RESUMO
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.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 8 , Genes myc , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Cromossomos Artificiais Bacterianos , Células Clonais , Citogenética , DNA de Neoplasias , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Hibridização de Ácido Nucleico , Valores de Referência , Transcrição GênicaRESUMO
Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite. This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.
Assuntos
Clonagem Molecular/métodos , Amplificação de Genes , Animais , Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Linhagem Celular , Cricetinae , DNA , DNA Recombinante , Resistência a Medicamentos , Regulação da Expressão Gênica , Mesocricetus , Mutação , Hibridização de Ácido Nucleico , Ácido Fosfonoacéticos/análogos & derivadosRESUMO
Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.
Assuntos
Interferon Tipo I/fisiologia , Oncogenes , Transcrição Gênica , Linhagem Celular , Células Clonais , Humanos , Cinética , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Queratinas/genética , Fator de Transcrição Sp1/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.
Assuntos
Aspartato Carbamoiltransferase , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , DNA/genética , Di-Hidro-Orotase , Amplificação de Genes , Complexos Multienzimáticos , Compostos Organofosforados/farmacologia , Ácido Fosfonoacéticos/farmacologia , Proteínas/genética , Animais , Ácido Aspártico/farmacologia , Linhagem Celular , Clonagem Molecular , Cricetinae , Resistência a Medicamentos , Genes , Mesocricetus , Modelos Genéticos , Hibridização de Ácido Nucleico , Ácido Fosfonoacéticos/análogos & derivadosRESUMO
Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.
Assuntos
Neoplasias da Mama/genética , Amplificação de Genes , Oncogenes , Animais , Neoplasias da Mama/patologia , Linhagem Celular , Cromossomos Humanos , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transcrição Gênica , Transplante HeterólogoRESUMO
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.
Assuntos
Neoplasias do Colo/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Neoplasias do Colo/genética , Fator 3 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Genes myc , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Células Tumorais Cultivadas/patologia , Animais , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Amplificação de Genes , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/etiologia , Fenótipo , Proteínas Proto-Oncogênicas c-myc , Transfecção , Células Tumorais Cultivadas/transplanteRESUMO
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.
Assuntos
Neoplasias do Colo/patologia , Genes myc , Células Clonais , Neoplasias do Colo/genética , Meios de Cultura Livres de Soro , Regulação Neoplásica da Expressão Gênica , Genes p53 , Humanos , Fenótipo , Transfecção , Células Tumorais CultivadasRESUMO
Proto-oncogenes are the genes which are most frequently found amplified in human tumor cells. Acquisition of a drug-resistant phenotype by gene amplification is frequent for in-vitro cultured cells but is very rare in human tumors. Proto-oncogenes amplified in human tumors belong essentially to one of three families (erbB, ras, myc) or to the 11q13 locus. Amplification is always specific for the tumor cells and is not found in constitutional DNA of the patient, indicating that amplification of the gene is selected for during tumor growth. For genes of the first three families, amplification results in overexpression in most of the cases. These are strong arguments in favor of a role of this amplification in tumor progression. The gene whose overexpression is the driving force for the selection of the amplification of the 11q13 locus is not known. The prad1 gene is presently a good candidate. Amplification of one type of proto-oncogene is generally not restricted to one tumor type. However, the N-myc gene is amplified mainly in tumors of neuronal or neuroendocrine origin and L-myc amplification is restricted to lung carcinomas. To understand the role of proto-oncogene amplification and overexpression in tumor progression it is necessary to know the function of the corresponding protein in the cell. erbB proteins are transmembrane receptors for growth factors. ras genes encode small GTP-binding proteins which are possibly involved in signal transduction. The myc proteins are transcription factors. The expression of the c-myc gene is induced a few hours after cells of various types have been induced to proliferate. The genes of these three families therefore encode proteins which appear to be involved in signal transduction. It is possible that overexpression of one of them, as a result of gene amplification, makes the cell a better responder to low levels of growth stimuli. For several genes which are found amplified in human tumors, it was shown that overexpression of the normal protein could confer a transformed or tumorigenic phenotype to in-vitro cultured cells. In addition, several studies on animal and human tumor-derived cell lines with an amplified proto-oncogene have established a relationship between proto-oncogene amplification and the tumorigenic phenotype. In neuroblastomas, it was proposed that down-modulation of MHC Class I antigens is a consequence of N-myc amplification and that this could be important in the progression toward a metastatic phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Amplificação de Genes , Neoplasias/genética , Proto-Oncogenes , Animais , Humanos , Neoplasias/patologia , Proto-Oncogene MasRESUMO
A cDNA corresponding to the beta subunit of the human translocon-associated protein was cloned and sequenced. The polypeptide is 183 amino acids long and 96% homologous to its canine counterpart. Both polypeptides contain a cleavable signal sequence, an NH2-terminal domain extruding in the endoplasmic reticulum lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm.
Assuntos
Proteínas de Ligação ao Cálcio , Glicoproteínas de Membrana , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Cães , Retículo Endoplasmático/química , Humanos , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/química , Receptores de Peptídeos/química , Homologia de Sequência de AminoácidosRESUMO
A rearranged tpr-met oncogene was identified in a MNNG-transformed human Xeroderma pigmentosum (XP) cell line (ASKMN). A 2016 bp cDNA was cloned and sequenced, disclosing an ORF with a coding capacity for a 523 aa protein. The sequence of this tpr-met cDNA was very similar to that previously reported in another human MNNG-transformed cell line (MNNG-HOS).
Assuntos
DNA Complementar/isolamento & purificação , Fibroblastos/metabolismo , Metilnitronitrosoguanidina , Proteínas de Fusão Oncogênica/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Transformada , Clonagem Molecular , DNA Complementar/biossíntese , Fibroblastos/efeitos dos fármacos , Humanos , Dados de Sequência MolecularRESUMO
We used polymerase chain reaction amplification of minisatellite sequences (33.6.3, MS51, YNZ22) and of a Y chromosome-specific sequence (DYZ1) to document prospectively chimerism in 23 leukemia patients grafted with non T-cell depleted marrows from HLA-identical sibling donors. Twenty-two patients had a complete hematopoietic chimerism (within the sensitivity limit of the method used: 1%) early (about 1 month) after transplantation and one had detectable residual host cells (partial chimerism). These cells were still present after 8 months, and this patient relapsed 16 months after transplantation. Two patients with early complete chimerism relapsed 16 and 17 months after transplantation. Seven patients died from toxicity or infections and 13 are in clinical remission with a follow-up of 16 to 48 months. Nine male patients grafted with the marrow from a female donor were also studied by amplification of the DYZ1 marker (0.01% sensitivity). In all nine cases, residual male nucleated cells were detected early and up to 1 year after transplantation. These results suggest that the detection of persistent residual recipient cells above a 1% level might be predictive of relapse but that the detection of such cells in a 0.01-1% range is probably unrelated to relapse and does not seem to influence the outcome of the transplantation procedure.
Assuntos
Transplante de Medula Óssea/patologia , Leucemia/terapia , Quimera , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Genético , Prognóstico , Sequências Repetitivas de Ácido Nucleico , Fatores de Tempo , Doadores de Tecidos , Cromossomo YRESUMO
Marrow transplantation from unrelated donors has been linked with an increased risk of graft-versus-host disease (GVHD). In an attempt to lower the risk of acute GVHD we used CD34 marrow cell selection for T cell depletion. Since T cell depletion has been linked to an increased risk of relapse and an increased risk of marrow failure, we used PCR amplification of minisatellite sequences to investigate donor cell engraftment and RT-PCR amplification of recurrent chromosomal translocations to investigate the residual disease post-transplant. Twenty-three patients who underwent BMT after positive selection of the CD34-positive cell population were studied. Results were then compared with those of 37 patients who underwent transplantation with unmanipulated marrow graft. Among the 23 patients who received CD34+ selected cell grafts, seven (30%) had evidence of full donor engraftment, 14 had evidence of residual recipient cells (61%), one had a non-take, and one autologous bone marrow recovery. Analysis of the chimaerism status post-transplant in 36 patients who received unmanipulated marrow grafts showed that 31 patients (86%) had evidence of full donor engraftment. The difference in the incidence of mixed chimaerism profile between patients who received unmanipulated marrow graft and those receiving CD34+ selected cell grafts was statistically significant (P< 0.01). Nine patients who received CD34+ selected cell grafts could be analysed for the presence of minimal residual disease post-transplant (one with t(9;22) acute lymphoblastic leukaemia and eight with CML). In the patient transplanted for a Ph-positive acute leukaemia, and in two out of the eight patients with CML, the search fora fusion transcript was consistently negative after transplantation. Among the six patients with evidence of residual disease, three patients also had a mixed chimaerism profile and were given donor lymphocyte infusions. Minimal residual disease study was performed post-transplant in 16 patients who received unmanipulated marrow grafts. In 10 of 14 patients with CML, and in two patients with acute leukaemia the search for a fusion transcript was consistently negative after transplantation. The difference in the incidence of minimal residual disease between patients who received an unmanipulated marrow graft and those receiving CD34+ selected cell grafts was not statistically significantly significant, but numbers of patients included in this analysis are still few. In conclusion, our study highlights the strong influence of graft manipulation on the incidence of mixed chimaerism after transplantation from an unrelated donor.
Assuntos
Purging da Medula Óssea/métodos , Transplante de Medula Óssea , Leucemia/terapia , Síndromes Mielodisplásicas/terapia , Proteínas de Fusão Oncogênica , Quimeras de Transplante , Adolescente , Adulto , Antígenos CD34 , Criança , Pré-Escolar , Subunidade alfa 2 de Fator de Ligação ao Core , Feminino , Proteínas de Fusão bcr-abl/análise , Humanos , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Reação em Cadeia da Polimerase , Proteína 1 Parceira de Translocação de RUNX1 , Proteínas Recombinantes de Fusão/análise , Fatores de Transcrição/análise , Transplante HomólogoRESUMO
Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.
Assuntos
Separação Celular/métodos , DNA/análise , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Iodeto Peroxidase/genética , Reação em Cadeia da Polimerase , Alelos , Sequência de Bases , DNA/química , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Humanos , Dados de Sequência Molecular , GravidezRESUMO
We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.
Assuntos
Clonagem Molecular , Genes Bacterianos , Vetores Genéticos , Plasmídeos , Regiões Promotoras Genéticas , Acetiltransferases/genética , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase , Dados de Sequência Molecular , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from P0 previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481-3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron 1 was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from P0 and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from P0 (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.
Assuntos
Processamento Alternativo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , DNA Complementar , Humanos , Células Tumorais CultivadasRESUMO
Secondary malignancies (lymphomas, leukemias, and solid tumors) occurring after bone marrow transplantation are now more frequently reported, as the patients surviving the early phase of the graft and remaining free of their original disease are more numerous. Besides early Epstein-Barr virus-associated B-cell lymphoproliferative diseases, which are the most common type and most often of donor origin, few late-occurring lymphomas have been described that might represent a distinct entity. We report here a case of Hodgkin's disease developing 8 years after allogeneic bone marrow transplantation for chronic myelogeneous leukemia. Only two Hodgkin's diseases after allogeneic bone marrow transplantation have been reported in the literature so far. The case we report is of interest because of its donor origin and its association with Epstein-Barr virus infection.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Doença de Hodgkin/etiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Neoplasias do Mediastino/etiologia , Segunda Neoplasia Primária/etiologia , Doadores de Tecidos , Adulto , Ciclofosfamida/uso terapêutico , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/transmissão , Herpesvirus Humano 4/isolamento & purificação , Doença de Hodgkin/patologia , Humanos , Terapia de Imunossupressão/métodos , Masculino , Neoplasias do Mediastino/patologia , Metotrexato/uso terapêutico , Segunda Neoplasia Primária/patologia , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/transmissão , Irradiação Corporal TotalRESUMO
We have developed a method to analyze the size distribution of the first-strand cDNA molecules corresponding to given mRNA species. First-strand molecules synthesized from cytoplasmic polyadenylated RNAs are separated by electrophoresis on an alkaline agarose gel, and a Southern blot hybridization is performed. As an example, we analyzed the first-strand molecules corresponding to the human c-myc mRNAs. This method can be used to determine whether full-length, first-strand molecules corresponding to an mRNA species to be cloned are synthesized efficiently. Interestingly, this method allows one to analyze full-length, first-strand cDNA molecules with a much higher resolution than Northern blot analysis of mRNA molecules. This method can therefore be used to discriminate between the multiple mRNA species transcribed from a given gene or the homologous mRNA species transcribed from a given gene family.
Assuntos
DNA Complementar/análise , RNA Mensageiro/genética , Southern Blotting , Clonagem Molecular , Sondas de DNA , DNA Complementar/biossíntese , Eletroforese em Gel de Ágar , Genes myc , Humanos , Hibridização de Ácido Nucleico , Poli A/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Transcrição GênicaRESUMO
Donor hematopoiesis or donor chimerism in the host following allogeneic bone marrow transplantation (BMT) has appeared crucial to the engraftment process. However, as molecular techniques exploiting neutral variation in human genetic material have been used in the study of chimerism, the detection of residual host cells or mixed hemopoietic chimerism has indicated that donor chimerism is not obligatory following BMT. This review focuses on the detection and significance of mixed chimerism (MC) in patients transplanted for both malignant and non-malignant hemopoietic disease and attempts to tease out the contribution of MC to engraftment, leukemia relapse, graft rejection and long-term disease-free survival.