RESUMO
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.
Assuntos
Dexametasona/farmacologia , Histonas/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Circadianas Period/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Linhagem Celular Tumoral , Genes Precoces , Camundongos , Células NIH 3T3 , Niacinamida/farmacologia , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Elementos de Resposta/fisiologia , Estresse Fisiológico , Ativação TranscricionalRESUMO
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.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Oxirredutases N-Desmetilantes , Proteínas Circadianas Period , Animais , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Metilação , Camundongos , Niacinamida/metabolismo , Niacinamida/farmacologia , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Regiões Promotoras Genéticas , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Triterpenos/farmacologia , Transporte Ativo do Núcleo Celular , Adenocarcinoma/genética , Ligação Competitiva , Neoplasias da Mama/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Células HeLa/patologia , Humanos , Mifepristona/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores de Glucocorticoides/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Triancinolona/farmacologia , Ácido UrsólicoRESUMO
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.
Assuntos
Envelhecimento/imunologia , Apoptose/efeitos dos fármacos , Doadores de Sangue , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Linfócitos/imunologia , Acetilação/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Antineoplásicos/agonistas , Antineoplásicos/farmacologia , Apoptose/imunologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Sinergismo Farmacológico , Inibidores Enzimáticos/agonistas , Inibidores Enzimáticos/farmacologia , Feminino , Histona Desacetilases/imunologia , Histona Desacetilases/metabolismo , Histonas/imunologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/agonistas , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/imunologiaRESUMO
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.
Assuntos
Clorambucila/farmacologia , Histonas/biossíntese , Autorradiografia , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Humanos , Hidroxiureia/farmacologia , Cinética , Biossíntese de Proteínas , Células Tumorais CultivadasRESUMO
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.
Assuntos
Apoptose/fisiologia , Inibidores de Histona Desacetilases , Histonas/metabolismo , Linfócitos/citologia , Linfócitos/enzimologia , Acetilação , Apoptose/efeitos dos fármacos , Northern Blotting , Western Blotting , Butiratos/farmacologia , Cromatina/enzimologia , Inibidores Enzimáticos/farmacologia , Histonas/análise , Histonas/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , RNA Mensageiro/análiseRESUMO
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.
Assuntos
Diploide , Fibroblastos/citologia , Fibroblastos/fisiologia , Variação Genética , Histonas/genética , Mitose/fisiologia , Mutagênese Insercional , RNA Mensageiro/metabolismo , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Humanos , Timidina/metabolismoRESUMO
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.
Assuntos
Diploide , Fibroblastos/fisiologia , Histonas/genética , Mitose , RNA Mensageiro/metabolismo , Butiratos/farmacologia , Diferenciação Celular/fisiologia , Linhagem Celular , Senescência Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , HumanosRESUMO
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.
Assuntos
Envelhecimento/imunologia , Histonas/sangue , Linfócitos T/química , Adulto , Idoso , Células Cultivadas , HumanosRESUMO
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.
Assuntos
Envelhecimento , Pesquisa/organização & administração , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Caenorhabditis elegans , Dano ao DNA , Grécia , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Presenilina-1RESUMO
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.
Assuntos
Transtorno Bipolar/imunologia , Histonas/biossíntese , Ativação Linfocitária , Linfócitos/imunologia , Adulto , Idoso , Análise de Variância , Biomarcadores/sangue , Transtorno Bipolar/sangue , Ciclo Celular , Eletroforese em Gel Bidimensional , Feminino , Fase G1 , Fase G2 , Histonas/sangue , Histonas/isolamento & purificação , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Pessoa de Meia-Idade , Valores de Referência , Fase de Repouso do Ciclo Celular , Fase SRESUMO
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.
Assuntos
Bombesina/farmacocinética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Peptídeos/farmacocinética , Pertecnetato Tc 99m de Sódio/farmacocinética , Idoso , Neoplasias da Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/diagnóstico por imagem , Feminino , Humanos , Linfonodos/metabolismo , Metástase Linfática , Pessoa de Meia-Idade , Cintilografia , Tecnécio Tc 99m Sestamibi/farmacocinéticaRESUMO
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.
Assuntos
Bombesina , Carcinoma de Células Pequenas/diagnóstico por imagem , Neoplasias do Colo/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Peptídeos , Pertecnetato Tc 99m de Sódio , Animais , Bombesina/síntese química , Bombesina/farmacocinética , Carcinoma de Células Pequenas/metabolismo , Neoplasias do Colo/metabolismo , Feminino , Humanos , Marcação por Isótopo , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Peptídeos/síntese química , Peptídeos/farmacocinética , Cintilografia , Ratos , Ratos Wistar , Receptores da Bombesina/metabolismo , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Clorambucila/farmacologia , Fase S/efeitos dos fármacos , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Cromatina/química , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Citometria por Imagem , Microscopia Eletrônica , Células Tumorais CultivadasRESUMO
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.
Assuntos
Anticorpos Monoclonais , Antígeno Carcinoembrionário/imunologia , Tecnécio , Animais , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Indicadores e Reagentes , Marcação por Isótopo/métodos , Masculino , Camundongos , Radioimunoensaio/métodos , Distribuição TecidualAssuntos
Senescência Celular/fisiologia , Fibroblastos/metabolismo , Histonas/biossíntese , Ciclo Celular , Células Cultivadas , Diploide , Fibroblastos/citologia , Histonas/genética , Histonas/isolamento & purificação , Humanos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificaçãoRESUMO
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.
Assuntos
Carcinoma/metabolismo , Clorambucila/farmacologia , Fase G2/efeitos dos fármacos , Histonas/biossíntese , Fase S/efeitos dos fármacos , Antineoplásicos Alquilantes/farmacologia , Afidicolina/farmacologia , Carcinoma/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , DNA de Neoplasias/efeitos dos fármacos , Eletroforese/métodos , Histonas/efeitos dos fármacos , Humanos , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
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.