil exerts its effect on total histone synthesis, the degree of this effect and its uncoupling with DNA synthesis inhibition, indicate that it is temporally linked with the onset of S phase histone transcription and not with DNA synthesis initiation as occurs with agents, such as hydroxyurea.

Radiochemical and radioimmunological data of 99Tcm-anti-CEA labelled by two diverse methods.

The aim of this study was to make a comparative evaluation of a direct and an indirect method for the labelling of anti-CEA with technetium-99m (99Tcm). With the direct method, disulphide bridges were cleaved by the use of 2-mercaptoethanol as reductant, whereas with the indirect method, the antibody was coupled to 2-iminothiolane. In both cases, a preformed intermediate chelate was used for 99Tcm exchange. The radiochemical and radiobiological behaviour of the 99Tcm-labelled species were studied. Furthermore, the influence of the labelling systems on the integrity of monoclonal antibodies, as well as the ability of 99Tcm-anti-CEA to tag onto human cancer cells, was investigated for the two labelling systems. Both methods showed a high labelling yield and resulted in immunoreactive and stable derivatives. However, detailed electrophoretical and radiochemical data, as well as the cysteine challenge trial, indicated relatively greater stability for the 2-mercaptoethanol reduction procedure.

The effect of chlorambucil on the biosynthesis of the HMG and histone H1 chromosomal proteins of HEp-2 cells.

The effect of chlorambucil, a bisalkylating agent, on the biosynthesis of the 5% PCA extractable protein fraction of the cancer cell line, HEp-2, has been analyzed. It was found that the synthesis of all the high mobility group proteins as well as that of the H1 and H1o histone proteins are inhibited by this agent. HMG 14 and the H1, H1o proteins are inhibited to the same extent as that reported for the core histones of the same cell line [7], while slightly higher levels of inhibition were found for the HMG 1, 2 and 17 proteins. The proteins, P1 and HMG I exhibited the highest level of inhibition of the entire fraction. These findings extend previous findings regarding the histone proteins and may be correlated to a dysfunction in the normal process of chromatin condensation and a potential cytotoxic effect of this agent during the G2 phase.

Influence of chlorambucil, a bifunctional alkylating agent, on the histone variant biosynthesis of HEp-2 cells.

The effect of chlorambucil on the synthesis of histone variants of a cancer cell line HEp-2 is analysed and compared to that of nontreated and hydroxyurea treated cells. Cell proteins were labelled with [14C]lysine and [14C]arginine and histone variants resolved by one- or two-dimensional electrophoresis. Chlorambucil shows no significant decrease in total protein synthesis but shows a significant decrease in histone biosynthesis. It does not selectively inhibit the synthesis of the S-phase variants, i.e., H2A.1, H2A.2, H3.2 or the G1/G2 phase (basal) histone variants, i.e., H2A.Z, H2A.X and H3.3. On the contrary, hydroxyurea treated cells, which also show no significant decrease in amino acid incorporation into total cellular protein but do exhibit a significant inhibition of histone biosynthesis, show a selective inhibition of the synthesis of S-phase variants, but have no effect on the synthesis of basal histone variants. On the basis of histone variants being synthesized in the presence of chlorambucil, it is shown that although chlorambucil shows a specificity for histone synthesis inhibition it has a general action over the whole variant complement and is not coupled to S-phase synthesis in a way typical for DNA synthesis inhibiting drugs.