RESUMEN
While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dioxoles/farmacología , Isoquinolinas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas , Prueba de Complementación Genética , Pérdida de Heterocigocidad , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Tetrahidroisoquinolinas , Trabectedina , Factores de Transcripción , UrocordadosRESUMEN
Nonmalignant diploid human fibroblast cells (GM3498B) derived from a skin biopsy of a patient with Bloom's syndrome have been transformed by transfection with DNA from a tumorigenic mouse cell line (Ha-8) carrying a single copy of the Harvey murine sarcoma virus (Ha-MuSV) genome. The transformed cell lines have an extended life-span, form colonies in agarose, and proliferate in nude mice--characteristics of neoplastic transformation. Like the parental cells, they also exhibit a high spontaneous level of sister chromatid exchanges. Finally, the transformed cells contain most, if not all, of the Ha-MuSV genome as well as the human rasH sequence. These experiments show that these diploid nonmalignant human cells can be used as recipients in transfection experiments for studying the genetic control of neoplastic transformation.
Asunto(s)
Síndrome de Bloom/genética , Transformación Celular Neoplásica , ADN de Neoplasias/genética , Adhesión Celular , Células Cultivadas , Humanos , Oncogenes , TransfecciónRESUMEN
A genomic sequence and cloned complementary DNA has been identified for a novel receptor-like gene of the PDGF receptor/CSF1 receptor subfamily (platelet-derived growth factor receptor/colony-stimulating factor type 1 receptor). The gene recognized a 6.4-kilobase transcript that was coexpressed in normal human tissues with the 5.3-kilobase PDGF receptor messenger RNA. Introduction of complementary DNA of the novel gene into COS-1 cells led to expression of proteins that were specifically detected with antiserum directed against a predicted peptide. When the new gene was transfected into COS-1 cells, a characteristic pattern of binding of the PDGF isoforms was observed, which was different from the pattern observed with the known PDGF receptor. Tyrosine phosphorylation of the receptor in response to the PDGF isoforms was also different from the known receptor. The new PDGF receptor gene was localized to chromosome 4q11-4q12. The existence of genes encoding two PDGF receptors that interact in a distinct manner with three different PDGF isoforms likely confers considerable regulatory flexibility in the functional responses to PDGF.
Asunto(s)
Cromosomas Humanos Par 4 , Genes , Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Células Cultivadas , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Tirosina Quinasas/genética , ARN Mensajero/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas , Distribución TisularRESUMEN
A DNA sequence related to the abl proto-oncogene was identified in human placenta. Molecular cloning and nucleotide sequence analysis revealed two putative exons whose predicted amino acid sequence was most homologous to the corresponding sequences of c-abl and v-abl but was related to other tyrosine kinase genes as well. The new sequence was localized by in situ hybridization and somatic cell genetic analysis to human chromosome 1q24-25, which differs from the location of any previously identified tyrosine kinase gene. The detection of a novel 12-kb transcript by this gene in human normal and tumor cells establishes it as a new member of the tyrosine kinase family that is closely related to but distinct from c-abl.
Asunto(s)
ADN/genética , Oncogenes , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Clonación Molecular , Exones , Humanos , Hibridación de Ácido Nucleico , Placenta/análisis , Proto-Oncogenes MasRESUMEN
Two related Rho GTPase-activating proteins, DLC-1 (deleted in liver cancer 1) and DLC-2, are emerging as bona fide tumor suppressor genes that inhibit cancer cell growth. In this report, we characterized a gene on chromosome Xq13 that encodes DLC-3 (also known as KIAA0189 and STARD8), a third member of the DLC family. The DLC-3 gene has transcripts with alternative 5' ends, one of which, DLC-3alpha, encodes an 1103-amino acid polypeptide highly similar to DLC-1 and DLC-2. A second isoform (DLC-3beta) would yield a protein lacking the N-terminal sterile alpha motif domain. The DLC-3 gene is widely expressed in normal tissues, but DLC-3 mRNA levels were low or absent in a significant number of breast, ovarian, liver and prostate cancer cell lines. Using a cancer profiling array to compare matched tumor and normal human tissues, downregulation of DLC-3 mRNA was observed in kidney, lung, ovarian, uterine and breast cancer samples. By quantitative reverse transcriptase-polymerase chain reaction, DLC-3 expression was reduced in primary prostate carcinomas relative to normal prostate tissue. Transfection of human breast and prostate cancer cells with a DLC-3alpha expression vector inhibited cell proliferation, colony formation and growth in soft agar. These results indicate that deregulation of DLC-3 may contribute to breast and prostate tumorigenesis.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Proliferación Celular , Regulación hacia Abajo , Femenino , Genes Supresores de Tumor , Humanos , Masculino , Datos de Secuencia Molecular , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.
Asunto(s)
Proteínas de Drosophila , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/efectos de los fármacos , Células 3T3/química , Células 3T3/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Secuencia de Bases , Northern Blotting , Química Encefálica/fisiología , Mapeo Cromosómico , Expresión Génica/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/química , Neuronas/citología , Neuronas/efectos de los fármacos , Neurturina , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/análisis , Ratas , Nervio Ciático/química , Nervio Ciático/citología , Nervio Ciático/lesiones , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Testículo/química , Regulación hacia Arriba/fisiologíaRESUMEN
T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.
Asunto(s)
Empalme Alternativo , Mucosa Gástrica/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Núcleo Celular/metabolismo , Mapeo Cromosómico , Cromosomas , Cromosomas Humanos Par 3 , Claudinas , Clonación Molecular , ADN Complementario/metabolismo , Regulación hacia Abajo , Exones , Eliminación de Gen , Biblioteca de Genes , Células HeLa , Humanos , Inmunohistoquímica , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Distribución Tisular , Transcripción Genética , TransfecciónRESUMEN
Previous work had identified a corepressor, NAB1, which represses transcriptional activation mediated by NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcription factors are encoded by immediate-early genes and have been implicated in a wide variety of proliferative and differentiative processes. We have isolated and characterized another corepressor, NAB2, which is highly related to NAB1 within two discrete domains. The first conserved domain of NAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of both NGFI-A and Krox20, and its expression is regulated by some of the same stimuli that induce NGFI-A expression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12 cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that is rearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of the Caenorhabditis elegans genome has identified a gene that bears high homology to both NAB1 and NAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role.
Asunto(s)
Evolución Biológica , Cromosomas Humanos Par 12 , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Proteínas de Neoplasias , Neoplasias/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Caenorhabditis elegans/genética , Diferenciación Celular , División Celular , Mapeo Cromosómico , Secuencia de Consenso , Secuencia Conservada , Cartilla de ADN , Proteína 1 de la Respuesta de Crecimiento Precoz , Proteína 2 de la Respuesta de Crecimiento Precoz , Femenino , Reordenamiento Génico , Genoma , Humanos , Hibridación Fluorescente in Situ , Leiomioma/genética , Lipoma/genética , Liposarcoma/genética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Placenta/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Activación Transcripcional , Neoplasias Uterinas/genética , Dedos de ZincRESUMEN
Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.
Asunto(s)
Cromosomas Humanos Par 2 , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Inmediatas-Precoces , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Células COS , Mapeo Cromosómico , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Datos de Secuencia Molecular , Proteínas de Unión al ARN/genética , Análisis de Secuencia , Activación Transcripcional , Dedos de ZincRESUMEN
Several chemical carcinogens and noncarcinogens were tested for their ability to induce sister chromatid exchanges (SCE) and structural chromosome aberrations in cultured V79-4 Chinese hamster cells. All of the direct-acting carcinogens induced a large increase in SCE frequency. Two chemicals, which are mutagenic in microorganisms but whose carcinogenicity is poorly documented, also increased the frequency of SCE. Carcinogenic polycyclic hydrocarbons caused an increased incidence of SCE only when a metabolizing feeder layer was used, whereas no increase was observed with noncarcinogenic polycyclic hydrocarbons. The other noncarcinogens also did not influence the SCE frequency. Although some chemicals increased the frequency of structural chromosome aberrations, no correlation was found between the frequencies of SCE and aberrations.
Asunto(s)
Carcinógenos/farmacología , Aberraciones Cromosómicas , Cromosomas/efectos de los fármacos , 2-Acetilaminofluoreno/farmacología , 4-Nitroquinolina-1-Óxido/farmacología , 9,10-Dimetil-1,2-benzantraceno/farmacología , Acridinas/farmacología , Benzopirenos/farmacología , Línea Celular , Evaluación Preclínica de Medicamentos , Hidroxiurea/farmacología , Metilcolantreno/farmacología , Metilnitronitrosoguanidina/farmacología , Fenantrenos/farmacología , Compuestos Policíclicos/farmacología , Pirenos/farmacologíaRESUMEN
BACKGROUND: Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have shown to have normal diploid characteristics are hyperplastic, and depends on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. PURPOSE: We investigated whether the chromosomal changes that occurred in these KS cell lines were random contribute to the pathogenesis of KS. METHODS: We used the conventional G-banding technique and fluorescence in sti hybridization to identify structural and numerical chromosomal changes in the KS cell lines. RESULTS: We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibits loss of heterozygosity of loci at region 3p14-ter. CONCLUSION: This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. IMPLICATIONS: Future research should focus on the identification cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 3 , Sarcoma de Kaposi/genética , Translocación Genética , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Animales , Línea Celular , Bandeo Cromosómico , Mapeo Cromosómico , Humanos , Huésped Inmunocomprometido , Terapia de Inmunosupresión/efectos adversos , Hibridación Fluorescente in Situ , Cariotipificación , Neoplasias Renales/inmunología , Ratones , Sarcoma de Kaposi/etiología , Células Tumorales CultivadasRESUMEN
Five foreskin-derived keratinocyte lines, immortalized by transfection of human papillomavirus (HPV16) DNA, were cytogenetically abnormal, exhibiting numerical deviations and altered chromosomes due to translocations, deletions, achromatic lesions, or partial duplications. Furthermore, all lines had cells with either homogeneously staining regions or double minute chromosomes, alterations associated with malignancy or drug resistance. None of these lines were tumorigenic in nude mice, showing that such alterations which are a manifestation of DNA amplification also occur in nonneoplastic cells. By in situ chromosome hybridization, viral sequences were identified on abnormal chromosomes at the junction of chromosome translocations, at achromatic lesions and within homogeneously staining regions and duplicated chromosome segments. Thus, for the first time in an experimental system, HPV16 integration into the cellular genome was associated with the induction of a subset of chromosome alterations. HPV16 integration that frequently occurred at fragile sites and near protooncogenes may be a critical alteration which confers a selective growth advantage and an indefinite proliferative potential to HPV-transfected cells.
Asunto(s)
Aberraciones Cromosómicas/genética , Bandeo Cromosómico , Trastornos de los Cromosomas , ADN Viral/genética , Biblioteca Genómica , Cariotipificación , Papillomaviridae/genética , Transfección , Línea Celular , Humanos , QueratinocitosRESUMEN
Sodium bisulfite, a nonmutagen at neutral pH, induces neoplastic transformation of cultured Syrian hamster fetal cells. Morphologically transformed fibroblast colonies were isolated, and derived cell lines formed anchorage-independent colonies in agarose and progressively growing s.c. fibrosarcomas in nu/nu mice. Five tumorigenic cell lines analyzed by G- and C-banding were chromosomally abnormal with numerical deviations and structural alterations. Three tumors that developed in nude mice had the chromosome constitution of the inoculated transformed cell as well as secondary changes associated with tumor progression. Transformed cell lines had either a predominantly near-diploid or a near-tetraploid population with consistent chromosome gain and loss. Monosomy of the chromosome 13 observed in three cell lines was a nonrandom numerical alteration. Four lines had abnormal chromosomes resulting from deletions, unbalanced translocations, or centric fusions, and one cell line had a chromosome with a homogeneously staining region. Changes of chromosomes 1 and X were observed in three lines. The breakpoints on X chromosome nonrandomly involved the region qa5 which is frequently affected in hamster cells transformed by other carcinogens and may result in loss of genes essential for the maintenance of a normal phenotype. The formation of abnormal chromosomes cannot be directly attributed to the initial DNA damage as bisulfite concentrations effective in causing neoplastic transformation induced a significant but minimal increase in sister chromatid exchanges and failed to cause chromosome aberrations. Bisulfite inhibition of DNA replication might be a contributing factor in the occurrence of abnormal chromosomes. This cytogenetic analysis provides the first evidence that neoplastically transformed cells by a nonclastogenic carcinogen exhibit persistent chromosome rearrangements, a genetic alteration essential to the process of malignant transformation.
Asunto(s)
Transformación Celular Neoplásica , Aberraciones Cromosómicas , Sulfitos , Animales , Células Cultivadas , Bandeo Cromosómico , Cricetinae , Cariotipificación , Mesocricetus , Ratones , Ratones Desnudos , Intercambio de Cromátides Hermanas , Cromosoma XRESUMEN
Malignant mesothelioma (MM) is a neoplasm closely associated with asbestos exposure, which has been implicated in 70-80% of the cases. In this study, nine MM (two fresh surgical specimens, two permanent cell lines, and five xenografts in nude mice) were examined cytogenetically. Six patients had a known history of asbestos exposure. Seven MM were chromosomally abnormal, the majority having complex structural alterations affecting different chromosomes, whereas two fresh surgical specimens had a normal chromosome constitution. Alterations of chromosome 3 were detected in seven cases and changes involving chromosomes 1 and 7 were observed in six cases. The breakpoints of translocations and deletions on chromosome 1 involved several bands; however, 50% of the breakpoints were near the locations of Blym, L-myc, and ski protooncogenes. Forty % of the breaks on chromosome 7 involved bands q11.1-11.2 and 20% were at q22, the location of the met protooncogene. Nonrandom changes on chromosome 3 were interstitial or terminal deletions, and translocations involving the region p14-21. The deleted 3p segment was identifiable as part of a chromosome translocation in one MM and was apparently lost in the other six. The deletions involving 3p are either spontaneous or asbestos-induced lesions at vulnerable genomic sites and are the most common and nonrandom chromosome alterations observed. Possibly 3p abnormalities are causally related to the development of this malignancy.
Asunto(s)
Aberraciones Cromosómicas , Mesotelioma/genética , Adulto , Anciano , Deleción Cromosómica , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 7 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proto-Oncogenes , Células Tumorales CultivadasRESUMEN
The human papillomavirus type 18 integrated in the HeLa cell genome is amplified on chromosome 8. E6, E7, and E1 open reading frames are amplified 5-fold, and the late viral DNA region, the viral long control region, and cellular flanking sequences are amplified 15-fold. The common 5'-flanking cellular DNA was localized by in situ hybridization of normal and HeLa cells only on chromosome 8 band q24. This flanking probe is included in the amplification unit of Colo320 cells, but in the case of HeLa the amplified region does not include the myc gene which is structurally conserved. Viral integration on chromosome 8 represents an independent event and not a rearrangement of viral DNA located on other chromosomes. The amplification of HPV-18 E6 and E7 open reading frames and the constitutive expression of the myc protooncogene may contribute to immortalization and/or proliferative capacity of HeLa cells.
Asunto(s)
Cromosomas Humanos Par 8 , Amplificación de Genes , Oncogenes , Papillomaviridae/genética , Proto-Oncogenes , Secuencia de Bases , Mapeo Cromosómico , ADN Viral/análisis , Reordenamiento Génico , Células HeLa , HumanosRESUMEN
The chromosomes of five chemical carcinogen-transformed strain 2 guinea pig fetal cell lines were identified by G and C banding techniques and were compared with the normal karyotype from secondary untreated cultures. One line transformed by benzo(a)pyrene had a diploid constitution with no G and C band alterations. Three lines were near diploid, one was near tetraploid, and each contained abnormal chromosomes. A 3-methylcholanthrene line had an abnormal metacentric chromosome formed by centric fusion of two nonhomologous autosomes. The three N-methyl-N'-nitrosoguanidine or diethylnitrosamine cell lines exhibited submetacentric or subtelocentric abnormal chromosomes originating from translocations between two No. 1 or a No. 1 and another autosome. The involvement of Chromosome 1 may be due to its association with nucleolar organization. The greater frequency of contact between such chromosomes, compared to other autosomes, creates an increased risk of chromatid exchange possibly explaining their frequent participation in abnormal chromosome formation or nondisjunction.
Asunto(s)
Carcinógenos/farmacología , Transformación Celular Neoplásica , Cromosomas/efectos de los fármacos , Benzopirenos/farmacología , Células Cultivadas , Aberraciones Cromosómicas , Mapeo Cromosómico , Dietilnitrosamina/farmacología , Metilcolantreno/farmacología , Metilnitronitrosoguanidina/farmacología , PoliploidíaRESUMEN
The relationship between the induction of DNA damage as reflected by sister chromatid exchange (SCE) formation and morphological transformation in exponentially growing Syrian hamster embryo cells was determined quantitatively after split doses of chemical or physical carcinogens. With split doses of carcinogen separated by 2 to 24 hr, only N-acetoxy-2-fluorenyl-acetamide (0.50 microgram/ml) enhanced both SCE induction and transformation when compared to single exposure. Split doses of N-methyl-N'-nitro-N- nitrosoguanidine (0.20 microgram/ml), mitomycin C (50 ng/ml), or ultraviolet light (3.0 J/sq m) were less effective than single exposures, while split doses of methyl methanesulfonate (40 micrograms/ml) caused transformation frequencies similar to a single treatment and decreased SCE frequencies with time intervals greater than 4 hr. Split or single exposures of X-irradiation (200 R) resulted in similar low frequencies of transformation and SCE. Contrasting with these results, a significant potentiation of SCE occurred after split doses of N-methyl-N'-nitro-N-nitrosoguanidine in cultures arrested in G1 with arginine-glutamine-deficient medium or by contact inhibition compared to a single treatment. This response was attributed to the interaction of carcinogen with DNA containing unrepaired damage and demonstrates the importance of the cell cycle phase of the target cell during carcinogen exposure for the induction of SCE by split doses of N-methyl-N'-nitro-N-nitrosoguanidine. The similarity of responses for transformation and SCE induction with split doses of carcinogens suggests that DNA lesions involved in SCE are essential for the initiation of neoplastic development.
Asunto(s)
Carcinógenos/toxicidad , Intercambio Genético/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de los fármacos , Rayos Ultravioleta , Animales , Línea Celular , Células Cultivadas , Cricetinae , ADN/genética , Embrión de Mamíferos , Cinética , Mesocricetus , Intercambio de Cromátides Hermanas/efectos de la radiación , Rayos XRESUMEN
Normal human foreskin fibroblasts treated in vitro with a chemical carcinogen or irradiated with ultraviolet light subsequently acquired anchorage independent growth and an extended but finite capacity for exponential growth. All cell lines were derived from cells recovered from colonies that had grown in semisolid medium; cell lines originally treated with a chemical carcinogen produced nodules after s.c. inoculation into nude mice. G-banding analysis of 10 cell lines (including one ultraviolet light line) revealed that seven were chromosomally abnormal with structural and numerical chromosome alterations, one was characterized by a consistent trisomy, and the other two were normal diploid. Structural alterations consisted of chromosome deletions, translocations, and partial chromosome duplications. Although no common structural or numerical abnormality was detected, several structural alterations were observed involving chromosomes 1, 7, 11, and 22, where fgr, erb-B, H-ras-1, and sis protooncogenes, respectively, are located. In one cell line trisomy 17 was a unique chromosome alteration. The induction of chromosome changes may have influenced the proliferative capacity of the treated cells relative to nontreated cells. However, the two cell lines without detectable chromosome changes also had an increased proliferative life span, suggesting that other submicroscopic genetic alterations may have affected cell multiplication. Although carcinogen induced chromosome abnormalities may represent a step in the process of neoplastic development, additional genetic and/or epigenetic changes, are required for indefinite growth and the expression of malignancy.
Asunto(s)
Carcinógenos/farmacología , Transformación Celular Neoplásica , Transformación Celular Neoplásica/efectos de los fármacos , Aberraciones Cromosómicas , Rayos Ultravioleta , Adhesión Celular , Ciclo Celular , Línea Celular , Transformación Celular Neoplásica/efectos de la radiación , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Cariotipificación , MasculinoRESUMEN
Genetic differences in aryl hydrocarbon hydroxylase (AHH) (flavoprotein-linked monoxygenase EC 1.14.14.1) activity in cultured lymphocytes have been linked with individual risk for certain environmentally caused cancers. Cytochrome P1-450 is the form of cytochrome P-450 most closely associated with AHH activity. In this study the chromosomal localization and the expression of human cytochrome P1-450 gene were determined in phytohemagglutinin-stimulated lymphocytes. In situ hybridization analysis provides assignment of the structural gene for human cytochrome P1-450 to chromosome 15q22-q24. Treatment of lymphocytes with benzanthracene increased the amount of mRNA hybridized to the cloned cytochrome P1-450 gene. The level of cytochrome P1-450 mRNA in these lymphocytes correlates well with the induced AHH activity indicating that non-cytochrome P1-450 enzymes contribute little to the individual differences in the level of AHH activity in the lymphocytes. Southern analyses of genomic DNA from individuals with high and low induced AHH activity demonstrated no detectable differences in the pattern or intensity of restriction fragments after treatment with benzanthracene from either individual. This finding together with the excellent correlation between the induced cytochrome P1-450 and AHH activity, suggests that transcriptional control rather than gene amplification or gross form of gene rearrangement accounts for cytochrome P1-450 induction in man. Measurements of cytochrome P1-450 mRNA content in cultured lymphocytes provide an alternative approach to the assay of AHH activity in assessing AHH phenotype and predicting different susceptibilities to deleterious environmental agents.
Asunto(s)
Cromosomas Humanos 13-15 , Sistema Enzimático del Citocromo P-450/genética , Hidrocarburo de Aril Hidroxilasas/genética , Mapeo Cromosómico , Clonación Molecular , Regulación de la Expresión Génica , Genes , Humanos , Activación de Linfocitos , Linfocitos/fisiología , Oxigenasas de Función Mixta/genética , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The majority of adult human epithelial cancers exhibit evidence of genetic instability, and it is widely believed that the genetic instability manifested by aneuploidy or microsatellite instability plays an essential role in the genesis of these tumors. Indeed, most experimental models of cancer also show evidence of genomic instability. The resulting genetic chaos, which has widespread effects on many genes throughout the genome, confounds attempts to determine the precise cohort of genetic changes that are required for the transformation of normal human cells to a tumorigenic state. Here we show that genetic transformation of human kidney epithelial cells can occur in the absence of extensive aneuploidy, chromosomal translocations, and microsatellite instability. These observations demonstrate that the in vitro oncogenic transformation of human cells can proceed without widespread genomic instability.