RESUMEN
Two dimensional gel electrophoresis (2-D PAGE) and automated image analysis were used to study the effects of bacterial lipopolysaccharide (LPS) activation on secreted and cellular rat alveolar macrophage proteins. Primary alveolar macrophages were cultured and exposed to LPS in the presence of [35S]-methionine for 24 h. Image analysis of 2-D PAGE revealed that LPS treatment primarily modulated the proportions of several alveolar macrophage cellular proteins versus control in addition to limited protein induction and repression. The differential effect of LPS was more pronounced on secreted proteins where qualitative and quantitative differences from control cells were found. Immunoblots of secreted proteins with anti-tumor necrosis factor alpha (TNF alpha) and anti-interleukin-1 alpha(IL-1 alpha) antibodies identified these monokines from protein fluorographic patterns. IL-1 alpha was detected as a single polypeptide of 17 kD at pI = 5. Use of recombinant TNF alpha and monoclonal and polyclonal antibodies for immunodetection revealed the 17 kD form of TNF alpha as well as higher molecular weight species at 18 kD and 22 kD. Thus, analysis of radiolabeled rat macrophages treated with LPS reveals quantitative modulation of several cellular proteins as well as a distinctive pattern of secreted proteins which contain multiple monokine forms resolvable by 2-D PAGE.
Asunto(s)
Lipopolisacáridos/toxicidad , Macrófagos Alveolares/efectos de los fármacos , Proteínas/metabolismo , Animales , Electroforesis en Gel Bidimensional , Femenino , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Macrófagos Alveolares/química , Fotofluorografía , Ratas , Ratas Endogámicas F344RESUMEN
Scurfy (sf), is an X-linked recessive lethal mutation that occurs spontaneously in the C3H mouse. The disease is characterized by lymphoid and hematopoietic dysfunction. Affected males are of small stature and exhibit scaliness and crusting of the eyelids, ears, tail, and feet, marked splenomegaly, moderate hepatomegaly, enlarged lymph nodes, and atrophy of the thymus. The average lifespan of the affected hemizygous males (sf/y) is 24 +/- 0.7 days. Total cellular proteins were extracted from pooled samples of thymus and spleen obtained from combined litters of mice. Tissue-specific protein profiles characteristic of either sf mutant or normal mice were analyzed by two dimensional polyacrylamide gel electrophoresis (2DPAGE) at different stages of the phenotypic expression of the sf mutation, to identify changes in protein patterns that might be associated with the progression of the disease. The resultant gels were silver stained, digitized, and analyzed, by image analysis utilizing a pipelined image processor connected to a host computer. At 14 +/- 1 days of age, protein patterns from sf mutant and normal mice control organs showed considerable homogeneity, although there were proteins identified unique to the sf mutant and to the normal controls. At 20 +/- 1 days of age, the pattern differences between the sf mutant and normal control increased markedly. Differences were expressed as the percent of proteins that were unique to either the sf mutant or the normal control from the total number of each type. The percent of proteins that increased or decreased in the three organs utilized in this study ranged between 21%-39% at 14 days and were between 25%-54% at 20 days. Differences in protein expression between the normal and sf mutant as the disorder progressed for each of the three tissues examined. In addition, thymus protein profiles from 9 day old littermates that were phenotypically normal but genotypically unknown were evaluated to determine if marker proteins could be identified for the sf mutation. Limited protein changes were noted at relative molecular weights of 66, 60, 54, 39, 37, 33, 25, 23, 27, and 11 kDa. These data suggest that the sf mutation follows a trackable pattern of protein expression and repression different than the normal control C3H mouse. Several potential marker proteins associated with the sf mutation were identified in 9 day thymus prior to the phenotypic expression of the disease. These putative biomarkers may be useful for characterizing the sf mutation and the mutant may act a possible model the Wiskott-Aldrich syndrome (WAS).
Asunto(s)
Anomalías Múltiples/genética , Electroforesis en Gel Bidimensional , Trastornos Linfoproliferativos/genética , Ratones Mutantes/genética , Proteínas/análisis , Vísceras/química , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Factores de Edad , Animales , Biomarcadores , Densitometría , Modelos Animales de Enfermedad , Femenino , Genes Letales , Genes Recesivos , Heterocigoto , Procesamiento de Imagen Asistido por Computador , Focalización Isoeléctrica , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Masculino , Ratones , Ratones Endogámicos C3H/genética , Tinción con Nitrato de Plata , Timo/química , Timo/patología , Síndrome de Wiskott-Aldrich , Cromosoma XRESUMEN
Total cellular proteins from mouse C3H10T1/2 fibroblasts were compared by two-dimensional (2-D) gel electrophoresis after radiolabeling with [35S]methionine (35S-Met) or 14C-amino acids (14C-AA). 35S-Met labeling of protein was three to four times greater than 14C-AA incorporation over a 24 h period. Automated comparative analysis of replicate fluorographs after 6, 12, and 24 h of labeling showed considerable homology between radiolabeling methods. More than 88% percent of 35S-Met and 14C-AA-labeled proteins were common at each time point. However, the total number of 35S-Met-labeled proteins dropped from 6 to 24 h while the number of 14C-AA-labeled proteins increased. Additionally, twenty-one proteins were uniquely labeled by 14C-AA that were not detectable by 35S-Met over the labeling period. Densitometric analysis showed that several 35S-Met and 14C-AA-labeled proteins exhibited time-related differences in radiolabel incorporation while most proteins remained relatively constant. Protein patterns of silver-stained gels from 6 to 24 h were highly registered and showed few qualitative differences. Proteins detected in radiolabeled gels were generally, but not always, found in silver-stained gels. Thus, 35S-Met appears better suited for short-term radiolabeling of cellular protein while more comprehensive labeling of protein occurs with 14C-AA during prolonged incubation of cell cultures under present experimental conditions.
Asunto(s)
Aminoácidos , Electroforesis en Gel Bidimensional , Metionina , Proteínas/análisis , Animales , Radioisótopos de Carbono , Línea Celular , Fibroblastos/química , Procesamiento de Imagen Asistido por Computador , Ratones , Fotofluorografía , Tinción con Nitrato de Plata , Radioisótopos de AzufreRESUMEN
The cytoplasmic proteins from three cell lines derived from the C3H mouse were compared after treatment with benzo(a)pyrene by two-dimensional gel electrophoresis and computerized image analysis. The three C3H lines were the transformable 10T1/2, the transformation resistant CVP and the methylcholanthrene transformed 10T1/2Cl line. Specialized algorithms were used to analyze gel images to record and compare individual proteins in the three cytoplasmic preparations. Multiple replicate gels were used to construct a master image to insure all the proteins were represented in the analytical system. In studies designed to examine the efficacy of utilizing image analysis, benzo(a)pyrene treatment caused significant alteration in the expression of numerous proteins in all three cell lines. Comparative analysis between cell types also showed most of the induced and repressed proteins were unique to a given cell line and did not match the induced or repressed proteins in either of the other cell lines. These results suggest that the response to chemical carcinogen treatment may be somewhat cell-specific and result in a variable response to the cells ability to respond to the chemical insult.
Asunto(s)
Proteínas de Neoplasias/aislamiento & purificación , Células Tumorales Cultivadas/química , Animales , Benzo(a)pireno/toxicidad , Línea Celular Transformada , Citoplasma/química , Electroforesis en Gel Bidimensional , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Neoplasias de la Próstata/química , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The proerythroblastoid Friend erythroleukemia cell (FELC) line, clone TR 19-9, was treated with 4 mM hexamethylene bisacetamide (HMBA) over a 6-day period. Greater than 94% of the FELC reacted positively to the benzidine assay for hemoglobin by Day 4 of treatment. Protein accumulation during the final 4 days of treatment (from Days 2 to 6) was monitored by labeling for 24-h periods with a 14C-labeled amino acid mixture. At the end of each radiolabeling time point, cells were harvested and cytoplasmic proteins were isolated and subjected to two-dimensional gel electrophoresis in triplicate. Short-term fluorographic exposures were made in the linear X-ray film response range to monitor those polypeptides which were most rapidly accumulated. Fluorographs were digitized for computer image analysis and gel data comparison rationales were used to combine the polypeptides contained on the replicate fluorographs into a single cytoplasmic polypeptide profile or Master Image for each of the two experimental conditions, control and HMBA-treated FELC. These two images were merged into a single Master Composite Image containing a total of 211 polypeptides so that those polypeptides common to both and/or unique to each of the experimental conditions could be viewed graphically in the same plane. A total of 98 polypeptides in HMBA-treated FELC were shown to have large accumulation rate differences from the control FELC;32 of these polypeptides were present in the HMBA Master Image which were not detected in the Control Master Image and 66 polypeptides were present in the Control Master Image but not detected in the HMBA Master Image. Five polypeptides, found in both Master Images, were shown to vary quantitatively in the HMBA-treated FELC from the corresponding polypeptides in the control. These quantitative data measurements on the rates of accumulation of various common polypeptides offer a mode for simultaneously monitoring the kinetics of induction and repression of many gene products throughout an experimental time course.
Asunto(s)
Citoplasma/análisis , Leucemia Eritroblástica Aguda/análisis , Proteínas de Neoplasias/análisis , Acetamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Electroforesis , Virus de la Leucemia Murina de Friend , Porfobilinógeno Sintasa/análisis , Proteínas Proto-Oncogénicas/análisisRESUMEN
Human mammary tumor T 47D cells were examined for their capacity to metabolize benzo[a]pyrene (BaP) to gain insight into potential metabolic pathways for BaP in human epithelial tissue. Confluent cultures metabolized 95% of 4 microM BaP after 24 h incubation. BaP metabolites were analyzed from culture medium since only residual amounts remained in cells. Tetraols/triols, dihydrodiols, quinones and phenols were either unconjugated or existed as sulfate conjugates. Glucuronide conjugation was minor. Remaining water-soluble (WS) metabolites could be extracted with butanol or removed from culture medium protein with methanol/water and suggestive evidence indicates these may be glutathione conjugates. A portion of BaP-WS metabolites were covalently bound to medium protein. This latter phenomenon is attributed to translocation of reactive BaP metabolites across cell membranes which could potentially occur in vivo during cellular processing of BaP.
Asunto(s)
Benzo(a)pireno/metabolismo , Neoplasias de la Mama/metabolismo , Sulfatos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Femenino , Glucuronatos/metabolismo , Glucuronidasa/metabolismo , Humanos , Sulfatasas/metabolismo , Factores de TiempoAsunto(s)
Benzopirenos/metabolismo , Carcinógenos/metabolismo , Especificidad de la Especie , Animales , Benzo(a)pireno , Biotransformación , Cricetinae , Humanos , Cinética , Ratones , RatasRESUMEN
In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12--96-h incubations of hamster embryo cells with 4 microM [3H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites (less than 0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [3H]B[e]P.
Asunto(s)
Benzopirenos/metabolismo , Carcinógenos/metabolismo , Dihidroxidihidrobenzopirenos , Animales , Benzo(a)pireno , Benzoflavonas/farmacología , Benzopirenos/farmacología , Células Cultivadas , Cricetinae , Embrión de Mamíferos , Isomerismo , CinéticaRESUMEN
Hamster embryo cells metabolize benzo(a)pyrene to derivatives that covalently modify the nuclear macromolecules including proteins. Not all proteins are modified to the same extent nor by the same metabolites. In particular, a protein of apparent molecular weight 32,000 is highly modified by derivatives of trans-9,10-dihydro-9,10-dihydroxy B(a)P. This protein is shown here to be preferentially lost from nuclei during purification by centrifugation through high molarity sucrose solutions followed by osmotic shock. It does not appear to be a cytoplasmic contaminant, but shares many properties of an abundant protein from Xenopus laevis oocytes, nucleoplasmin.
Asunto(s)
Benzopirenos/metabolismo , Proteínas Portadoras/aislamiento & purificación , Núcleo Celular/metabolismo , Proteínas Nucleares , Fosfoproteínas , Animales , Benzo(a)pireno , Fraccionamiento Celular/métodos , Núcleo Celular/análisis , Células Cultivadas , Cricetinae , Citoplasma/análisis , Embrión de Mamíferos , Histonas/metabolismo , Nucleoplasminas , Nucleoproteínas , Octoxinol , Polietilenglicoles/farmacología , Proteínas/metabolismo , Sacarosa/farmacologíaRESUMEN
In hamster embryo cells incubated for 24 hr with 4 microM [3H]benzo(a)pyrene, a major portion of the nonextractable radioactivity in nuclear preparations copurifies with the protein fraction. When these proteins are analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis, significant variations in the labeling intensities of the various proteins are seen. Control experiments demonstrate that the labeling is due to covalent binding to protein. Histones H3 an H2A are heavily labeled while the other histones of the nucleosome core, H2B and H4, are devoid of radioactivity. Large amounts of label are associated with proteins with mobilities similar to the very lysine-rich histones H1. However, the results of differential extraction experiments suggest that the labeled proteins do not belong to either the H1 or the high-mobility-group class of chromosomal proteins. During 6 hr of inhibition of protein synthesis by cycloheximide, the metabolism of [3H]benzo(a)pyrene, as monitored by high-pressure liquid chromatography, remained normal. Patterns of labelling of nuclear proteins after 3 or 6 hr were identical in the presence and absence of cycloheximide. This finding strongly suggests that binding of benzo(a)pyrene derivatives to nuclear proteins occurs in situ.
Asunto(s)
Benzopirenos/metabolismo , Carcinógenos/metabolismo , Nucleoproteínas/metabolismo , Animales , Benzo(a)pireno , Células Cultivadas , Cricetinae , Cicloheximida/farmacología , Histonas/metabolismo , Unión ProteicaRESUMEN
Benzo[a]pyrene (B[a]P), 7,8-dihydroxy-7,8-dihydro-B[a]P, and 9,10-dihydro-B[a]P are metabolized by hamster embryo cells to derivatives that bind to nuclear macromolecules. The selectivity for different classes of macromolecules varies depending on the compound analyzed. The ratio of DNA specific activity to protein specific activity (pmol bound/mg of macromolecules) is high (1.51) for 7,8-dihydroxy-7,8-dihydro-B[a]P, extremely low (0.03) for 9,10-dihydroxy-9,10-dihydro-B[a]P, and intermediate (0.26) for B[a]P. Histones H3 and H2A are the major targets of 7,8-dihydroxy-7,8-dihydro-B[a]P; a protein(s) with a mobility similar to that of histone H1 is heavily labeled by 9,10-dihydroxy-9,10-dihydro-B[a]P, with minor labeling of other (nonhistone) bands. The labeling pattern seen with B[a]P is a combination of the patterns seen with the two dihydrodiol metabolites studied. Analysis of the ethyl acetate-soluble metabolites suggests that hamster embryo cells produce 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydro-B[a]P from 9,10-dihydroxy-9,10-dihydro-B[a]P and raise the possibility that this vicinal diol epoxide is an intermediate in the binding of 9,10-dihydroxy-9,10-dihydro-B[a]P to nuclear proteins. The differences seen suggest that factors other than the intrinsic chemical reactivity of the epoxide group are extremely important in the interaction of potential ultimate carcinogens with biological systems.
Asunto(s)
Benzopirenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Dihidroxidihidrobenzopirenos , Histonas/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Cricetinae , ADN/metabolismo , Embrión de Mamíferos , Relación Estructura-ActividadRESUMEN
Metabolites of benzo(e)pyrene (B[e]P) formed upon incubation of [3H]-B[e]P with hepatic microsomes from control and induced rats have been separated by high-pressure liquid chromatography and identified by comparison of retention times, absorbance and fluorescence spectra with those of synthetic standards. The major metabolite produced was B[e]P-4,5-dihydrodiol, accounting for 20-30% of the total metabolism depending on the source of the microsomes. This was followed by a phenolic metabolite (shown not to be 4-OH-; 9-OH-; or 10-OH-B[e]P). A possible proximate carcinogenic derivative of B[e]P, B[e]P-9,10-dihydrodiol, was identified, but was found to constitute less than 1% of the total metabolites. Similar results were obtained with a purified and reconstituted mixed-function oxidase system. In these later incubations, production of the dihydrodiols was dependent on the addition of purified epoxide hydrase to the incubation mixtures. These results suggest that formation of the reactive diol-epoxide, 9,10-dihydroxy-11,12-epoxy-9,10,11,-12-tetrahydro-B[e]P, a potential ultimate carcinogenic metabolite of B[e]P, is not favored by rat liver enzymes. This provides a partial explanation for the lack of carcinogenicity of B[e]P within the framework of the bay region theory of chemical carcinogenesis.