RESUMEN
A conditional expression system was established whereby the human K-ras, v-src, and v-mos genes were cloned into a conditional expression vector downstream of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Rat-1 fibroblasts were transfected with these constructs and selected in medium containing G418. Cloned transfectants were isolated and characterized for absolute dependence on dexamethasone for expression of oncogene products and anchorage-independent growth in soft agar. Expression of activated p21K-ras(val12) enabled the fibroblasts to degrade extracellular matrix collagen secreted by murine microvessel endothelial cells. Concurrent with p21K-ras(val12) induction a proteinase with the characteristic size and substrate specificity of transin, the murine homologue of the human matrix metalloproteinase stromelysin, was expressed and secreted. Induction of v-mos and v-src oncogenes resulted in little or no detectable transin expression respectively coinciding with a relative or absolute failure to increase degradation of extracellular matrix collagen. This study suggests that in this system the expression of the ras oncogene can contribute to the in vitro invasive behavior of tumor cells by upregulating the production of a metalloproteinase capable of degrading collagen synthesized by vascular endothelial cells.
Asunto(s)
Colágeno/metabolismo , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Genes ras , Metaloendopeptidasas/biosíntesis , Animales , División Celular/fisiología , Células Cultivadas , Dexametasona/farmacología , Endotelio Vascular/fisiología , Inducción Enzimática/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Expresión Génica , Vectores Genéticos , Virus del Tumor Mamario del Ratón/genética , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/metabolismo , Ratones , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Oncogénicas v-mos/genética , Proteínas Oncogénicas v-mos/metabolismo , ARN Mensajero/genética , Ratas , Transfección , TritioRESUMEN
The ability of tumor cells to express elevated levels of proteinases capable of degrading tissue matrix and basement membrane components in vitro has been correlated to their invasive and metastatic potential. Many in vitro invasion assays have been performed either in the presence of serum or with tumor cells that had been previously grown in serum. Since serum contains large amounts of active proteinase inhibitors, their presence could complicate interpretations. We have, therefore, attempted to measure the amounts of serine proteinase inhibitors released into culture medium by two rat mammary adenocarcinoma metastatic variants selected in vitro for serum-independent growth and differing in their in vivo metastatic behavior. Concentrated spent media (CSM) derived from cultures of poorly metastatic MTLn2(T42D) and highly metastatic MTLn3(T17D) tumor cells, grown in the presence and absence of fetal bovine serum (FBS) for 20-24 h, were compared for the presence of serine proteinase inhibitors capable of inactivating alpha-chymotrypsin. Our results show that when MTLn2(T42D) and MTLn3(T17D) tumor cells were exposed to FBS, the CSM of MTLn2(T42D) exhibited nearly 5-fold greater amounts of active proteinase inhibitors than that of MTLn3(T17D). The amount of proteinase inhibitory activity detected in the CSM of tumor cells not exposed to FBS was not eliminated but declined by 82% and 37% for MTLn2(T42D) and MTLn3(T17D), respectively. Analysis for enzyme-inhibitor (E-I) complex formation by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography confirmed the kinetic results and revealed that the major inhibitor present in CSM/FBS of both variants forms a heat- and sodium dodecyl sulfate-stable E-I complex with an apparent molecular weight of approximately 79,000, identical to that formed when FBS or purified alpha 1-proteinase inhibitor is incubated with [alpha-125I]chymotrypsin. E-I complexes with apparent molecular weights of 44,000 and 50,000 were formed from CSM/bovine serum albumin of MTLn3(T17D) and MTLn2(T42D), respectively, that were not detected when [alpha-125I]chymotrypsin was incubated with bovine serum albumin. We infer from these observations that, in culture, poorly metastatic MTLn2(T42D) tumor cells, as compared to their highly metastatic MTLn3(T17D) counterparts, exhibit an increased capacity to retain and subsequently release significantly greater amounts of serum-derived active proteinase inhibitors. Moreover, the detection of proteinase activity by kinetic analysis and E-I complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography in CSM prepared from cultures not exposed to FBS indicates that both variants have the capacity to produce their own inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Metástasis de la Neoplasia/fisiopatología , Inhibidores de Serina Proteinasa/metabolismo , Animales , Quimotripsina/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Femenino , Neoplasias Pulmonares/secundario , Invasividad Neoplásica , Ratas , Ratas Endogámicas F344RESUMEN
The pharmacokinetics and tissue distribution in mice of several recombinant human alpha-interferons [rHuIFN-alpha A, D, I, and A/D(Bgl)] as well as natural mouse alpha-interferon (MuIFN-alpha) were assessed following single intravenous injections. The serum profiles of rHuIFN-alpha A, rHuIFN-alpha D, rHuIFN-alpha A/D(Bgl), and MuIFN-alpha were similar, whereas those following rHuIFN-alpha I showed a much longer terminal elimination phase. Differences in elimination half-life, volume of distribution, and total body clearance between these IFNs were observed. There was appreciable uptake of IFN in the kidney: the amount of each interferon per gram of tissue in the kidney ranges from 1 to 9 times the amount found in the serum. The greatest uptake appeared with rHuIFN-alpha D, followed by rHuIFN-alpha A, rHuIFN-alpha A/D(Bgl), and MuIFN-alpha. The only exception was rHuIFN-alpha I which showed no uptake into the kidney.
Asunto(s)
Interferón Tipo I/metabolismo , Animales , Transporte Biológico Activo , Humanos , Interferón Tipo I/sangre , Riñón/metabolismo , Cinética , Ratones , Especificidad de la Especie , Distribución TisularRESUMEN
The in vivo fate of radiolabeled recombinant human leukocyte A interferon (125I-rIFN-alpha A) and intramolecular hybrid A/D (125I-rIFN-alpha A/D Bgl) were studied, following iv injection into CD1 mice. Trichloroacetic acid (TCA) precipitable radioactivity, as well as antiviral activity, were measured in sera and several organ extracts. The results obtained by bioassay were very similar to those obtained by measuring the TCA precipitable radioactivity. The kidney and liver showed preferential uptake of these 125I-IFNs. These studies indicate the feasibility of using radioiodinated IFNs in tissue distribution and pharmacokinetic evaluations.
Asunto(s)
Interferón Tipo I/metabolismo , Animales , Mucosa Gástrica/metabolismo , Humanos , Interferón Tipo I/sangre , Radioisótopos de Yodo , Riñón/metabolismo , Cinética , Hígado/metabolismo , Ratones , Distribución TisularRESUMEN
A new rapid assay has been developed for measurement of the binding of [3H]retinoic acid to cellular retinoic acid-binding protein. The assay, which uses activated charcoal for the separation of bound from unbound retinoic acid, was used to determine the concentration required to inhibit the binding of [3H]retinoic acid to cellular retinoic acid-binding protein by 50% for 18 retinoids with free carboxylic acid groups. Partially purified cellular retinoic acid-binding proteins isolated from rat testes and carcinogen-induced rat mammary tumors were used for these determinations. The following parameters were also determined for some or all of the retinoids: hypervitaminosis A doses; activity against carcinogen-induced mouse skin papillomas; inhibition of growth of a rat chondrosarcoma; inhibition of growth of 3T6 cells; and differentiation of the embryonal carcinoma cell line PCC4.azaIR. While all retinoids that are potent in these biological test systems bind tightly to cellular retinoic acid-binding protein, the converse is not true. The lack of a consistent quantitative correlation between 50% inhibitory concentration and biological activity is probably due to insufficient concentrations of the retinoid in the target tissue or celll, which is a consequence of factors such as absorbability, metabolism, tissue distribution, and pharmacokinetics.
Asunto(s)
Proteínas de Unión al Retinol/metabolismo , Tretinoina/metabolismo , Vitamina A/análogos & derivados , Animales , Unión Competitiva , Condrosarcoma/prevención & control , Femenino , Técnicas In Vitro , Masculino , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Neoplasias Experimentales/prevención & control , Papiloma/prevención & control , Ratas , Neoplasias Cutáneas/prevención & control , Testículo/metabolismo , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
(4-Methoyx-2,3,6-trimethylphenyl)nonatetraenoic acids, esters, and amides (analogues of retinoic acid) bearing a fluorine atom(s) or a trifluoromethyl group on the polyene side chain were synthesized. The biological activities of these compounds and of 10-, 12-, and 14-fluororetinoic acid esters were evaluated in vivo in a chemically induced mouse papilloma test; the toxicities were assessed in an in vivo mouse hypervitaminosis A test. Antipapilloma activity greater than the parent nonfluorinated ester was found for 1c (ethyl 12-fluororetinoate) and 23 and 39 (aromatic 4- and 6-fluororetinoid esters, respectively). A similar increase in antipapilloma activity was observed for 71 and 72, the aromatic 4- and 6-fluororetinoic acids, respectively, relative to 2 and for 73 (aromatic 4-fluororetinoid amide) relative to 4.