RESUMO
ß decay of proton-rich nuclei plays an important role in exploring isospin mixing. The ß decay of ^{26}P at the proton drip line is studied using double-sided silicon strip detectors operating in conjunction with high-purity germanium detectors. The T=2 isobaric analog state (IAS) at 13 055 keV and two new high-lying states at 13 380 and 11 912 keV in ^{26}Si are unambiguously identified through ß-delayed two-proton emission (ß2p). Angular correlations of two protons emitted from ^{26}Si excited states populated by ^{26}P ß decay are measured, which suggests that the two protons are emitted mainly sequentially. We report the first observation of a strongly isospin-mixed doublet that deexcites mainly via two-proton decay. The isospin mixing matrix element between the ^{26}Si IAS and the nearby 13 380-keV state is determined to be 130(21) keV, and this result represents the strongest mixing, highest excitation energy, and largest level spacing of a doublet ever observed in ß-decay experiments.
RESUMO
OBJECTIVE: To investigate the therapeutic effect of gene silencing peptidyl arginine deaminase 4 (PAD4) on pulmonary interstitial lesions induced by collagen-induced arthritis (CIA) mice, and possible mechanisms. METHODS: A CIA mouse model was established in DBA/1 mice, followed by a tail vein injection of the virus solution prepared by the PAD4-siRNA expression vector once a week for 8 times. The mice were sacrificed at the end of the experiment. The expression of PAD4 mRNA in lungs was detected by real-time quantitative PCR (qRT-PCR). The expression of PAD4 protein was detected by tissue immunohistochemistry. Cell culture was performed by spleen tissue. Flow cytometry changes in the ratio of Tfh cells to Tfr cells were examined; lung staining was performed in the lungs to observe changes in lung pathology. RESULTS: (1) Compared with the blank group, the expression of PAD4 mRNA in the lung tissue of the model group increased, the difference was statistically significant (P < 0.05). PAD4 mRNA in the lung tissue of the CIA mice after PAD4-siRNA treatment. The expression level was significantly lower than that of the model group and the negative control group, and the difference was statistically significant (P < 0.05). (2) Red fluorescence was less in the lung tissue of the blank group, while more red fluorescence was observed in the inflammatory cell infiltration area and trachea around the lung tissue of the model group and the negative control group, and the red fluorescence of the three groups after PAD4-siRNA treatment was significantly reduced; (3) Compared with the blank group, the proportion of Tfh cells in the model group increased, the difference was statistically significant (P < 0.05), the proportion of Tfh cells in spleen cells of the CIA mice after PAD4-siRNA treatment was significantly lower than that of the model group and the negative control group, the difference was statistically significant (P < 0.05); compared with the blank group, in the mouse spleen cells in the model group the proportion of Tfr cells was slightly decreased, but the difference was not statistically signifi-cant. The proportion of Tfr cells in the spleen cells of the mice increased after PAD4-siRNA treatment, but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group (P < 0.05); (4) The proportion of Tfh/Tfr in the spleen cells of the model group was increased, compared with the blank group, the difference was statistically significant (P < 0.05); the ratio of Tfh/Tfr in the three groups after PAD4-siRNA treatment all decreased, the difference was statistically significant (P < 0.05); (5) Compared with the blank group, the alveolar wall of the lung tissue of the model group was thickened, the inflammatory cell infiltration was increased, and the lung tissue destruction and inflammatory infiltration of the CIA mice were decreased after PAD4-siRNA treatment. The degree of reduction was reduced. CONCLUSION: Gene silencing of PAD4 can reduce the proportion of Tfh cells, increase the proportion of Tfr cells, reverse the proportion of Tfh/Tfr, and reduce the degree of interstitial lesions and inflammatory infiltration of lung tissue.
Assuntos
Artrite Experimental , Animais , Arginina , Artrite Experimental/genética , Artrite Experimental/terapia , Inativação Gênica , Pulmão , Camundongos , Camundongos Endogâmicos DBARESUMO
OBJECTIVE: To evaluate the prognostic significance of serum GGT in patients with hepatitis C virus-related hepatocellular carcinoma (HCV-HCC) treated with transcatheeter arterial chemoembolization (TACE). METHODS: The clinicopathological data of 110 patients with hepatitis C virus-related stage B hepatocellular carcinoma, who received TACE treatment from January 2008 to May 2011, were retrospectively analyzed. The patients were divided into two groups: the normal GCT group (GGT<50 U/L, 41 cases) and high GCT group (GGT≥50 U/L, 69 cases). The Kaplan-Meier method was used to analyze the survival rates, log-rank test was used for univariate analysis, and Cox regression model was used for multivariate analysis. The factors affecting survival and prognosis of the patients were analyzed. RESULTS: The pretreatment GGT level was (160.0±120.2) U/L in the high GGT group and (40.1±8.5) U/L in the normal GGT group (P<0.001). After TACE treatment, the 1-, 2- and 3-year survival rates were 90.2%, 45.9% and 24.6% in the high GGT group, and 90.2%, 75.6% and 58.5%, respectively, in the normal GGT group (P=0.002). The univariate analysis showed that the pretreatment GGT level, ECOG score, α-fetoprotein, tumor size, tumor number, and Child grade are factors affecting the prognosis of HCV-related hepatocellular carcinoma patients (P<0.05 for all). The Cox multivariate survival analysis revealed that the tumor size, tumor number, Child grade, and serum GGT level are independent prognostic factors for patients with stage B HCV-related hepatocellular carcinoma. CONCLUSION: The level of serum GGT before TACE is an independent prognostic factor for patients with stage B HCV-related hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/terapia , gama-Glutamiltransferase/sangue , Análise de Variância , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/virologia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Carga Tumoral , alfa-Fetoproteínas/análiseRESUMO
OBJECTIVE: To construct sphingosine 1-phosphate receptor-1 (S1P1)-small interfering RNA (siRNA) lentiviral vectors and infect human salivary gland cells (HSG), and to investigate its possible therapy on Sjogren's syndrome. METHODS: HSG cells were divided into blank group, empty vector group, scramble-siRNA group and S1P1-siRNA group. The lentiviral vectors expressing siRNA against S1P1 and the pLL3.7 were respectively transfected into 293T cells with pMD2.G, pMDL g/p RRE, pRSV-REV to produce virus, and then infect HSG cells. The efficiency was observed by flow cytometry after the transfection for 48 h. The expression levels of S1P1 mRNA of HSG were detected by real-time RT-PCR and the expression of S1P1 protein was detected by immunohistochemistry method. The expression levels of interferon-γ (IFN-γ) and interleukin (IL)-17 in the supernatant of the cells were detected by ELISA method. RESULTS: (1) The scramble-siRNA, S1P1-siRNA lentiviral vector was successfully constructed, and the lentivirus titer was about 3.5×108 TU/mL. (2) The level of S1P1 mRNA was lower in S1P1-siRNA group than those in the blank group, empty vector group, and scramble-siRNA group 48 h after infection, there were significant differences between them (P<0.05). (3) The expression of S1P1 protein was lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (4) The levels of IL-17 were lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (5) The levels of IFN-γ in S1P1-siRNA group were lower than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). CONCLUSION: The lentiviral vector targeting S1P1 was successfully constructed. S1P1 siRNA could suppress the levels of S1P1 mRNA and protein, and decrease the expression of IL-17 and IFN-γ. S1P1 siRNA could infect HSG cells stably and inhibit the expression of S1P1 gene specifically and efficiently, and reduce the levels of inflammatory cytokines.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/farmacologia , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/farmacologia , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/fisiologia , Transfecção/métodos , Citocinas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Humanos , Técnicas In Vitro/métodos , Interferon gama/efeitos dos fármacos , Interferon gama/genética , Interleucina-17/genética , Lentivirus , RNA Mensageiro , Glândulas Salivares/efeitos dos fármacosRESUMO
Phorbol-12-myristate-13-acetate (PMA) treatment induces erythroblastoma D2 cells kept in suspension to undergo RhoA-dependent contraction and to become proapoptotic, while attached cells are induced to differentiate accompanied by the reduction of RhoA activity. In this study, we found that guanine exchange factor H1 (GEF-H1) is highly expressed in D2 cells. Depletion of GEF-H1 expression in D2 cells decreased RhoA activity and prevented PMA-induced contraction and apoptosis. Upon PMA stimulation, GEF-H1 became associated with microtubules in cells that were induced to differentiate. As a contrast, in the proapoptotic population of cells GEF-H1 stayed in the cytoplasm without showing PMA-responsive microtubule translocation. Given that GEF-H1 is inactivated when associated with microtubules and its release into cytosol due to depolymerization of microtubules activates RhoA, our results demonstrated that nonmicrotubule-associated GEF-H1 in D2 cells contributes to the sustained activation of RhoA/ROCK signaling in suspension cells, making cells susceptible to PMA-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microtúbulos/fisiologia , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We have used the sequence of the cell-cycle regulatory region of human thymidine kinase promoter to study the DNA-protein interaction in human tumor and normal cells. By performing band-shift assays and DNase I footprint analysis, we have demonstrated that human tumor cells exhibited an elevated level of binding activity to the distal CCAAT box of human thymidine kinase promoter. The expression of human thymidine kinase CCAAT-binding activity was serum or independent in human tumor cells but serum dependent in normal human diploid fibroblasts. Our results present the fact that the constitutive interaction of CCAAT-binding factor with the promoter of a cell growth-controlled gene, such as thymidine kinase, is consistent with the loss of stringent cell growth regulation associated with tumorigenic phenotype.
Assuntos
Ciclo Celular/genética , DNA/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/fisiologia , Timidina Quinase/genética , Sequência de Bases , Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Timidina Quinase/metabolismo , Células Tumorais CultivadasRESUMO
Expression of a mRNA cross-hybridized to human cytomegalovirus immediate-early gene promoter-enhancer was detected in the human promyelocytic leukemia cell line HL-60. The 0.6 kilobase of NruI/SacI DNA fragment of eukaryotic expression vector pCDM8 representing human cytomegalovirus immediate-early gene promoter-enhancer was used as the probe to hybridize with polyadenylated RNA by the Northern blot analysis. A 3.7-kilobase strand of polyadenylated RNA was visualized in the cytoplasmic fraction of HL-60 promyelocytes. In contrast, other human hematopoietic cell lines, hepatoma cells, and normal human fibroblasts did not show such a transcript by cross-hybridization. This transcript was called CMVE RNA. The expression of CMVE mRNA was also detectable in the fresh blast cells from patients with acute myeloid leukemia, and particularly from a patient with acute myeloid leukemia of the M3 type. Taken together, these findings suggest that the CMVE RNA-encoded gene plays an important role in the pathogenesis of acute promyelocytic leukemia.
Assuntos
Citomegalovirus/genética , Elementos Facilitadores Genéticos/genética , Leucemia Mieloide/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Doença Aguda , Northern Blotting , DNA de Neoplasias/análise , Humanos , Hibridização de Ácido Nucleico , Sondas RNA , RNA Mensageiro/ultraestrutura , RNA Neoplásico/ultraestrutura , Células Tumorais CultivadasRESUMO
Translational control is one of the mechanisms that regulate thymidine kinase (TK) expression in the cell cycle. Evidence for the TK mRNA sequence that is involved in its own translation has been lacking. In this report, we show that TK-deficient mouse fibroblasts transfected with pFLAG-TK express a TK mRNA containing the 5'-untranslated region (5'UTR) and produce two polypeptides, FLAG-TK and TK, resulting from an alternative initiation of translation. Most interestingly, the 5'UTR of TK allowed the translation of FLAG-TK mRNA to become cap-independent in an in vitro translation system. Furthermore, this 5'UTR sequence decreased significantly the efficiency of translation from the AUG codon of FLAG when the concentration of FLAG-TK RNA was low. Here, we also show that in normal human IMR-90 fibroblasts the induction of TK polypeptide by serum stimulation is insensitive to rapamycin treatment, which is known to inhibit the translations of transcripts of some growth-controlled genes by affecting the cap-binding efficiency. Taken together, we propose that the 5'UTR in TK mRNA might actually confer a secondary structure to regulate ribosome binding during translation in a cap-independent manner.
Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas/genética , Capuzes de RNA , RNA Mensageiro/genética , Timidina Quinase/genética , Sequência de Bases , Primers do DNA , Humanos , PlasmídeosRESUMO
Aging of IMR-90 human diploid fibroblasts in culture is accompanied by specific changes of polyamine metabolism including: (a) a fivefold decrease of serum-induced activity of ornithine decarboxylase (ODC1 EC 4.1.1.17); (b) a six to tenfold increase of polyamine catabolism; and (c) a reduction of putrescine uptake. These changes apparently led to a significant reduction of putrescine accumulation in senescent cells following serum stimulation. Since the induction of ODC is a mid-G1 event, the change of polyamine metabolism may be related to changes of expression of other cell-cycle-dependent genes during cellular aging. In addition to ODC gene, we have examined the expression of two early G1 genes, c-erbB and c-myc, and one late G1/S gene thymidine kinase, at mRNA levels, in both young and old IMR-90 cells. We have also compared the enzyme activities of two late G1/S genes, thymidine kinase and thymidylate synthetase, in young and old cells following serum stimulation. We did not observe significant changes of c-erbB, c-myc, and ODC mRNA levels during cellular senescence. However, we found that serum-induced mRNA level of thymidine kinase gene in old IMR-90 cells was significantly reduced compared to that in the young cells. Results also demonstrate that aging of IMR-90 cells was accompanied by significant decrease of both thymidine kinase and thymidylate synthetase activities. In view of the recognized importance of polyamines in growth regulation, it is possible that alteration of polyamine metabolism may contribute to the impairment of expression of some key G1/S genes and such impairment may contribute to the ultimate loss of dividing potential in senescent cells.
Assuntos
Sobrevivência Celular , Regulação da Expressão Gênica/fisiologia , Poliaminas/metabolismo , Transporte Biológico , Northern Blotting , Ciclo Celular , Sobrevivência Celular/fisiologia , Fibroblastos/fisiologia , Humanos , Técnicas In Vitro , Modelos Biológicos , Ornitina Descarboxilase/genética , Putrescina/metabolismo , Timidilato Sintase/biossínteseRESUMO
The effects of acetone and isopropanol on the microsomal monooxygenase system have been investigated to study the role of this enzyme system in the metabolism of nitrosamines. Treatment of rats with acetone or isopropanol (2.5-5 ml/kg, i.g.) causes a 3-4.5-fold enhancement in the NADPH-dependent nitrosodimethylamine demethylase (NDMAd) activity. This is accompanied by only moderate increases in the gross cytochrome P-450 (P-450) content and NADPH-cytochrome c reductase activity. Several other monooxygenase activities were increased to different extents from an 8% increase in aryl hydrocarbon hydroxylase to a 261% increase in ethoxycoumarin O-dealkylase activities. Kinetic analysis indicates that a low Km form of NDMAd (Km = 0.07 mM) is induced by these treatments. In the microsomes of the treated rats, this high affinity form becomes predominant, in contrast to control microsomes which possess at least three Km-values for NDMAd. The treatment also enhances the metabolism of nitrosomethylethylamine, nitrosomethylbenzylamine and nitrosomethylaniline although to lesser extents than with nitrosodimethylamine. Several lines of observations suggest that the enhanced NDMAd is due to the induction of one or more specific P-450 isozyme(s) by pretreatment with acetone or isopropanol: (a) The treatment induces proteins with molecular weights (Mr) of 50 000 and 52 000 which are in the range of known P-450 isozymes. (b) The induction of these proteins and NDMAd activity was inhibited by CoCl2 and cycloheximide. (c) The induced microsomes had a peak at 450.6 nm different from the 450.0 nm peak of control microsomes. When added to the incubation mixture, both acetone and isopropanol inhibit NDMAd activity. Isopropanol is more potent than acetone and is shown to be a competitive inhibitor with a Ki-value of 0.151 mM.
Assuntos
1-Propanol/farmacologia , Acetona/farmacologia , Microssomos Hepáticos/enzimologia , Nitrosaminas/metabolismo , Oxirredutases N-Desmetilantes/biossíntese , Animais , Citocromo P-450 CYP2E1 , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Cinética , Masculino , NADPH-Ferri-Hemoproteína Redutase/análise , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
Normal human diploid fibroblasts cannot divide indefinitely in culture. At the end of their lifespan they withdraw from the cell cycle permanently by a process termed cellular senescence. Recent molecular studies indicate that upregulation of two inhibitors of cyclin-dependent kinases, p16 and p21, is responsible for blocking the G1/S transition in senescent cells. Although the state of senescence resembles terminal differentiation in that both exhibit irreversible growth arrest and resistance to apoptosis, other molecular changes are seen only in senescent cells. This suggests that the signal pathway specific for senescence is present in normal cells. Changes in chromosomes, such as progressive shortening of the telomeres and erosive damage by detrimental by-products in metabolism, may be the signals that trigger senescence, leading to the inactivation of cell cycle progression. On the other hand, it seems that a dominant genetic program is intrinsically preset to ensure a growth limit in the normal cell. This notion is supported by cell fusion and microcell transfer experiments which show that escaping from senescence requires recessive mutations in senescence-specific genes. Identification of these participating genes and clarification of their mode of action will provide the basis for understanding the mechanisms governing the differences between mortality in normal cells and immortality in cancer cells.
Assuntos
Senescência Celular , Animais , Apoptose , Divisão Celular , Aberrações Cromossômicas , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/fisiologia , Fase G1 , HumanosRESUMO
Deregulation of Shp2, a non-receptor tyrosine phosphatase, causes hyperactivation of extracellular signal-regulated kinase (ERK), leading to growth abnormality. Here, we show that inhibition of RhoA-Dia is sufficient to upregulate ERK activation in epithelial cells. Oncogenic Shp2 expression attenuates RhoA-Dia signaling, by which microtubule (MT) is destabilized with reduced level of acetylation. Either MT stabilization, silencing of histone deacetylase 6 (HDAC6) or enforcing RhoA-Dia signal prevents oncogenic Shp2-induced ERK hyperactivation. We provide evidence that downregulation of RhoA-Dia-EB1 pathway by oncogenic Shp2 leads to HDAC6-mediated reduction in MT acetylation, in turn affecting ERK regulation. In response to serum stimulation, cells expressing wild-type Shp2 display transient ERK activation. In contrast, cells expressing oncogenic Shp2 have prolonged ERK activation. HDAC6 inhibition diminishes sustained activation of ERK and slows down the growth of these cells. Likewise, in human cancer cells, blocking Shp2 increases MT acetylation and decreases ERK phosphorylation, which are reversed by inhibition of Dia. As such, HDAC6 inhibition in these cells also reduces ERK activity. Our findings link MT regulation by HDAC6 to oncogenic Shp2 and ERK regulation, implicating the therapeutic potential of HDAC6 inhibitor in diseases involving Shp2 deregulation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histona Desacetilases/metabolismo , Microtúbulos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Acetilação , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática , Inativação Gênica , Desacetilase 6 de Histona , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismoAssuntos
DNA de Neoplasias/metabolismo , Regulação da Expressão Gênica , Leucemia Eritroblástica Aguda/metabolismo , Sequências Repetitivas de Ácido Nucleico , Acetamidas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/patologia , Metilação , Camundongos , RNA Neoplásico/biossínteseRESUMO
Chronic myeloid leukemia (CML) is a lethal hematological disorder caused by the p210(Bcr-Abl) oncogene. Previous studies have suggested that p210(Bcr-Abl) transformation contributes to homing and retention defects, typical of immature myeloid cells in CML, by attenuating chemotactic response to stromal-derived factor-1alpha (SDF-1alpha). As Rho family GTPases are key regulators of the cytoskeleton and have been previously found to interact with p210(Bcr-Abl), this study aimed to determine whether p210(Bcr-Abl) signaling affects SDF-1alpha chemotaxis through Rho GTPase signaling. We found that SDF-1alpha stimulated Cdc42 GTPase activation in myeloid progenitor 32D, but not in p210(Bcr-Abl)-transformed (32Dp210) cells. In fact, the basal level of active Cdc42 was elevated in 32Dp210 cells and mononuclear cells isolated from bone marrow of CML patients. Inhibition of p210(Bcr-Abl) kinase activity decreased basal Cdc42 activity and restored SDF-1alpha-induced Cdc42 and migration responses. Transduction of active Tat-Cdc42V12 abolished this reconstituted chemotactic response. As Cdc42 is particularly important in cytoskeletal remodeling and directional sensing, these results suggest that sustained activation of Cdc42 GTPase through p210(Bcr-Abl) tyrosine kinase signaling in CML cells contributes to defects in SDF-1alpha-chemotactic response due to desensitization of the actin polarization signal required for directional migration.
Assuntos
Movimento Celular , Quimiocina CXCL12/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Ativação Enzimática , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica/fisiologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were performed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Genes , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Timidina Quinase/genética , Células Tumorais Cultivadas/enzimologia , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Sondas de DNA , Dactinomicina/farmacologia , Humanos , Cinética , Leucemia Promielocítica Aguda , Monócitos/citologia , Monócitos/enzimologia , Hibridização de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas/citologiaRESUMO
We have investigated the DNA-protein complex formation in nuclear extracts of human cells using the sequence of cell-cycle regulatory unit (CCRU) of human thymidine kinase (TK) promoter. It appeared that a distinct DNA-protein complex was present in three human tumor cell lines and that the CCAAT box within the sequence of CCRU was a necessary element for complex formation. Upon 4 days of serum deprivation, this DNA-protein complex remained unchanged in HeLa cells, but the expression of TK mRNA was decreased. Furthermore, DNA methylation of the Hhal site of the CCRU sequence of the TK promoter greatly reduced the binding activity of nuclear proteins from different human tumor cell lines. On the basis of these data, we proposed a possible role for DNA methylation in the regulation of TK transcription during late G1/S phase progression of the cell cycle.
Assuntos
Ciclo Celular/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Timidina Quinase/genética , Sequência de Bases , Sítios de Ligação , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Células HeLa , Humanos , Leucemia Promielocítica Aguda , Metilação , Dados de Sequência Molecular , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Células Tumorais CultivadasRESUMO
Possible changes of glycoproteins in IMR-90 human embryonic lung fibroblasts during senescence in vitro were studied by the metabolic labeling technique using radioactive precursors for carbohydrate moieties of glycoproteins. IMR-90 fibroblasts at three different population doubling level (PDL) were incubated with [3H]fucose and [3H]glucosamine for various periods of time. The radioactively labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The results indicated a marked increase, by more than eight-fold on per mg protein basis, of labeling by [3H]fucose in old IMR-90 fibroblasts (PDL = 45) as compared to young (PDL = 22) and middle-age (PDL = 30) IMR-90 fibroblasts. In contrast, no significant difference in [3H]glucosamine labeling was observed in young and old IMR-90 cells.
Assuntos
Fibroblastos/metabolismo , Fucose/metabolismo , Glicoproteínas/metabolismo , Linhagem Celular , Sobrevivência Celular , Fibroblastos/citologia , Glucosamina/metabolismo , Fatores de TempoRESUMO
Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.
Assuntos
Fibroblastos/enzimologia , Regulação da Expressão Gênica , Ornitina Descarboxilase/genética , Sangue , Linhagem Celular , Sobrevivência Celular , Indução Enzimática , Fibroblastos/citologia , Humanos , Ornitina Descarboxilase/biossíntese , Timidina Quinase/biossínteseRESUMO
When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.
Assuntos
Aminoácidos/metabolismo , Sobrevivência Celular , Fibroblastos/metabolismo , Poliaminas/metabolismo , Linhagem Celular , Diploide , Eflornitina , Indução Enzimática/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Inibidores da Ornitina Descarboxilase , Poliaminas/biossíntese , Processamento de Proteína Pós-Traducional , Putrescina/metabolismoRESUMO
The nuclear protein interacting with the distal CCAAT box of human thymidine kinase (TK) gene promoter has been suggested to be a specific TK-CCAAT-binding protein, which is responsible for the serum-dependence of TK transactivation in normal human IMR-90 fibroblasts. By biochemical characterization, TK-CCAAT-binding protein was found to be distinct from other known CCAAT-binding proteins (Pang, J. H., and Chen, K. Y. (1993) J. Biol. Chem. 268, 2909-2916). In this study, we identify NF-Y, which is composed of Ya and Yb subunits, to be responsible for the TK-CCAAT binding activity in the crude nuclear extract from HL-60 cells. The interaction of NF-Y with the distal CCAAT box of the TK promoter in the crude extract appeared to be more heat-sensitive than that in the DNA affinity chromatography purified fraction. We have further established that the serum dependence of TK-CCAAT binding activity in normal IMR-90 fibroblasts is due to the decrement of NF-Ya, but not NF-Yb expression following serum-deprivation, and that such serum dependence is absent in HL-60 cells.