RESUMEN
BACKGROUND/AIMS: We have developed a gene therapy strategy based on the observation that insulin-like growth factor I (IGF-I) is necessary for the acquisition and maintenance of the transformed phenotype in hepatocarcinoma. This strategy consists in transfecting the rat hepatoma cell line with an episomal vector expressing the antisense IGF-I c-DNA under the control of the metallothionein I promoter inducible by zinc, decreasing therefore the level of IGF-I in these cells. The transfected clones lost their tumorigenic properties, and were able to induce, in vivo, the regression of an established tumor in syngeneic rats. To understand the loss of tumorigenic properties of these transfected clones, we have quantified, by different approaches, the number of apoptotic cells according to the level of IGF-I expression. METHODS: IGF-I antisense synthesis in transfected cells was stimulated using zinc. We then characterized and quantified apoptosis, in these transfected clones, by morphological and DNA fragmentation analyses, flow cytometry and comet assay. RESULTS: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clones are able to restore the spontaneous apoptotic programme, and that apoptosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. CONCLUSION: The present results allow us to conclude that the level of apoptotic pathway in liver cell lines is directly related to the amount of IGF-I deficiency.
Asunto(s)
Apoptosis/genética , Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas/patología , Oligonucleótidos Antisentido , Animales , Carcinoma Hepatocelular/genética , ADN Complementario/genética , Citometría de Flujo , Neoplasias Hepáticas/genética , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
By using the PCR-SSCP technique we characterized various ER-specific RNA species present in a series of primary breast cancers, as well as in cell lines established from breast carcinomas and in mammary gland tissues from healthy specimens. A series of six truncated messenger RNAs generated by alternative splicing was characterized. These RNAs correspond to specific deletions of one (exons 2-7, except exon 6) or two (exons 3 + 4) exons. All these RNA variants are observed in each one of the analyzed RNAs, regardless of origin. In addition, the relative amount of these different variants in ER + tumors is comparable to that measured in ER - tumors and healthy mammary gland tissues. This data suggests that tumor progression is not related to the emergence of any of the ER mRNA variants.
Asunto(s)
Neoplasias de la Mama/genética , Mama/química , Eliminación de Gen , ARN Mensajero/análisis , Receptores de Estrógenos/genética , Empalme Alternativo , Secuencia de Bases , ADN Complementario , Electroforesis en Gel de Agar , Exones , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , ADN Polimerasa Dirigida por ARN , Moldes Genéticos , Células Tumorales CultivadasRESUMEN
The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).
Asunto(s)
Proteínas Portadoras/farmacología , Somatomedinas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Animales , División Celular/efectos de los fármacos , Embrión de Pollo , ADN/biosíntesis , ADN/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Insulina/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Ratones , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.
Asunto(s)
Proteínas Portadoras/farmacología , Replicación del ADN/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Animales , Proteínas Portadoras/aislamiento & purificación , Células Cultivadas , Embrión de Pollo , Embrión de Mamíferos , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Insulina/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Cinética , Ratones , Somatomedinas/fisiologíaRESUMEN
IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn2+ (10(-3)M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10(-8)M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface.
Asunto(s)
Proteínas Portadoras/metabolismo , Inhibidores de Crecimiento/metabolismo , Oligopéptidos/metabolismo , Animales , Sitios de Unión , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Inhibidores de Crecimiento/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Cinética , Manganeso/farmacología , Oligopéptidos/farmacología , Somatomedinas/metabolismoRESUMEN
Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.
Asunto(s)
Fibroblastos/metabolismo , Inhibidores de Crecimiento/farmacología , Mitógenos/farmacología , Oligopéptidos/farmacología , Proteína Oncogénica pp60(v-src)/fisiología , ARN/biosíntesis , Animales , Sangre , Línea Celular Transformada , Embrión de Pollo , ADN/biosíntesis , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteína Oncogénica pp60(v-src)/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transformación GenéticaRESUMEN
Density-dependent inhibition (DDI) of growth is assumed to be the result of diffusion in the medium of growth inhibitory molecules. In this work, we demonstrate the presence of inhibitory molecules (IDFc: chicken inhibitory diffusible factor) in the medium of chick embryo fibroblasts (CEF) cultures. IDFc partially purified by Bio-Gel P150 chromatography followed by reverse phase FPLC. The dose-response curve showed that 250 ng/ml IDFc inhibited 50% DNA synthesis. IDFc was also able to inhibit the growth of sparse cultures of CEF; this inhibition was reversible. IDFc was unable to prevent the DNA synthesis in cells transformed by v-src gene expression. These results suggest that IDFc is involved in the DDI of CEF growth.
Asunto(s)
Fibroblastos/metabolismo , Inhibidores de Crecimiento/metabolismo , Animales , Virus del Sarcoma Aviar , Transformación Celular Viral , Células Cultivadas , Embrión de Pollo , Cromatografía Líquida de Alta Presión , ADN/biosíntesis , Difusión , Electroforesis en Gel de Poliacrilamida , Inhibidores de Crecimiento/farmacologíaRESUMEN
An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio-Gel P150 chromatography and 2 reverse-phase FPLC. After the final step of purification, 60 ng/ml of IDF45 inhibited 50% of alpha-globulin-stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by alpha-globulin.
Asunto(s)
Inhibidores de Crecimiento/aislamiento & purificación , Interfase/efectos de los fármacos , alfa-Globulinas/farmacología , Animales , Línea Celular , ADN/biosíntesis , Inhibidores de Crecimiento/farmacología , Ratones , ARN/biosíntesisRESUMEN
Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.