RESUMO
The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15-20 min after MC (0.5 microM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hepatócitos/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Inibidores de Caspase , Caspases/metabolismo , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavanonas/farmacologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Masculino , Microcistinas , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Tireoglobulina/biossíntese , Glândula Tireoide/fisiologia , Tireotropina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/biossíntese , Cães , Ativação Enzimática , Regulação da Expressão Gênica , Iodeto Peroxidase/biossíntese , Microinjeções , Fosforilação , Receptores da Tireotropina/biossíntese , Transdução de Sinais , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacosRESUMO
Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.
Assuntos
Apoptose/fisiologia , Polirribossomos/metabolismo , RNA Ribossômico 28S/metabolismo , Ribonucleases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Bovinos , Permeabilidade da Membrana Celular , Células Cultivadas , AMP Cíclico/farmacologia , Dano ao DNA/fisiologia , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Ribossômico 28S/genética , Ratos , Ribossomos/químicaRESUMO
AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.
Assuntos
Permeabilidade Capilar/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de TransmissãoRESUMO
Fetal brain cells from rats given a transplacental pulse of N-ethyl-N-nitrosourea progressively acquire malignant characteristics and dedifferentiate when grown in vitro. One aspect of this dedifferentiation is a decreased morphological response to cyclic adenosine 3':5'-monophosphate (cAMP). In the present study, we have characterized and compared the isozymes (I, II) of cAMP-dependent protein kinase in fetal brain cells and in the neoplastically transformed, dedifferentiated BT5C glioma cell line. This is a first approach to find the mechanism behind the subresponsiveness of such cells towards cAMP. It is also part of a broader investigation of the cAMP effector system in cells showing various rates of normal and malignant growth. We found the regulatory and catalytic subunits of cAMP-dependent protein kinase to be expressed to a similar degree in both cell types. Sixty % of the enzyme was located in the 30,000 X g supernatant. The glioma cell line had a significantly higher ratio (1.2) between protein kinase I and II than did the normal fetal cells (0.5). This difference in isozyme distribution was not apparent using conventional methods for enzyme separation and detection, the use of specific antibodies being essential for that purpose. Of the chromatographically separated forms (a, b) of protein kinase II, Form IIa was selectively decreased in the glioma cell line. The alterations of the protein kinases in the glioma cell line described above may be of importance for some of the neoplastic properties of these cells. However, the subdued response of such cells towards cAMP is not explained since the concentrations of cAMP or its analogues required for activation of the kinases were similar for the enzymes from normal and neoplastically transformed cells.
Assuntos
Neoplasias Encefálicas/enzimologia , Encéfalo/enzimologia , Feto/enzimologia , Glioma/enzimologia , Isoenzimas/análise , Proteínas Quinases/análise , Animais , Células Cultivadas , Feminino , Gravidez , RatosRESUMO
We have compared the properties of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases I and II in hormone-dependent/cAMP-sensitive (DMBA tumor) and hormone-independent/cAMP-resistant (DMBA 1 tumor) rat mammary carcinomas. cAMP-resistance was not due to less total kinase in the hormone-independent tumor, grossly altered distribution between soluble and particulate forms of the kinase (80% soluble in either tumor), alteration in the relative proportion of isozymes I and II of the protein kinase (the soluble and the particulate fraction from both tumors contained about 50% of either isozyme), or a decreased sensitivity towards cAMP (both isozymes had affinities for cAMP and its derivatives that corresponded closely with those of isozymes from normal tissues). Furthermore, the sensitivity of the enzymes towards thermal denaturation was identical for samples from the two tumor types. Subtle differences did, however, exist between the regulatory moieties [regulatory subunit of cAMP-dependent protein kinase II (RII)] of isozyme II from the two tumors: autophosphorylated RII from the hormone-independent tumor migrated as a doublet corresponding to Mrs 54,000 and 52,000 on sodium dodecyl sulfate-polyacrylamide gels, against Mrs 53,000 and 52,000 for RII from the hormone-dependent tumor; RII from the two tumors showed different elution profiles upon DEAE-cellulose chromatography; a considerable proportion of the soluble RII in the hormone-independent tumor formed supramolecular aggregates as judged by size-exclusion chromatography. No such microheterogeneity was noted for isozyme I. This study thus shows that the lack of cAMP-responsiveness of one tumor is related either to a defect distal to the cAMP-dependent protein kinases or to the appearance of the new subtype of RII in the resistant tumor. If the latter explanation is correct, it means that the part of the RII molecule responsible for interaction with other proteins rather than that responsible for cAMP-binding and control of protein kinase activity modulates the growth-inhibiting response to cAMP.
Assuntos
AMP Cíclico/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Quinases/análise , 9,10-Dimetil-1,2-benzantraceno , Animais , Sítios de Ligação , AMP Cíclico/análogos & derivados , Feminino , Substâncias Macromoleculares , Peso Molecular , Fosforilação , Coelhos , Ratos , Ratos Endogâmicos F344 , Ratos EndogâmicosRESUMO
BACKGROUND: Obesity is still considered a risk factor for cardiovascular disease, although more recent knowledge also suggests obesity to be associated with reduced morbidity and mortality - the "obesity paradox". This study explores if long-term feeding of an obesogenic high fat diet renders the myocardium less susceptible to ischemic-reperfusion induced injury via Epac-dependent signaling. METHODS: Wild type (wt), Epac1 (Epac1-/-) and Epac2 (Epac2-/-) deficient mice were fed a high fat (HFD) or normal chow diet (ND) for 33 ± 1 weeks. Six experimental groups were included: (1) control wt ND (wt ND), (2) control wt HFD (wt HFD), (3) Epac1-/- mice on ND (Epac1-/-ND), (4) Epac1-/- mice on HFD (Epac1-/-HFD), (5) Epac2-/- mice on ND (Epac2-/-ND), and (6) Epac2-/- mice on HFD (Epac2-/-HFD). Isolated ex vivo mice hearts were perfused in a constant pressure Langendorff mode, and exposed to 30min of global ischemia (GI) and 60min of reperfusion. Endpoints were infarct size and functional recovery. RESULTS: All groups fed a HFD presented with significantly enhanced body weight, visceral fat content and reduced glucose clearance compared to corresponding ND groups. Although the HFD cohorts presented with an overall comparable systemic capability to clear glucose, the Epac1-/- HFD group presented with glucose levels slightly above the human diabetes criteria at the end of the intraperitoneal glucose tolerance test (ipGTT). Moreover, the HFD significantly reduced infarct size in both wild type (wt HFD 41.3 ± 5.5% vs. wt ND 58.0 ± 9.8%, p < 0.05) and Epac2-/- cohorts (Epac2-/-HFD 34.4 ± 7.2% vs. Epac2-/-ND 56.5 ± 3.8%, p < 0.05). Interestingly, however, the HFD did not reduce infarct size in Epac1-/- deficient mice hearts (Epac1-/-HFD 65.1 ± 5.1% vs. Epac1-/-ND 56.1 ± 3.5%, ns.). CONCLUSION: Epac1-dependent signaling is involved in mediating the cardioprotection afforded by long-term feeding of an obesogenic high fat diet in mice hearts.
RESUMO
The inhibition of rat liver adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Pb2+ could be separated into an irreversible and a reversible component. Evidence was obtained that both types of inhibition were due to free Pb2+, rather than Pb/ATP, and that Pb2+ did not act via the site wherein Mg2+ and Mn2+ activate the cyclase. Guanine nucleotides strongly counteracted the reversible inhibition of cyclase by Pb2+, providing another example of guanine nucleotide effects on adenylate cyclase function. It is suggested that the Pb2+-inhibited cyclase may be of value in the study of guanine nucleotide-cyclase interactions.
Assuntos
Inibidores de Adenilil Ciclases , Nucleotídeos de Guanina/farmacologia , Chumbo/farmacologia , Trifosfato de Adenosina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Ativação Enzimática , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Fígado/enzimologia , Magnésio/farmacologia , Manganês/farmacologia , RatosRESUMO
S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) was purified to apparent homogeneity from bovine liver, bovine adrenal cortex and mouse liver. All enzymes were tetramers, composed of two types of subunit present in the proportion 1:1, as judged by SDS-polyacrylamide gel electrophoresis. The partition coefficient was exactly the same for these enzymes on high-performance gel permeation chromatography, and they co-sedimented in density gradients, suggesting the same molecular size and form of S-adenosylhomocysteine hydrolase from these sources. The bovine enzymes differed from the mouse liver enzyme with respect to isoelectric point (pI = 5.35, versus pI = 5.7), affinity for DEAE-cellulose, and migration of subunits on SDS-polyacrylamide gel electrophoresis with SDS from some commercial sources. The enzymes were not substrates for cAMP-dependent protein kinase. The apparent Km values for adenosine (0.2 microM) and S-adenosylhomocysteine (0.75 microM) were the same for all three enzymes. The ratio between Vmax for the synthesis and hydrolysis of S-adenosylhomocysteine was about 4 for the mouse liver enzyme, and about 6 for the bovine enzymes. It is concluded that only subtle kinetic and physicochemical differences exist between S-adenosylhomocysteine hydrolase from these bovine and mouse tissues. This suggests that differences in experimental procedures rather than species- and organ-differences of S-adenosylhomocysteine hydrolase are responsible for the variability in kinetic and physicochemical parameters reported for the mammalian hydrolase.
Assuntos
Córtex Suprarrenal/enzimologia , Hidrolases/metabolismo , Fígado/enzimologia , Adenosil-Homocisteinase , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , AMP Cíclico/farmacologia , Eletroforese em Gel de Poliacrilamida , Hidrolases/isolamento & purificação , Focalização Isoelétrica , Cinética , Camundongos , Proteínas Quinases/metabolismo , S-Adenosil-Homocisteína/metabolismo , Especificidade da EspécieRESUMO
A number of physiochemical properties of the cyclic AMP-adenosine binding protein of mouse liver (Ueland, P.M. and Døskeland, S.O. (1977) J. Biol. Chem. 252, 677--686) have been studied. 1. The specific extinction coefficient, E1%280nm, was estimated to 13.0. 2. Amino acid and amide group analyses confirmed the acidic properties of the protein as determined by electrofocusing (pI = 5.7). Based on the estimated partial specific volume (v = 0.74 cm3/g) the minimum molecular weight of the native, tetrameric protein was recalculated to be 185 000 (s20,w = 8.8 . 10(-13) s and Stokes radius = 48 A). 3. No NH2-terminal amino acid was found by the dansyl method using [14C]-dansyl chloride, indicating that the NH2-terminal groups are blocked. 4. Amino acid analyses gave 6 half-cystine residues per subunit, and the same number of free sulfhydryl groups was found by titration of the denatured protein with 5,5'-dithiobis (2-nitrobenzoic acid). 5. The reactivity of the SH groups in the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) revealed rapidly reacting (SHI), sluggishly reacting (SHII) and "masked" (SHIII) SH groups. ATP, adenosine, Mg2+ and KCl, factors known to affect the activation of cyclic AMP binding sites (Ueland, P.M. and Døskeland, S.O. (1978) Arch. Biochem. Biophys., in press) changed the reactivity of separate SH groups.
Assuntos
Adenosina/metabolismo , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Fígado/metabolismo , Amidas/análise , Aminoácidos/análise , Animais , Sítios de Ligação , Carboidratos/análise , Camundongos , Peso Molecular , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/metabolismoRESUMO
The mechanism whereby the universal apoptogen and serine/threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selected for OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one clones were selected. Two of these (OAR1, OAR2) were studied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporine or microinjected Cytochrome c, thus, indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OA-induced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OA-resistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Resistência a Medicamentos/genética , Biblioteca Gênica , Ácido Okadáico/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Caspases/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/metabolismo , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/farmacologia , Resistência a Múltiplos Medicamentos/genética , Inibidores Enzimáticos/farmacologia , Fibroblastos , Humanos , Células Jurkat , Camundongos , Dados de Sequência Molecular , Fenótipo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/análise , Estaurosporina/farmacologia , TransfecçãoRESUMO
Discrete cleavages within 28S rRNA divergent domains have previously been found to coincide with DNA fragmentation during apoptosis. Here we show that rRNA and DNA cleavages can occur independently in apoptotic cells, i.e. that the previously observed correlation is likely to be coincidental. In HL-60 cells, apoptosis with massive DNA fragmentation could be induced without any signs of rRNA cleavage. The opposite situation; rRNA cleavage without concomitant internucleosomal DNA fragmentation, was found in okadaic acid-treated Molt-4 cells. Other leukemia cell lines underwent apoptosis either without (K562 and Molt-3) or with (U937) both forms of polynucleotide cleavage. In K562 cells transfected with a temperature-sensitive p53 mutant, internucleosomal DNA fragmentation but not 28S rRNA cleavage was inducible by wild-type p53 expression. The absence of apoptotic rRNA cleavage in some cell types suggests that this phenomenon is tightly regulated and unrelated to DNA fragmentation or a presumed scheme for general macromolecular degradation in apoptotic cells.
RESUMO
Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was characterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastructural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of the cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl-2 and these all showed a markedly decreased sensitivity towards injected cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. They also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, but can also interfere with caspase activation induced by cytochrome c microinjected directly into the cytoplasm.
Assuntos
Apoptose , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Células 3T3 , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Linhagem Celular Transformada , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Camundongos , Microinjeções , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Células Tumorais CultivadasRESUMO
The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.
Assuntos
Apoptose , Diferenciação Celular , Núcleo Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Granulócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Ciclo Celular/fisiologia , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Granulócitos/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Transdução de Sinais , Tionucleotídeos/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.
Assuntos
Apoptose , Caspases/metabolismo , Inibidores Enzimáticos/farmacologia , Peptídeos Cíclicos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Piranos , Compostos de Espiro , Células 3T3 , Animais , Antifúngicos/farmacologia , Caspase 3 , Inibidores de Caspase , Linhagem Celular , Linhagem Celular Transformada , Expressão Gênica , Humanos , Líquido Intracelular , Toxinas Marinhas , Camundongos , Microcistinas , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Peptídeos Cíclicos/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
Co-chaperone p23 is a component of the heat-shock protein (Hsp)90 multiprotein-complex and is an important modulator of Hsp90 activity. Hsp90 client proteins involved in oncogenic survival signaling are frequently mutated in leukemia, and the integrity of the Hsp90 complex could therefore be important for leukemic cell survival. We demonstrate here that p23 is cleaved to a stable 17 kDa fragment in leukemic cell lines treated with commonly used chemotherapeutic drugs. The cleavage of p23 paralleled the activation of procaspase-7 and -3 and was suppressed by the caspase-3/-7 inhibitor DEVD-FMK. In vitro translated 35S-p23 (in reticulocyte lysate) was cleaved at D142 and D145 by caspase-7 and -3. Cleavage of p23 occurred in caspase-3-deficient MCF-7 cells, suggesting a role for caspase-7 in intact cells. The Hsp90 inhibitor geldanamycin enhanced caspase-dependent p23 cleavage both in vitro and in intact cells. Geldanamycin also enhanced anthracycline-induced caspase activation and apoptosis. We conclude that p23 is a prominent target in leukemic cell apoptosis. Geldanamycin enhanced p23 cleavage both by rendering p23 more susceptible to caspases and by enhancing chemotherapy-induced caspase activation. These findings underscore the importance of the Hsp90-complex in antileukemic treatment, and suggest that p23 may have a role in survival signaling.
Assuntos
Apoptose , Caspases/metabolismo , Inibidores Enzimáticos/farmacologia , Leucemia/metabolismo , Chaperonas Moleculares/metabolismo , Fosfoproteínas/metabolismo , Quinonas/farmacologia , Benzoquinonas , Caspase 3 , Caspase 7 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas , Leucemia/patologia , Chaperonas Moleculares/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Fosfoproteínas/efeitos dos fármacos , Prostaglandina-E Sintases , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Cyclic AMP-dependent protein kinase subunit expression was studied during the first 35 h of primary culture of hepatocytes isolated from rats fed a protein restricted diet. In the absence of elevated cAMP the ratio between regulatory (RI + RII) and catalytic (C) subunits was constant. There was an increase of RI and a decrease of RII, the RI/RII ratio rising from 1 to 2.4 during the 35 h of culturing studied. This disproportionate expression of RI was reflected in an increase of RI alpha mRNA relative to RII alpha mRNA. The increase of liver RI previously noted after amino acid feeding of protein starved rats was thus reproduced when hepatocytes from such animals were cultured in an amino acid rich medium. When the cell cAMP level was chronically elevated by adding glucagon/isobutylmethylxanthine at the time of seeding, the C level decreased by more than 50% in a few hours. The concentration of C alpha mRNA was not affected. The elevated cAMP also led to a transient increase of RI alpha- and RII alpha mRNA. The effects of glucagon could be reproduced by cAMP analogs. The cAMP-induced down-regulation of C without concomitant down-regulation of R led to an increased R/C ratio. The decreased C and the increased R/C ratio both ensure that the hepatocytes will show a reduced kinase activation in response to a second challenge with cAMP (i.e. show hysteresis).
Assuntos
Proteínas de Transporte/genética , Inibidores Enzimáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/citologia , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , AMP Cíclico/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Expressão Gênica , Genes Reguladores/fisiologia , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The amount of an immunological marker (CVA) in mucified cells of the mouse vaginal epithelium was quantified by a mixed hemagglutination technique for tissue sections. Immature mice, adult mice which had been estrogenized neonatally (5 mug diethylstilbestrol daily for the first five days after birth), and adult non-estrogenized mice were studied. All adult animals were castrated 7-10 days before starting the experiments. Injections of 5 mug estradiol-17beta (48 and 24 h before killing the animals) increased the amount of CVA in all three groups of animals, but most markedly in the neonatally estrogenized mice. The amount of CVA found following estradiol treatment was decreased in adult animals injected with the ergot alkaloid CB154 (0.5 mg twice daily for 6 days) in addition to the hormone. This partial block of the estradiol-induced CVA response by CB154 was relieved by exogenous rat prolactin. The CVA content in immature animals was not influenced by CB154, given alone or together with estradiol. Combined treatment with estradiol and rat prolactin (3 mug every 8 h for 6 days) increased more efficiently than estradiol alone the amount of CVA in immature and adult nonestrogenized animals. Prolactin injected alone had no effect on the CVA content. These data strongly suggest a synergistic action of estradiol and prolactin in augmenting the epithelial CVA content. Explants of the vaginal wall from normal and neonatally estrogenized mice were grafted into the thigh muscles of newborn mice, every host carrying one graft from both types of animals. The CVA content in the epithelium of the two grafts increased to the same level in response to estradiol. When the hosts were injected with estradiol and prolactin, the CVA content was higher in grafts from estrogenized donors than in those from nonestrogenized animals. Our results demonstrate that the mucified vaginal cells in adult, neonatally estrogenized mice have a content of CVA which is higher than in nonestrogenized animals. This difference may be ascribed to hormonal factors (estradiol-prolactin) as well as to persistent effects in the vaginal cells as a result of the neonatal estrogen treatment.
Assuntos
Colo do Útero/imunologia , Estradiol/farmacologia , Prolactina/farmacologia , Vagina/imunologia , Fatores Etários , Animais , Animais Recém-Nascidos , Antígenos/análise , Bromocriptina/farmacologia , Colo do Útero/efeitos dos fármacos , Dietilestilbestrol/farmacologia , Feminino , Camundongos , Ovário/fisiologia , Vagina/efeitos dos fármacos , Vagina/transplanteRESUMO
cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbazóis , AMP Cíclico/farmacologia , Indóis , Interleucina-1/farmacologia , Fígado/citologia , Fígado/enzimologia , Peptídeos Cíclicos/farmacologia , Vasopressinas/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Alcaloides/farmacologia , Animais , Benzilaminas/farmacologia , Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , DNA/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fígado/fisiologia , Masculino , Microcistinas , Fenilalanina Hidroxilase/antagonistas & inibidores , Fenilalanina Hidroxilase/metabolismo , Fosfoproteínas/análise , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologiaRESUMO
Human mammary carcinoma cells (MCF-7) were arrested in late G1-phase after treatment with agents (forskolin, interleukin-1 beta 3-isobutyl-1-methylxanthine) that increased the endogenous concentrations of cAMP. The effect of elevated cAMP was mimicked by microinjected catalytic (C alpha) cAMP-dependent protein kinase (cAK) subunit and reversed by the injection of a dominant negative cAK regulatory mutant (RID199). Further evidence that activation of cAK induced growth arrest was provided by the use of pairs of stable cAMP analogs known to synergistically activate isolated cAK isozymes. Furthermore, the effect of cAMP was not potentiated by serine/threonine phosphatase inhibitors that profoundly restricted MCF-7 growth. Some 8-substituted cAMP analogs, e.g. 8-Cl-cAMP and 8-NH2-cAMP, induced cell death rather than reversible inhibition of growth. Their effect was not synergized with complementary cAMP analogs. Furthermore, their potency was decreased rather than increased in the presence of an inhibitor of degradation (3-isobutyl-1-methylxanthine). Finally, their effect could be mimicked by degradation products unable to activate cAK. We concluded that 8-Cl-cAMP (and 8-NH2-cAMP) induced irreversible growth arrest by a mechanism not involving cAK, whereas activation of cAK resulted in a transient and fully reversible inhibition of cell proliferation.