RESUMEN
During their replication, infectious retroviruses insert a reverse-transcribed cDNA copy of their genome, a "provirus", into the genome of their host. If the infected cell belongs to the germline, the integrated provirus can become "fixed" within the host genome as an endogenous retrovirus and be transmitted vertically to the progeny in a Mendelian fashion. Based on the numerous proviral sequences that are recovered within the genomic DNA of vertebrates--up to ten percent in the case of mammals--such events must have occurred repeatedly during the course of millions of years of evolution. Although most of the ancient proviral sequences have been disrupted, a few "endogenized" retroviral genes are conserved and still encode functional proteins. In this review, we focus on the recent discovery of genes derived from the envelope glycoprotein-encoding (env) genes of endogenous retroviruses that have been domesticated by mammals to carry out an essential function in placental development. They were called syncytins based on the membrane fusogenic capacity that they have kept from their parental env gene and which contributes to the formation of the placental fused cell layer called the syncytiotrophoblast, at the materno-fetal interface. Remarkably, the capture of syncytin or syncytin-like genes, sometimes as pairs, was found to have occurred independently from different endogenous retroviruses in diverse mammalian lineages such as primates--including humans--, muroids, leporids, carnivores, caviids, and ovis, between around 10 and 85 million years ago. Knocking out one or both mouse syncytin-A and -B genes provided evidence that they indeed play a critical role in placentation. We discuss the possibility that the immunosuppressive domain embedded within retroviral envelope glycoproteins and conserved in syncytin proteins, may be involved in the tolerance of the fetus by the maternal immune system. Finally, we speculate that the capture of a founding syncytin-like gene could have been instrumental in the dramatic transition from egg-laying to placental mammals.
Asunto(s)
Productos del Gen env/genética , Productos del Gen env/fisiología , Placentación/fisiología , Proteínas Gestacionales/genética , Proteínas Gestacionales/fisiología , Retroviridae/genética , Integración Viral/genética , Animales , Secuencia de Bases , Evolución Biológica , Secuencia Conservada , Retrovirus Endógenos/genética , Femenino , Humanos , Tolerancia Inmunológica , Ratones , Ratones Noqueados , Placentación/genética , Embarazo , Proteínas Gestacionales/deficiencia , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiologíaRESUMEN
High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 8 , Genes myc , Neoplasias de la Mama/patología , Línea Celular Tumoral , Aberraciones Cromosómicas , Cromosomas Artificiales Bacterianos , Células Clonales , Citogenética , ADN de Neoplasias , Femenino , Amplificación de Genes , Humanos , Hibridación Fluorescente in Situ , Hibridación de Ácido Nucleico , Valores de Referencia , Transcripción GenéticaRESUMEN
A series of 13 sporadic renal cell carcinomas was analyzed for the specific chromosome rearrangements after serial xenografting into immunodeficient mice. Seven tumors displayed genetic traits of the conventional subtype and 5 showed genetic features of the papillary subtype. In all the xenografted conventional tumors, we observed loss of 3p, as well as loss of the 9p21 region and of the long arm of chromosome 14, both considered as markers of a poor prognosis. In the xenografted papillary tumors, a duplication of chromosome arm 8q was observed concomitant with the duplication of the 7q31 region. The association of the 7q31 and 8q22 approximately qter duplicated regions was also observed for one conventional tumor. The latency of tumor take was found to be reduced and the median time to passage statistically shorter for all tumors which presented the associated duplication of the 7q31 and 8q22 approximately qter regions. The proto-oncogene NOV (nephroblastoma overexpressed gene) maps to 8q24.1 and is overexpressed in some Wilms tumors. It could be an interesting candidate gene, since its level of expression and release in the culture medium was found to be increased in all of the fast growing tumors analyzed.
Asunto(s)
Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/genética , Deleción Cromosómica , Proteínas Inmediatas-Precoces , Péptidos y Proteínas de Señalización Intercelular , Neoplasias Renales/diagnóstico , Neoplasias Renales/genética , Mutación , Animales , Northern Blotting , Western Blotting , Línea Celular , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 7 , Cromosomas Humanos Par 8 , Cromosomas Humanos Par 9 , Factor de Crecimiento del Tejido Conjuntivo , Duplicación de Gen , Humanos , Cariotipificación , Ratones , Ratones SCID , Trasplante de Neoplasias , Proteína Hiperexpresada del Nefroblastoma , Proteínas Oncogénicas Virales/genética , Pronóstico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The FGF-3 gene is constitutively expressed in tumorigenic clones from the SW613-S human colon carcinoma cell line but is silent in non-tumorigenic clones. We have investigated the transcriptional mechanisms responsible for this differential expression. Mapping of DNase I-hypersensitive sites throughout the FGF-3 gene and the region extending 15 kilobases upstream disclosed differences in the patterns obtained between tumorigenic and non-tumorigenic cells. Transient expression assays carried out with a reporter gene driven by FGF-3 promoter fragments of various lengths (0.143 to 11 kilobases) did not reproduce the differential regulation of the resident gene between the two cell types. The same constructs did exhibit a differential activity in stable transfectants, suggesting the involvement of a chromatin-based mechanism in this regulation. Under these conditions, even the 143-base pair minimal promoter fragment was able to drive the differential expression of the reporter gene. During the course of these analyses, several transcriptional modulatory elements (mainly activators) were identified in the FGF-3 upstream region and were found to colocalize with DNase I-hypersensitive sites. Moreover, a putative new promoter was discovered 6 kilobases upstream of FGF-3. Altogether, these data provide a basis for the elucidation of the complex regulation of the human FGF-3 gene.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Células Clonales , Neoplasias del Colon , Desoxirribonucleasa I , Factor 3 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Transfección , Células Tumorales CultivadasRESUMEN
Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31. We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours. This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis. The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level. However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours. Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.
Asunto(s)
Carcinoma Papilar/genética , Carcinoma de Células Renales/genética , Cromosomas Humanos Par 7/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas c-met/biosíntesis , Proto-Oncogenes , Animales , Southern Blotting , Carcinoma Papilar/patología , Carcinoma de Células Renales/patología , Pintura Cromosómica , Cromosomas Artificiales de Levadura , Humanos , Neoplasias Renales/patología , Ratones , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The keratin 18 (K18) gene is overexpressed in cells of tumorigenic clones isolated from the SW613-S human colon carcinoma cell line, compared to cells of nontumorigenic clones. The isolated minimal promoter (TATA box and initiation site) of the K18 gene has by itself a differential activity in tumorigenic and nontumorigenic cells. An Sp1 binding site located upstream of the TATA box contributes to the high level of expression of the gene in tumorigenic cells. We report here that the Sp1 gene is not differentially expressed between the two cell types and that this is also the case for genes coding for factors of the preinitiation complex known to directly interact with the Sp1 protein. Further, DNase I footprinting experiments and mutagenesis analysis indicated that the mechanism responsible for the differential activity of the minimal K18 promoter apparently does not involve the binding of a factor to a specific sequence. During the course of these experiments, it was found that the initiation site of the K18 promoter is actually located 11 bp upstream of the +1 position previously reported and that the TATA box is the only essential element of the minimal promoter. Treatment of the cells with histone deacetylase inhibitors was more efficient at stimulating the activity of the K18 promoter in nontumorigenic cells than in tumorigenic cells. We propose that overexpression of the K18 gene in tumorigenic cells could result from of a high level of acetylation of histones and/or of factors controlling the activity of the transcription complex.
Asunto(s)
Regulación de la Expresión Génica , Queratinas/genética , Secuencia de Bases , Sitios de Unión , Butiratos/farmacología , Neoplasias del Colon , Huella de ADN , ADN Complementario , Desoxirribonucleasa I , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Mutagénesis , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética , Factores de Transcripción/genética , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.
Asunto(s)
Neoplasias del Colon/patología , Genes myc , Células Clonales , Neoplasias del Colon/genética , Medio de Cultivo Libre de Suero , Regulación Neoplásica de la Expresión Génica , Genes p53 , Humanos , Fenotipo , Transfección , Células Tumorales CultivadasRESUMEN
With the purpose of studying the transcriptional regulation of the human FGF-3 gene, we have cloned and determined the nucleotide sequence of the 11-kbp region flanking its 5' end. Analysis of the sequence disclosed the presence of multiple repetitive elements. Remarkably, all of them were found to have inserted in the same orientation as the FGF-3 gene, suggesting that the whole upstream region could play a role in the control of its transcription. Unique regions within the sequence were scanned for the presence of transcriptional regulatory elements. A potential "Initiator" sequence preceded by several motifs homologous to binding sites for transcription factors pinpointed a putative promoter, 6 kbp upstream of the ATG codon for the FGF-3 protein. A 250-nt sequence stretch surrounding the "Initiator" was found to display punctate homology with the first (P1) of the three promoters (P1, P2 and P3) of the mouse Fgf-3/int-2 gene, specifically in the region of the transcriptional start sites. These data should be useful in studying the mechanisms of regulation of the FGF-3 transcription unit.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Proteínas Proto-Oncogénicas/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Clonación Molecular , Codón , Factor 3 de Crecimiento de Fibroblastos , Humanos , Ratones , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
The human colon carcinoma cell line, SW613-S, is composed of cells with a high-level amplification of the MYC proto-oncogene that are tumorigenic in nude mice and of cells with a low-level amplification of MYC that are not tumorigenic. Transcripts from FGF-3, a member of the fibroblast growth factor gene family, accumulate in cells from tumorigenic clones, but are undetectable in those from non-tumorigenic clones. Nuclear run-on analyses indicate that this differential FGF-3 expression is regulated at the level of transcription initiation. Determination of the structure of the FGF-3 transcripts indicates that they are generated by splicing of the three exons and termination at the single polyadenylation site predicted from the genomic sequence. Their size heterogeneity is due to multiple initiation sites spanning a 700 base-pair long promoter region. FGF-3 is activated in tumors induced in nude mice by MYC-transfected cells from non-tumorigenic clones. However, in most of the cell lines established from these tumors, FGF-3 expression tends to be lost upon in vitro propagation. Thus, in these transfectant cell lines, the presence of exogenous MYC gene copies is not sufficient to activate FGF-3 expression and in vivo growth is also required.
Asunto(s)
Neoplasias del Colon/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Actinas/metabolismo , Animales , Secuencia de Bases , Neoplasias del Colon/genética , Factor 3 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Genes myc , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.
Asunto(s)
Queratinas/genética , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
Clonal analysis has shown that the SW613-S human colon-carcinoma cell line is heterogeneous: some cell clones display a high level of amplification of the c-myc gene and are tumorigenic in nude mice, whereas others have a small number of copies of this gene and are non-tumorigenic. Tumorigenic clones can proliferate in a chemically defined serum-free medium, whereas non-tumorigenic clones cannot. Suramin, like anti-insulin-like growth factor (IGF) or anti-IGF-I receptor antibodies, efficiently inhibits the growth of tumorigenic clones in defined medium. Inhibition by suramin or by anti-IGF antibodies can be reversed by pure IGF-I or IGF-2. Pure IGF-1 or IGF-2 or culture medium conditioned by tumorigenic clones can stimulate DNA synthesis in cells of non-tumorigenic clones. Co-culture with cells of tumorigenic clones sustains the growth of non-tumorigenic clones in defined medium. Cells of both tumorigenic and non-tumorigenic clones express high-affinity IGF-1 receptors at their surface but tumorigenic clones produce on average 5 times more IGF-1 and 25 times more IGF-2 than non-tumorigenic ones. These results indicate that autocrine growth stimulation of tumorigenic clones by IGFs through the IGF-1 receptor is essential for their ability to grow in defined medium. Since cells of tumorigenic clones produce IGF-2 at levels 80 times higher than IGF-1 and since an antibody strictly specific for IGF-1 has no effect on DNA synthesis in cells of tumorigenic clones grown in defined medium, IGF-2 is very likely the main effector in the autocrine loop.
Asunto(s)
Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Factor II del Crecimiento Similar a la Insulina/fisiología , División Celular/fisiología , Células Clonales , Neoplasias del Colon/genética , Medio de Cultivo Libre de Suero , Amplificación de Genes , Genes myc , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/fisiología , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Estimulación Química , Células Tumorales CultivadasRESUMEN
Two types of clones have been isolated from the SW613-S human colon carcinoma cell line. Clones with a high level of amplification of the c-myc gene are tumorigenic in nude mice and can proliferate in chemically defined, serum-free medium, whereas clones with a low level of amplification are nontumorigenic and cannot multiply in defined medium. The expression level of the insulin-like growth factor type 1 (IGF-1) gene is low in tumorigenic clones and undetectable in nontumorigenic clones. Tumorigenic clones produce high levels of IGF-2 (and IGF-binding proteins), compared to nontumorigenic clones. This is the consequence of a differential transcriptional regulation of the IGF-2 gene between the two types of clones. This regulation consists of a modulation of the activity of promoters P3 and P4. Overexpression of the IGF-2 gene is constitutive in tumorigenic clones: it is stably maintained during in vitro propagation of the cells. Tumorigenic cell lines obtained after transfer of c-myc gene copies into the cells of nontumorigenic clones exhibit a high level of expression of the IGF-2 gene when they are grown in vivo, as subcutaneous tumors in nude mice. This high level of expression is lost in most of these cell lines when they are returned to in vitro culture conditions indicating that, in these cells, IGF-2 overexpression is not constitutive but inducible by in vivo growth conditions. We had previously shown that tumorigenic clones use the overproduced IGF-2 as an autocrine growth factor. The results reported here suggest than IGF-2 overexpression has an important role in the tumorigenic phenotype of these cells.
Asunto(s)
Carcinoma/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Transcripción Genética , Animales , Secuencia de Bases , Carcinoma/metabolismo , Proteínas Portadoras/biosíntesis , Neoplasias del Colon/metabolismo , Citoplasma/química , Exones/genética , Amplificación de Genes , Genes myc/genética , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Hígado/química , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Regiones Promotoras Genéticas/genética , Células Tumorales CultivadasRESUMEN
The c-myc gene is amplified and the c-Ki-ras gene is mutated in the SW613-S human colon carcinoma cell line. Two cell types with different levels of c-myc amplification are present in the SW613-S cell population and representative cell clones can be isolated. The clones with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice whereas those with a low level are not. The tumorigenic clones secrete transforming growth factor alpha (TGF-alpha) in the culture medium whereas the non-tumorigenic clones do not produce any detectable amount. Accordingly the level of TGF-alpha mRNA is higher and the transcription rate of the gene is increased in the tumorigenic clones. The acquisition of the tumorigenic phenotype by cells of non-tumorigenic clones, following introduction of c-myc gene copies by transfection, is accompanied by an increase in the steady-state level of TGF-alpha mRNA. These findings suggest a role for an elevated level of TGF-alpha production in the tumorigenic phenotype of SW613-S cells. The possibility that this role is indirect is discussed.
Asunto(s)
Carcinoma/patología , Neoplasias del Colon/patología , Factor de Crecimiento Transformador alfa/biosíntesis , Animales , Carcinoma/metabolismo , Transformación Celular Neoplásica , Células Clonales , Neoplasias del Colon/metabolismo , Amplificación de Genes , Genes myc , Humanos , Ratones , ARN Mensajero/análisis , Transcripción Genética , Factor de Crecimiento Transformador alfa/análisis , Factor de Crecimiento Transformador alfa/genética , Células Tumorales CultivadasRESUMEN
Subclones of the SW 613-S human colon carcinoma cell line differ by their ability to induce tumors in nude mice and by their level of amplification of the c-myc gene. Clones with a high level of amplification are tumorigenic in nude mice whereas those with a low level are not. Genes overexpressed in the tumorigenic clones as compared to the nontumorigenic ones were searched by differential screening of a cDNA library. Two cDNA clones corresponding to cytokeratin K18 and ferritin-H chain were isolated. The steady state level of the corresponding mRNAs is higher in cells of all tumorigenic clones. The level of cytokeratin K8 mRNA, the specific partner of cytokeratin K18 in intermediate filaments of epithelial cells, is also elevated in these cells. For all three genes, this is mainly due to an increase in the transcription rate, as shown by a nuclear run-on assay. Immunoblotting experiments showed that cytokeratins K8, K18, and K19 are more abundant in cells of tumorigenic clones. The mRNA of the other subunit of apo-ferritin (ferritin-L chain) is expressed at the same level in both types of clones. The mRNAs of cytokeratins K18 and K8 and of ferritin-H chain are also overexpressed in cells of nontumorigenic clones which have acquired a tumorigenic phenotype after transfection of c-myc gene copies.
Asunto(s)
Ferritinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Queratinas/biosíntesis , ARN Mensajero/biosíntesis , Secuencia de Bases , Células Clonales , Neoplasias del Colon/genética , Ferritinas/química , Ferritinas/genética , Genes myc/genética , Humanos , Queratinas/genética , Datos de Secuencia Molecular , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Amplification of the c-myc gene has been frequently reported in breast carcinomas. However the precise function of the c-myc protein is still unknown and the nature of the selective advantage offered to a cell by an overexpression of such a protein is unclear. We are addressing this question using the SW 613-S human breast carcinoma cell line as a model system. This cell line harbours an amplified c-myc gene and a mutated c-Ki-ras gene. By various criteria the amplified c-myc gene of SW613-S cells appears undistinguishable from a normal human c-myc gene. The SW613-S cell line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. DM-containing cells are progressively lost upon in vitro cultivation but are selected for during in vivo growth, as tumors in nude mice, or by cultivating the cells in a chemically defined, serum-free medium or under conditions preventing anchorage. Clones with different levels of amplification and different chromosomal localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice, whereas those with a low level are not. Introduction of c-myc gene copies by transfection confers tumorigenicity to the nontumorigenic clones, indicating that a high level of amplification of the c-myc gene contributes to the tumorigenic phenotype of SW 613-S cells. Tumorigenic clones grow unattached, are able to proliferate in a chemically defined medium, and produce high levels of several growth factors (e.g. TGF-alpha, IGF2). Nontumorigenic clones are more dependent upon anchorage for growth, show a restricted growth in defined medium, and produce low or undetectable level of the growth factors tested. We have identified several genes, besides c-myc, the expression level of which is markedly different in the two types of clones. TGF-alpha, IGF2, PDGF-A, int-2, cytokeratins K8 and K18 and ferritin H chain are overexpressed in tumorigenic clones. In contrast, c-erbB1 (EGF receptor), c-jun, vimentin and p53 are expressed at a higher level in the nontumorigenic clones. Finally the major histocompatibility class I antigens, ferritin L chain, TGF-beta and c-Ki-ras, are examples of genes expressed at the same level in both types of clones.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Neoplasias de la Mama/genética , Amplificación de Genes , Marcadores Genéticos/análisis , Sustancias de Crecimiento/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Línea Celular , Femenino , Sustancias de Crecimiento/genética , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas c-mycRESUMEN
The human HBL-100 cell line harbours SV40 DNA integrated in tandem at a unique site. The SV40 T-antigen expressed in these cells is defective in a function essential to the replication of the viral genome. The integrated SV40 sequences were molecularly cloned in a bacteriophage, and a subclone (plasmid pSVHBI) containing a complete SV40 DNA was isolated. As compared to SV40 wild-type strain 776, sequence analysis of pSVHBI early region revealed the presence of several DNA alterations. Among these, a point mutation at position 3199, predicting a change at amino-acid 540 of arginine to isoleucine, was shown by marker rescue to be responsible for the deficiency of T-antigen. This novel mutation further delimits one of the T-antigen domains involved in SV40 DNA replication. Transfection experiments demonstrated that the transforming activity of the SV40 genome from HBL-100 cells is still preserved. Moreover, several transformed human cell clones thus obtained could be permanently established in culture.
Asunto(s)
Clonación Molecular , ADN Viral/análisis , Virus 40 de los Simios/genética , Secuencia de Bases , Línea Celular , Replicación del ADN , Humanos , Mutación , TransfecciónRESUMEN
The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.
Asunto(s)
Neoplasias de la Mama/genética , Mapeo Cromosómico , Amplificación de Genes , Proto-Oncogenes , Southern Blotting , Pruebas de Carcinogenicidad , Bandeo Cromosómico , Femenino , Humanos , Immunoblotting , Hibridación de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
The c-myc gene is amplified in the SW 613-S cell line which was established from a human breast carcinoma. This line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. Clones with different levels of amplification and different cytological localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene were highly tumorigenic in nude mice whereas those with a low level were not. Introduction of c-myc gene copies by transfection into the cells of several non-tumorigenic clones restored the tumorigenic phenotype. Our results indicate that a high level of amplification of the c-myc gene is a requirement for the tumorigenicity of SW 613-S cells in animals.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Células Tumorales Cultivadas/patología , Animales , Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/etiología , Fenotipo , Proteínas Proto-Oncogénicas c-myc , Transfección , Células Tumorales Cultivadas/trasplanteRESUMEN
In vitro transfection experiments have shown that cooperation between two different oncogenes can confer a fully malignant phenotype to primary rodent cells. We have previously reported that SW 613-Tul cells, derived from a tumor induced in a nude mouse by the human breast carcinoma cell line SW 613-S, showed a 30-fold amplification of the c-myc gene. In the present work, we show that these cells also harbor an activated c-Ki-ras gene capable of inducing the formation of foci upon transfection of NIH 3T3 cells with SW 613-Tul genomic DNA. Our results suggest that both the c-myc and c-Ki-ras oncogenes, activated by two different mechanisms, may cooperate in the full expression of the tumorigenic phenotype of SW 613-Tul cells.
Asunto(s)
Neoplasias de la Mama/genética , Amplificación de Genes , Regulación de la Expresión Génica , Genes ras , Proto-Oncogenes , Transfección , Animales , Línea Celular , Células Cultivadas , ADN de Neoplasias/genética , Femenino , Humanos , Ratones , Ratones Desnudos , Hibridación de Ácido NucleicoRESUMEN
The human epithelial HBL-100 cell line harbors SV40 genetic information and has an unlimited growth potential. Despite displaying properties characteristic of transformation since its early in vitro passages, it is capable of producing progressively growing tumors in nude mice only after long-term culture. This is a reproducible phenomenon and apparently not the consequence of a selection of preexisting malignant cells. Superinfection of early passage nontumorigenic HBL-100 cells with Kirsten murine sarcoma virus, which contains a Ki-ras oncogene having undergone multiple activating events, induces morphologic alterations and rapidly converts the cells to neoplastic cells, further supporting the hypothesis of multistep carcinogenesis. The HBL-100 cell line might be useful in defining the oncogenes representative of different families, which are able to complement SV40 in this system.