RESUMEN

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-alpha and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-kappa B, STAT3, PI3K, and beta-catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.

Asunto(s)

Andrógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Transducción de Señal , Acetilcisteína/farmacología , Androstadienos/farmacología , Proteínas Reguladoras de la Apoptosis , División Celular/fisiología , Supervivencia Celular/fisiología , Citocinas/metabolismo , Citocinas/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Factor de Unión 1 al Potenciador Linfoide , Masculino , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Próstata/tratamiento farmacológico , Factor de Transcripción STAT3 , Ligando Inductor de Apoptosis Relacionado con TNF , Timidina/metabolismo , Transactivadores/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Wortmanina , beta Catenina

RESUMEN

Expression and structural organization of tyrosine aminotransferase (TAT) gene in Morris hepatoma cell line 7777 with active and glucocorticoid-inducible TAT gene and in hepatoma 8994, where TAT gene does not function were analysed. No differences in the number of receptor macromolecules, translocation and nuclear binding of hormone-receptor complexes in hormone sensitive (7777) and resistant (8994) cell lines were demonstrated. Dexamethasone increases TAT gene transcription in 7777 cell line but not 8994. Restriction analysis of TAT gene does not reveal any differences either in structural or in regulatory regions. Gel retardation assay with cloned TAT fragment (-400 b.p.) from normal hepatocytes showed identical shift of mobility in 7777 and 8994 cell lines. Moreover, 5'-flanking sequence (-890 b.p.) of TAT gene linked to the bacterial CAT gene is transiently expressed in both cell lines. We have shown that HpaII site (-105 b.p.) of TAT gene is methylated in those cells where TAT gene does not function (thymus, spleen, Zajdela ascites hepatoma) and is demethylated in TAT gene expressing hepatoma 7777 and normal rat hepatocytes. In hepatoma 8994 there are no DNAse I hypersensitive regions, typical to functioning TAT gene from hepatoma 7777 and normal hepatocytes.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas Experimentales/enzimología , Tirosina Transaminasa/genética , Animales , Cloranfenicol O-Acetiltransferasa/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Genes tat , Metilación , Plásmidos , ARN Mensajero/genética , Ratas , Mapeo Restrictivo , Transcripción Genética , Transfección

RESUMEN

The expression of the tyrosine aminotransferase (TAT) mRNA after cycloheximide treatment was analysed by Northern blotting method in Morris rat hepatoma cell lines. The level of TAT mRNA increased after 6-8 h of cycloheximide treatment only in the McA-RH 7777 cell line. McA-RH 7777 nuclear run-off assay showed that TAT transcription was induced by cycloheximide treatment. Both glucocorticoid and cycloheximide modulated TAT gene transcription in a synergistic way. There was no induction of TAT expression following cycloheximide or cycloheximide glucocorticoid simultaneous treatment in another cell line (McA-RH 8994), while c-myc and c-fos expression was superinduced by cycloheximide treatment. The possible mechanism of transcription regulation and its damage in hepatoma cells is discussed.

Asunto(s)

Cicloheximida/farmacología , Neoplasias Hepáticas Experimentales/enzimología , Transcripción Genética/efectos de los fármacos , Tirosina Transaminasa/genética , Animales , Northern Blotting , Southern Blotting , ADN de Neoplasias/genética , Electroforesis en Gel de Poliacrilamida , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Ratas , Células Tumorales Cultivadas

RESUMEN

Glucocorticoids control protein synthesis and [3H]thymidine incorporation into DNA in resting and mitogen-stimulated in vitro human peripheral blood lymphocytes. This effect depends on the dose and time of hormone addition to the culture medium. Using two-dimensional electrophoresis, it was demonstrated that the steroids can regulate the expression of various proteins in intact and mitogen-stimulated human peripheral blood lymphocytes. This is suggestive of a dynamic control by glucocorticoids of the synthesized protein transcription during the cell cycle of a given population. These results also indicate that such a dynamic control is not due to the fluctuations in the number of receptor macromolecules, the presence or absence of the acceptor site on the DNA but, rather, to the modulation of the biological activity of specific nuclear factors capable of regulating the transcription of definite genes.

Asunto(s)

Dexametasona/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Biosíntesis de Proteínas , Células Cultivadas , ADN/biosíntesis , Electroforesis en Gel Bidimensional , Humanos , Interleucina-2/farmacología , Cinética , Linfocitos/citología , Linfocitos/metabolismo , Mitógenos

RESUMEN

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5. No other specific binding activities were found in the rat-liver nuclear fractions tested under these conditions. As the metal ions became inaccessible to chelation in excess of EDTA and EGTA when protein factor A5 was complexed to DNA we assumed that factor A5 is metalloprotein which requires Zn or Ca to maintain a structure of its DNA-binding domain. To identify the polypeptide possessing this domain, a protein gel blotting procedure was employed. By incubating gel blots with the 3 2P-DNA-probe in the buffer containing Zn2+, specific binding to the only polypeptide with approximate Mr 30 kDa was clearly revealed. Both gel retardation and gel blotting assays consistently showed that nuclear factor A5 is present in the liver, but not in the spleen extracts.

Asunto(s)

Proteínas de Unión al ADN/aislamiento & purificación , Hígado/metabolismo , Metaloproteínas/aislamiento & purificación , Metales/metabolismo , Proteínas Nucleares/aislamiento & purificación , Regiones Promotoras Genéticas , Tirosina Transaminasa/genética , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Metaloproteínas/genética , Metaloproteínas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Bazo/metabolismo

RESUMEN

Kinetics of tyrosine aminotransferase (TAT) mRNA synthesis in Morris rat hepatoma cell lines 7777 and 8994 after dexamethasone treatment (10(-7) M) was studied by molecular hybridization of the RNA with cloned fragments of TAT gene from rat liver cells. It was demonstrated that initiation of TAT gene transcription increased 20 minutes after glucocorticoid treatment. The level of TAT mRNA was not induced by dexamethasone in rat hepatoma cell line 8994. Actinomycin D prevented the deinduction of TAT by stabilization of TAT mRNA.

Asunto(s)

Genes , Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/genética , Transcripción Genética/efectos de los fármacos , Tirosina Transaminasa/genética , Animales , Dactinomicina/farmacología , Inducción Enzimática , Neoplasias Hepáticas Experimentales/enzimología , Ratas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Tirosina Transaminasa/biosíntesis

RESUMEN

Glucocorticoids induce tyrosine aminotransferase synthesis in 7777 Morris hepatoma but fail to do so in Zajdela hepatoma. This internal property indicates the resistance to the hormone. However, both hepatoma cell lines do respond to the triamcinolone acetonide in a similar way, as judged by some other criteria, e. g. interaction with the immobilized hormone on the inert carrier, adhesion to glass and kinetic parameters of alkaline phosphodiesterase I activity. Moreover, both cell types respond to glucocorticoids by modification of synthesis of some proteins, as revealed previously by two-dimensional electrophoresis. The results show that in case of tumour cells which retain their specific receptor apparatus but do not respond to glucocorticoids by usual criteria, the conclusion whether tumour cells are hormone-sensitive or not has to be drawn from the analysis of their multiple response judging by several assays.

Asunto(s)

Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hormono-Dependientes , Animales , Línea Celular , Inducción Enzimática/efectos de los fármacos , Técnicas In Vitro , Neoplasias Hepáticas Experimentales/enzimología , Tirosina Transaminasa/metabolismo

RESUMEN

The mechanisms of reversible decrease of hormone-dependent induction of tyrosine aminotransferase (TAT) by rat liver cells after prolonged administration of the glucocorticoid was studied. It was shown that the main links of the glucocorticoid action mechanism (i.e., the formation of a cytoplasmic hormone-receptor complex and the hormone accumulation in the nuclei) do not change under these conditions. It was found also that one of the necessary prerequisites for the decrease of the hormone-dependent induction of TAT is the constant production by liver cells of large amounts of TAT irrespective of whether this process is induced by the glucocorticoid or by a non-hormonal inducer, e.g., tryptophan. Using the dot-hybridization technique, it was demonstrated that the inhibition of hormone-dependent induction of TAT is correlated with the reduction of mRNA TAT. It was supposed that the main links in the mechanism of inhibition of the hormone-dependent induction are the formation of a large excess of the inducible protein--TAT--in the cells as well as the accumulation of end products of the TAT-catalyzed transamination reaction which cause a feed-back repression of the de novo synthesis of TAT. Studies with cell cultures of Morris hepatoma which is known to be sensitive to glucocorticoids revealed the ability of glucose, the end product of gluconeogenesis reactions, to provide for selective inhibition of the hormone-induced accumulation of mRNA TAT in hepatoma cells.

Asunto(s)

Hidrocortisona/farmacología , Hígado/efectos de los fármacos , Tirosina Transaminasa/biosíntesis , Adrenalectomía , Animales , Inducción Enzimática , Hidrocortisona/metabolismo , Hígado/enzimología , Neoplasias Hepáticas Experimentales/enzimología , Masculino , Ratas , Ratas Endogámicas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Factores de Tiempo , Triptófano/farmacología

RESUMEN

The transfer of Morris hepatoma cells induced by the hormone within 10-60 min in to a hormone-free medium is associated with the augmentation of tyrosine aminotransferase synthesis. The kinetics of this process does not differ from that of the hormone-induced enzyme. The return of tyrosine aminotransferase synthesis to the basal level occurs 15-20 hours after the hormone withdrawal from the medium, although the concentration of the intranuclear hormone sharply decreases already after 3 hours. It was demonstrated that the presence in the hepatoma cell nuclei of 20-25% of the initially bound hormone for at least 20 hours after the cell transfer to the hormone-free medium is not sufficient for maintaining a high level of tyrosine aminotransferase gene expression. Using two-dimensional electrophoresis of 3H-labeled hepatoma cell proteins, it was demonstrated that the observed high activity of tyrosine aminotransferase is due to the de novo synthesis of enzyme molecules rather than to the existence of preformed long-living tyrosine aminotransferase molecules inside the cell. Study of [14C]uridine incorporation into non-ribosomal nuclear RNA of hepatoma cells showed a long-term presence of the label in the RNA throughout the chase experiment. It was assumed that the high activity of the enzyme for 10-15 hours after the hormone release from the hepatoma cell nuclei is due to the accumulation in the nuclei of long-living pre-mRNA molecules synthesized after the hormone addition to the cells and during the first hours after the cell transfer to the hormone-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Neoplasias Hepáticas Experimentales/enzimología , ARN Neoplásico/biosíntesis , Triamcinolona Acetonida/farmacología , Tirosina Transaminasa/biosíntesis , Animales , Línea Celular , Dactinomicina/farmacología , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática/efectos de los fármacos , Neoplasias Hepáticas Experimentales/metabolismo , Ratas

RESUMEN

Some regularities of [3H]triamcinolone acetonide (TA)binding to glucocorticoid-sensitive Morris hepatoma cell nuclei were studied. It was shown that part of the hormone incorporated into the nuclei form highly stable complexes with nuclear structures that are not destroyed during nuclei lysis with 0.25% SDS. Such complex formation is not practically suppressed by a 500-fold excess of non-labeled TA. As the time of incubation of Morris hepatoma cells with the hormone rises from 10 min to 24 hours, the specific binding of TA to the nuclei decreases, while the specific radioactivity of the [3H]TA-nuclei complexes resistant to 0.25% SDS increases. The stable complexes are eluted from Sepharose 6B together with the bulk of the nuclear proteins and do not contain DNA. Actinomycin D extrudes, to some extent, the [3H]TA from the complexes having specific binding sites that are localized in the nuclei and induces the accumulation of the steroid in the firmly bound nuclear complexes resistant to 0.25% SDS. The ability to suppress hormonal induction of tyrosine aminotransferase was detected only in the antibiotics with a high affinity for the GC-pairs of DNA. i.e., actinomycin D and mitramycin. It was assumed that high concentrations of TA specifically bound to the nuclei are necessary only at initial steps of hormonal induction. At later stages, gradual dissociation of the complexes takes place and the hormone is accumulation within the composition of the SDS-resistant firmly bound complexes.

Asunto(s)

Dactinomicina/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Triamcinolona Acetonida/metabolismo , Animales , Sitios de Unión , Células Cultivadas , ADN de Neoplasias/metabolismo , Inducción Enzimática/efectos de los fármacos , ARN Neoplásico/biosíntesis , Tirosina Transaminasa/biosíntesis

RESUMEN

The resistance of Morris hepatoma cells strain 8994 to glucocorticoids which lack hormonal induction of tyrosine aminotransferase synthesis was studied. The cells of Morris hepatoma 7777 were used as a sensitive strain by a criterion of the enzyme synthesis. Using two-dimensional polyacrylamide gel electrophoresis, it was demonstrated that, i) in the cells of the "resistant" Morris hepatoma strain 8994 glucocorticoids change the rate of synthesis of at least five proteins, ii) two of these proteins are common for both cell strains, while the other ones are individual for each line, iii) in the cells of Morris hepatoma 8994 tyrosine aminotransferase is not controlled by glucocorticoids, iiii) glucocorticoids may not only control the activated genes but also the genes whose expression is suppressed. It was assumed that in the absence of disturbances in the receptor apparatus the resistance of any cell population to glucocorticoids can only be established by the use of a great variety of experimental approaches. The resistance of a cell population to the hormones cannot be judged upon by an analysis of the intensity of synthesis of one or even several proteins.

Asunto(s)

Glucocorticoides/farmacología , Neoplasias Hepáticas Experimentales/enzimología , Tirosina Transaminasa/genética , Animales , Línea Celular , Resistencia a Medicamentos , Inducción Enzimática , Genes/efectos de los fármacos , Cinética , Neoplasias Hepáticas Experimentales/fisiopatología , Ratas

RESUMEN

The maximal amount of specifically bound triamsinolone acetonide (TA) penetrates into Morris hepatoma cells at initial steps of hormonal induction. This amount is gradually decreased during incubation of the cells with the hormone. As the incubation time rises, the inhibition of [3H]TA binding to the nuclear fraction induced by an excess of the non-labelled hormone is further decreased, which is paralleled with deceleration of the [3H]TA efflux from the nuclei to a hormone-free medium. After removal of the hormone the cells retain their ability to induce the synthesis of tyrosine aminotransferase for 10 hours, although after 3 hours the amount of the bound hormone falls down to 20-25% of the original level. The rate of further deinduction of tyrosine aminotransferase synthesis depends on the incubation time, i.e. in the cells preincubated with TA for 10 min the deinduction occurs at a slower rate than in the cells preincubated with the hormone for 48 hours. The increase in the amount of specifically bound TA 10 min after hormone addition leads to augmented synthesis of tyrosine aminotransferase which surpasses the synthesis providing for the maintenance of maximal induction. An addition of actinomycin D to a hormone-free medium containing the cells preincubated with TA for 48 hours prevents the deinduction. It is assumed that Morris hepatoma cells contain an actinomycin D-sensitive feed-back mechanism which controls the concentration and distribution of specifically bound intranuclear TA depending on hormonal response.

Asunto(s)

Neoplasias Hepáticas Experimentales/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Triamcinolona Acetonida/metabolismo , Animales , Transporte Biológico , Línea Celular , Dactinomicina/farmacología , Inducción Enzimática , Cinética , Ratas , Triamcinolona Acetonida/farmacología , Tirosina Transaminasa/genética

RESUMEN

Resistance of Djungarian hamster cells to colchicine and adriablastin is connected with gene amplification and decreased plasma membrane permeability for cytostatic drugs. Overproduction of protein (mol. weight about 22 Kd and pI about 5.7) was identified in colchicine- and adriablastin-resistant cell lines by means of two-dimensional gel electrophoresis. Obviously, the amplification of this protein genes leads to the changes in plasma membrane permeability and to the development of drug resistance.

Asunto(s)

Colchicina/farmacología , Cricetinae/genética , Doxorrubicina/farmacología , Proteínas/genética , Animales , Permeabilidad de la Membrana Celular , Resistencia a Medicamentos , Amplificación de Genes

RESUMEN

Using the DNA filter binding assay, the effects of ionic strength and pH on SV40 T-antigen interaction with viral DNA were studied. The apparent association constants for T-antigen binding to SV40 DNA in Scatchard coordinates in the presence of 40 mM NaCl are equal to 0.67 . 10(6) M-1 (pH 6.0) and 0.86 x 10(7) M-1 (pH 7.4). These data indicate that the interaction between T-antigen and SV40 DNA is more specific at pH 7.4. The coincident values of association constants for T-antigen binding to viral and cellular DNAs (Ka = 0.9 x 10(7) M-1 for cellular DNA) at pH 7.4 and the absence of competition between the two DNA species upon binding with T-antigen suggest that viral and cellular DNAs possess similar sites for T-antigen binding. Denatured DNA competes with viral DNA only at pH 6.0, when the T-antigen--SV40 DNA interaction is less specific.

Asunto(s)

Antígenos de Neoplasias/inmunología , Antígenos Virales/inmunología , Transformación Celular Viral , ADN Viral/inmunología , ADN/inmunología , Virus 40 de los Simios/inmunología , Complejo Antígeno-Anticuerpo , Antígenos Virales de Tumores , Cinética

RESUMEN

The [3H]hydrocortisone transfer into intact target cells and into non-target cells, e. g. isolated hepatocytes, hormone-sensitive Morris hepatoma 7777 cells, hormone-resistant Zajdela ascite hepatoma cells, was studied. In target cells the glucocorticoid transfer is temperature-dependent, i. e. the curve for hormone binding by the cells at increasing concentrations shows a plateau Phospholipase A2 and neuraminidase decrease the ability of the cells to incorporate the labelled hormone. The experimental data differ significantly from those obtained for target cell cytosol and for intact Zajdela hepatoma cells. It was assumed that in target cells the crucial role in hormone transfer into the cells belongs to plasma membranes, while in non-target cells the hormone penetrates inside the cells by passive diffusion.

Asunto(s)

Hidrocortisona/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Cinética , Masculino , Ratas

RESUMEN

SV40 T-antigen was isolated from hamster tumors and purified about 1600-fold by the procedure including successive ammonium sulfate precipitation, chromatography on DEAE-cellulose, preparative polyacrylamide gel electrophoresis and elimination of the bulk of contaminating cell proteins by the interaction with antibodies to the tissues of normal hamsters. The resulting preparation was not quite homogenous being contaminated with some of cell proteins left. T-antigen in the tumor extract was revealed at least in three distinct forms with molecular weight of 100 000, 200 000, and 400 000. It is proposed that these forms correspond to mono-, di-, and tetramers of the basal protein of T-antigen, although the alternative explanation, the existence of complex of T-antigen with cell proteins, cannot be ruled out.

Asunto(s)

Antígenos de Neoplasias/aislamiento & purificación , Antígenos Virales/aislamiento & purificación , Virus 40 de los Simios/inmunología , Animales , Cricetinae , Sustancias Macromoleculares , Peso Molecular , Proteínas de Neoplasias , Neoplasias Experimentales/análisis , Neoplasias Experimentales/inmunología

RESUMEN

Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.

Asunto(s)

ADN Circular/metabolismo , ADN Viral/metabolismo , Desoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Animales , Bacteriófagos , Cromatina/enzimología , Reparación del ADN , Hígado/enzimología , Neurospora crassa/enzimología , Pseudomonas , Ratas , Virus 40 de los Simios