RESUMEN
Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 microgram ml-1), between fucose-labeled and mannose-labeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of MO4 cells with SW (0.4 microgram ml-1). The shift induced by dNM (10 mM) + SW (0.4 microgram ml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; 1 mM) had major effects on both mannose and fucose-labeled structures and so did N-methyl-1-deoxynojirimycin (MdNM; 2 mM) and castanospermine (CS; 100 micrograms ml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucose-labeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Indolizinas , Invasividad Neoplásica , Péptidos/metabolismo , Polisacáridos/metabolismo , Pirrolidinas , 1-Desoxinojirimicina , Alcaloides/farmacología , Animales , Antivirales/farmacología , Transformación Celular Viral/fisiología , Embrión de Pollo , Glucosamina/análogos & derivados , Glucosamina/farmacología , Glucosilceramidasa/antagonistas & inhibidores , Glicosilación , Iminofuranosas , Manitol/análogos & derivados , Manosidasas/antagonistas & inhibidores , Ratones , Ratones Endogámicos C3H , Swainsonina , Células Tumorales CultivadasRESUMEN
Rat2 cells are thymidine kinase-deficient derivatives from the immortalized rat embryo cell line Rat1. They show no phenotypic correlates of malignancy in vitro and produce tumors in syngeneic Fischer rats after long latency periods. We have investigated how transfection with oncogenes would alter the in vitro and in vivo behavior of Rat2 cells. Thus we have manipulated Rat2 cultures in various ways. The cell lines obtained were categorized as parental, in vitro subclones, untransfected in vivo derivatives, non-oncogene (neor and tk) transfectants, oncogene (mutated c-Ha-ras, polyoma middle-T, FBR v-gag-fos-fox) transfectants, and in vivo derivatives of transfectants. They were tested in vitro for morphotype, colony formation in soft agar, growth in organ culture, invasion in organ culture, and in vivo for latency period of tumor formation, tumor growth rate, invasiveness, and metastasis. Differences between the consequences of various manipulations were found in the number of malignancy-related phenotypic alterations. The following trend could be deduced from our data: induction of invasiveness in organ culture by all manipulations; morphotypic transformation and shortening of tumor-latency period by all oncogene transfections and by passage with tumor formation in vivo; growth in organ culture and increased tumor growth rate in vivo by transfection with ras-, or fos-oncogenes and by passage in vivo. Metastatic capability (present in parental Rat2 cell tumors) and colony formation in soft agar (absent in Rat2 cells) were not affected by the present manipulations. We concluded that differences between the oncogene-transfectants and the untransfected in vivo derivatives do not lie in the expression of malignancy-related phenotypes but in the time needed to acquire them.
Asunto(s)
Transformación Celular Neoplásica , Oncogenes , Timidina Quinasa/genética , Transfección , Animales , División Celular , Línea Celular , Cósmidos , ADN/genética , Embrión de Mamíferos , Genes ras , Fenotipo , RatasRESUMEN
Estramustine (EM) is a conjugate of estradiol and nor-nitrogen mustard (nor-HN2), which is effective in the treatment of prostate cancer. We have compared the effect of EM with that of the known microtubule inhibitor vinblastine (VLB) on the following functions of malignant MO4 mouse cells and of DU-145 human prostate cancer cells in vitro: directional migration, invasion; and the organization and the assembly/disassembly equilibrium of microtubule complexes. The circular area covered by cells migrating from an aggregate explanted on a solid substrate was taken as an index of directional migration. Invasion was studied through confrontation of MO4 or DU-145 cells with fragments of embryonic chick heart in organ culture. Microtubules were investigated immunocytochemically and through immunodetection on protein blots. VLB and EM inhibited directional migration and invasion of MO4 and DU-145 cells in a dose-dependent manner; equimolar combinations of estradiol plus nor-nitrogen mustard did not mimic these effects. At anti-invasive concentrations VLB led to partial disassembly of microtubule complexes, whereas EM resulted in an abnormal pattern of microtubule complexes without alteration of the overall assembly/disassembly equilibrium. Combined treatment with VLB and EM resulted in an enhanced VLB effect, namely complete disassembly. In all tests DU-145 cells were more sensitive to both VLB and EM than were MO4 cells, and the effects were less reversible. The present experiments showed that EM shares an anti-invasive activity with other microtubule inhibitors.
Asunto(s)
Carcinoma/patología , Estramustina/farmacología , Compuestos de Mostaza Nitrogenada/farmacología , Neoplasias de la Próstata/patología , Animales , Movimiento Celular/efectos de los fármacos , Estradiol/farmacología , Humanos , Masculino , Mecloretamina/farmacología , Ratones , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Técnicas de Cultivo de Órganos , Tubulina (Proteína)/metabolismo , Vinblastina/farmacologíaRESUMEN
Invasion in vitro and in vivo and spontaneous metastasis was investigated in cell lines before and after introduction of immortalizing (polyoma large-T and activated myc) genes and of transforming (polyoma middle-T and activated ras) genes in Fischer rat cells. Invasion in vitro was tested by confrontation of rat cells with embryonic chick heart fragments in organ culture. Invasion in vivo and metastasis was evaluated in nude mice and in syngeneic rats after injection of cells i.p. or s.c. in the flank and after implantation of cell aggregates s.c. in the tail. Rat cells were also analyzed for the presence of myc oncogenes, and for the expression of ras oncogenes. Cells from primary or low passage rat embryo (REF) cells were not invasive in vitro and did not produce tumors in vivo. Cell lines (LTRAT1, LTaRAT1) derived from REF cultures after transfection with plasmids encoding polyoma large-T antigens, behaved like REF cells. Cell lines (REFpEJgpt4, REFpEJmycN7) established from REF cultures after transfection with either a plasmid encoding an activated human ras protein or with the latter plasmid plus one containing an activated myc gene, were invasive in vitro and in vivo and produced invasive and metastatic tumors in syngeneic rats. Cell lines (FR3T3) established in an apparently spontaneous way were invasive in vitro and produced invasive tumors in vivo without metastasis. Derivatives of FR3T3 (FRLT1, MTT4, MMC1, and PyT21) transfected with plasmids encoding one or more of the polyoma antigens, differed from FR3T3 cells by a shorter latency period of tumor formation (less than 1 versus 1 to 3 weeks). Like FR3T3 tumors, FRLT1, MTT4, MMC1, and PyT21 tumors were invasive but not metastatic. Other spontaneously established lines (Rat1) were invasive and metastatic. Cells (Rat1pEJ6.6) derived from Rat1 cultures after transfection with a plasmid encoding an activated ras protein, showed shorter tumor latency periods (less than 1 versus 7 weeks). A thymidine kinase deficient Rat1 derivative (Rat2) was not invasive in vitro but produced invasive and metastatic tumors in vivo with long (9 to 21 weeks) latency periods. Rat2pT24B4 cells derived by us from Rat2 cells after transfection with a plasmid containing a mutated human ras gene (pT24), were invasive in vitro and in vivo as were cells derived from Rat2 tumors. We conclude from our experiments that invasiveness and metastatic capability are often acquired by established REF-derived cell lines in an apparently spontaneous way.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Antígenos Virales de Tumores/genética , Metástasis de la Neoplasia , Neoplasias Experimentales/patología , Oncogenes , Animales , Ciclo Celular , Movimiento Celular , Transformación Celular Viral , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Desnudos , Neoplasias Experimentales/genética , Poliomavirus/genética , Ratas , TransfecciónRESUMEN
Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO4 cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasion in vivo. Tunicamycin (1.0 microgram/ml), 2-deoxy-D-glucose (100 mM), beta-OH-norvaline (1.0 mM), and Monensin (0.1 microgram/ml) reversibly inhibited the invasion of MO4 cells. At these concentrations the drugs also inhibited the growth of MO4 cells. 1-Deoxynojirimycin (10mM), swainsonine (0.4 microgram/ml), and Marcellomycin (0.1 microgram/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO4 cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO4 cells in vitro.
Asunto(s)
Antraciclinas , Metabolismo de los Hidratos de Carbono , Membrana Celular/efectos de los fármacos , Glicoproteínas/metabolismo , 1-Desoxinojirimicina , Alcaloides/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Comunicación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Desoxiglucosa/farmacología , Glucosamina/análogos & derivados , Glucosamina/farmacología , Corazón , Ratones , Monensina/farmacología , Naftacenos/farmacología , Neoplasias , Sacarasa/antagonistas & inhibidores , Swainsonina , Treonina/análogos & derivados , Treonina/farmacología , Tunicamicina/farmacologíaRESUMEN
Light and electron microscopic investigations on mammalian cells in vitro and in vivo showed that tubulozole-C (R 46 846), the cis-isomer of tubulozole, a new synthetic anticancer drug, interfered with the structure and function of microtubules in both interphase and mitotic cells. The activity of this compound in experimental tumor systems can thus be explained partly by a direct antimitotic effect and partly by the disintegration of the normal subcellular organization of the nondividing cells. At concentrations which affect the microtubule system, tubulozole-C arrested directional migration of transformed cells and malignant invasion in a three-dimensional organ culture system. Investigations in vivo show that malignant L1210 leukemia cells are more susceptible to the antimicrotubular effect of tubulozole-C than are the normal leukocytes of the host. The trans-isomer of tubulozole (tubulozole-T, R 48 265), which has no antitumor activity in vivo, did not affect the microtubule system of cells in vitro or their capacity for directional migration or for malignant invasion.
Asunto(s)
Dioxolanos/farmacología , Dioxoles/farmacología , Microtúbulos/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Pollos , Dipodomys , Femenino , Humanos , Leucemia L1210/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Microscopía Electrónica , Miocardio/metabolismo , Polímeros/metabolismo , Embarazo , Tubulina (Proteína)/metabolismoRESUMEN
Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40 degrees C. Invasion was absent for at least 7 days at or below temperatures of 29 degrees C. Invasion was invariably observed at or above 30.5 degrees C. Differences in invasion between 29 and 30.5 degrees C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28 degrees C (MO(4)28 cell line) did not differ in invasiveness from the parent MO4 cells. We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.
Asunto(s)
Fibrosarcoma/patología , Invasividad Neoplásica , Neoplasias Experimentales/patología , Temperatura , Animales , Línea Celular , Embrión de Pollo , Matriz Extracelular/ultraestructura , Glicoproteínas/análisis , Pulmón/ultraestructura , Ratones , Microscopía Electrónica , Miocardio/ultraestructura , Técnicas de Cultivo de ÓrganosRESUMEN
Spheroids of MO4 mouse fibrosarcoma cells were irradiated with single doses of photons between 1 and 50 Gy. Growth of irradiated spheroids was followed in suspension culture and after explantation on glass. We found that MO4 spheroids recovered from higher doses of irradiation when they were explanted on glass than when they were kept in suspension culture. These results suggest that irradiated MO4 cell populations may become anchorage-dependent for growth.
Asunto(s)
Adhesión Celular , División Celular/efectos de la radiación , Animales , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Electrones , Fibrosarcoma , Ratones , MitosisRESUMEN
Podophyllotoxin (PPT) and its glycosylated congeners, 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-beta-D-glucopyranoside] (VP-16-213) and 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-thenylidene-beta-D-glucopyranoside] (VM-26), were studied for their effects on the following activities of MO4 mouse fibrosarcoma cells in vitro: growth as an aggregate in culture on a gyrotory shaker, directional migration from an aggregate explanted on glass, organization of the cytoplasmic microtubule complex as inferred from immunostaining with an antiserum against tubulin, and invasion into fragments of 9-day-old embryonic chick cardiac muscle. At concentrations that inhibited growth (greater than or equal to 0.03 microgram/ml), PPT arrested directional migration, abolished the cytoplasmic microtubule complex, and interfered with invasion. VM-26 (0.1-1.0 microgram/ml) and VP-16-213 (1-30 micrograms/ml) interfered with growth but permitted directional migration, organization of the cytoplasmic microtubule complex, and invasion. These observations imply that microtubule inhibitors are anti-invasive because they interfere with the assembly of the cytoplasmic microtubule complex and not because they inhibit growth.