RESUMO
Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2. 1->1000 microM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 microM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized byic value in neuroblastoma.
Assuntos
Apoptose/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Glutationa/metabolismo , Neuroblastoma/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Butionina Sulfoximina/agonistas , Butionina Sulfoximina/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Amplificação de Genes , Genes myc/genética , Glutationa/farmacologia , Humanos , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Vitamina E/farmacologiaRESUMO
PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.
Assuntos
Anuros/embriologia , Fase de Clivagem do Zigoto/citologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Quinases/metabolismo , Animais , Blastômeros/enzimologia , Blastômeros/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Oócitos/enzimologia , Proteínas rac de Ligação ao GTPRESUMO
Highly purified cerebroside sulfate activator from pig kidneys was characterized by a number of chemical and biological procedures. Methods for chemical modifications were evaluated in an attempt to obtain biologically active derivatives. Iodination, dabsylation, and to a lesser degree reductive methylation provided useful products with good retention of cerebroside sulfate activator activity. Other procedures resulted in largely inactive derivatives or losses in both protein and biological activities. Attempts at renaturation of cerebroside sulfate activator subjected to various denaturing conditions appeared to be successful in many instances, but it was uncertain if the protein structure had actually been disrupted. The binding of cerebroside sulfate by activator was estimated by gel filtration under conditions similar to those of its assay. The formation of a relatively stable 1:1 complex was observed, collaborating results with the human protein. The complex was stable enough to be isolated and shown to be an efficient substrate for arylsulfatase A. The effectiveness of the pig kidney cerebroside sulfate activator for correcting the metabolic defect in activator-deficient human fibroblasts was compared with human materials. The pig kidney protein was taken up more efficiently by the cells and resulted in a better metabolic correction than material from human liver, but was somewhat less effective than a preparation from human urine.
Assuntos
Cerebrosídeos/metabolismo , Glicoproteínas/química , Rim/química , Animais , Cerebrosídeo Sulfatase/metabolismo , Cromatografia em Gel , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Humanos , Iodo/metabolismo , Metilação , Desnaturação Proteica , Saposinas , Proteínas Ativadoras de Esfingolipídeos , Suínos , p-Dimetilaminoazobenzeno/análogos & derivados , p-Dimetilaminoazobenzeno/químicaRESUMO
The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.
Assuntos
Glicoproteínas/isolamento & purificação , Rim/química , Sequência de Aminoácidos , Animais , Carboidratos/análise , Cromatografia , Cromatografia Líquida de Alta Pressão , Concanavalina A/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Saposinas , Homologia de Sequência do Ácido Nucleico , Proteínas Ativadoras de Esfingolipídeos , SuínosRESUMO
It had been shown previously that arylsulfatase A activity was attenuated in pseudo arylsulfatase A deficiency fibroblasts and that subunits of the enzyme were smaller than subunits of the enzyme in normal fibroblasts. Attenuated enzyme activity has now been affirmed in other tissues. Subunits of the enzyme from these sources were also found to be smaller with apparent molecular size 59 and 56 kdaltons. Subunits of enzyme in corresponding control tissues were larger and there was heterogeneity in apparent molecular size as follows: fibroblasts, 63 and 59 kdaltons; liver, 63 and 59 kdaltons; kidney, 63 and 58 kdaltons; spleen, 63 and 58 kdaltons; placenta, 62 and 58 kdaltons; and urine, 61 and 57 kdaltons. Attenuated enzyme activity and structurally altered enzyme in pseudo arylsulfatase A deficiency appears to be systemic. However, the reason for reduced amounts of structurally altered enzyme with normal catalytic activity is unresolved.
Assuntos
Cerebrosídeo Sulfatase/deficiência , Feminino , Fibroblastos/enzimologia , Humanos , Peso Molecular , Gravidez , Distribuição TecidualRESUMO
Two children in a family with five siblings were investigated because of low levels of fibroblast arylsulfatase A activity. Neither child had metachromatic leukodystrophy (MLD) and they were diagnosed as having benign pseudo arylsulfatase A deficiency trait (PD). Analysis of arylsulfatase A subunit profiles in fibroblasts provided data for genotype assignments for each family member. Father and mother were assigned an n/pd and n/mld phenotype, respectively. The low enzyme siblings were both assigned pd/mld; two of the three normal enzyme siblings were assigned an n/n phenotype and one an n/mld.
Assuntos
Cerebrosídeo Sulfatase/deficiência , Genótipo , Leucodistrofia Metacromática/genética , Sulfatases/deficiência , Adulto , Cerebrosídeo Sulfatase/genética , Criança , Fibroblastos/enzimologia , Humanos , Leucodistrofia Metacromática/enzimologia , LinhagemRESUMO
Analysis of arylsulfatase A from pseudo arylsulfatase A deficiency fibroblasts by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoradiochemical nitrocellulose blot radiography revealed two subunit bands which migrated faster than subunit bands of enzyme from normal fibroblasts. Immunoreactive material was present only at levels comparable to enzyme activity. These findings imply that arylsulfatase A in pseudodeficiency is structurally altered, but it is catalytically equivalent to normal arylsulfatase A. This altered enzyme must be the product of the pseudodeficiency gene since no immunoreactive product of the metachromatic leukodystrophy gene could be detected in metachromatic leukodystrophy cells by the procedure employed. It is not clear from the present data if the attenuated arylsulfatase A activity in pseudodeficiency results from a decreased rate of synthesis or an increased lability of the mutant enzyme.