RESUMEN

High-performance liquid chromatography (HPLC) is a recently introduced high-capacity automated method for detecting unknown mutations (denaturing HPLC) or for sizing DNA fragments under nondenaturing conditions. We have adapted the HPLC method for detection of loss of heterozygosity (LOH) and used glial tumours as a model to evaluate its sensitivity and specificity in comparison to conventional denaturing polyacrylamide gel electrophoresis. A total of 20 oligodendrogliomas (grades II and III), and five astrocytic tumours (grades III and IV) were analysed using 14 polymorphic microsatellite markers mapping to regions on chromosomes 1p, 19q, and 10q using both DNA-HPLC and denaturing gel electrophoresis. This study demonstrated complete concordance between both methods. However, unlike gel electrophoresis, HPLC is automated, does not require post-PCR processing, and does not require hazardous radioactive or expensive fluorescent labelling. Our data suggest that HPLC is a reliable and sensitive method for detection of allelic losses in tumour samples and it is a favourable alternative for high-sensitivity LOH detection in both research and diagnostic environments.

Asunto(s)

Neoplasias Encefálicas/genética , Cromatografía Líquida de Alta Presión , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Electroforesis en Gel de Poliacrilamida , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad

RESUMEN

The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype. We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin. FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively. In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells. Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836). YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.

Asunto(s)

Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 2/genética , Mieloma Múltiple/genética , Translocación Genética/genética , Células 3T3 , Adulto , Animales , Cromosomas Artificiales de Levadura , Progresión de la Enfermedad , Resultado Fatal , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Ratones , Mieloma Múltiple/terapia , Mapeo Físico de Cromosoma , Células Tumorales Cultivadas

RESUMEN

Allelic deletions of 10q25-26 and 19q13.3-13.4 are the most common genetic alterations in glial tumors. We have identified a balanced t(10;19) reciprocal translocation in the A172 glioblastoma cell line which involves both critical regions on chromosomes 10 and 19. In addition, loss of an entire copy of chromosome 10 has occurred in this cell line suggesting that the translocation event may provide a highly specific critical inactivating event in a gene responsible for tumorigenesis. Positional cloning of this translocation breakpoint resulted in the identification of a novel chromosome 10 gene, WDR11, which is a member of the WD-repeat gene family. The WDR11 gene is ubiquitously expressed, including normal brain and glial tumors. WDR11 is composed of 29 exons distributed over 58 kilobases and oriented towards the telomere. The translocation resulted in deletion of exon 5 and consequently fusion of intron 4 of WDR11 to the 3' untranslated region of a novel member, ZNF320, of the Krüppel-like zinc finger gene family. Since ZNF320 is oriented toward the centromere of chromosome 19, both genes appeared on the same derivative chromosome der(10). The chimeric transcript encodes the WDR11 polypeptide, which is truncated after the second of six WD-repeats. ZNF320 is also expressed in A172 cells, although it is not clear if the translocation affects the expression of the altered gene because of the presence of another unrearranged gene on chromosome 19. We suggest that, because of its localization in a region frequently showing LOH and the observation of inactivation of this gene in glioblastoma cells, WDR11 is a candidate gene for the frequently proposed tumor suppressor gene in 10q25-26 which is involved in tumorigenesis of glial and other tumors showing frequent alterations in the distal 10q region.

Asunto(s)

Cromosomas Humanos Par 10 , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Glioblastoma/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Translocación Genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cromosomas Humanos Par 19 , ADN Complementario/metabolismo , Exones , Eliminación de Gen , Glioma/genética , Glioma/metabolismo , Humanos , Hibridación Fluorescente in Situ , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Análisis de Secuencia de ADN , Telómero , Distribución Tisular , Células Tumorales Cultivadas , Dedos de Zinc

Asunto(s)

Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/fisiología , Ciclo Celular/fisiología , Genoma Humano , Humanos , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología

RESUMEN

Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, "nonpermissive" REF52 cells do not develop resistance to N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification of cad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, as cad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

Asunto(s)

Antineoplásicos/farmacología , Aspartato Carbamoiltransferasa/genética , Ácido Aspártico/análogos & derivados , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Ciclo Celular/genética , Dihidroorotasa/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Genes myc , Complejos Multienzimáticos/genética , Ácido Fosfonoacético/análogos & derivados , Proteína p53 Supresora de Tumor/genética , Animales , Ácido Aspártico/farmacología , Ácido Fosfonoacético/farmacología , Células Tumorales Cultivadas

RESUMEN

Loss of heterozygosity for 10q23-26 is seen in over 80% of glioblastoma multiforme tumors. We have used a positional cloning strategy to isolate a novel gene, LGI1 (Leucine-rich gene-Glioma Inactivated), which is rearranged as a result of the t(10;19)(q24;q13) balanced translocation in the T98G glioblastoma cell line lacking any normal chromosome 10. Rearrangement of the LGI1 gene was also detected in the A172 glioblastoma cell line and several glioblastoma tumors. These rearrangements lead to a complete absence of LGI1 expression in glioblastoma cells. The LGI1 gene encodes a protein with a calculated molecular mass of 60 kD and contains 3.5 leucine-rich repeats (LRR) with conserved flanking sequences. In the LRR domain, LGI1 has the highest homology with a number of transmembrane and extracellular proteins which function as receptors and adhesion proteins. LGI1 is predominantly expressed in neural tissues, especially in brain; its expression is reduced in low grade brain tumors and it is significantly reduced or absent in malignant gliomas. Its localization to the 10q24 region, and rearrangements or inactivation in malignant brain tumors, suggest that LGI1 is a candidate tumor suppressor gene involved in progression of glial tumors.

Asunto(s)

Neoplasias Encefálicas/genética , Cromosomas Humanos Par 10/genética , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Proteínas/genética , Translocación Genética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Neoplasias Encefálicas/secundario , Secuencia Conservada , Regulación hacia Abajo , Genes Supresores de Tumor/genética , Glioblastoma/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Repetidas Ricas en Leucina , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Mapeo Físico de Cromosoma , Proteínas/química , Células Tumorales Cultivadas

RESUMEN

Rodent cells resistant to N-phosphonacetyl-L-aspartate (PALA) invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells. In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD. These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts.

Asunto(s)

Aspartato Carbamoiltransferasa/genética , Ácido Aspártico/análogos & derivados , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Aberraciones Cromosómicas/genética , Cromosomas Humanos Par 2/genética , Dihidroorotasa/genética , Resistencia a Medicamentos/genética , Amplificación de Genes/genética , Complejos Multienzimáticos/genética , Ácido Fosfonoacético/análogos & derivados , Aspartato Carbamoiltransferasa/metabolismo , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Línea Celular , Centrómero/genética , Sondas de ADN , Dosificación de Gen , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ácido Fosfonoacético/metabolismo , Ácido Fosfonoacético/farmacología , Telómero/genética , Células Tumorales Cultivadas

RESUMEN

p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.

Asunto(s)

Genes p53 , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos , Embrión de Mamíferos , Etopósido/toxicidad , Fibroblastos , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/biosíntesis , Ratas , Retroviridae , Supresión Genética , Transactivadores/metabolismo

RESUMEN

Cells often acquire resistance to the antiproliferative agents methotrexate (MTX) or N-phosphonacetyl-L-aspartate (PALA) through amplification of genes encoding the target enzymes dihydrofolate reductase or carbamylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), respectively. We showed previously that Syrian hamster BHK cells resistant to selective concentrations of PALA (approximately 3 x ID50) arise at a rate of approximately 10(-4) per cell per generation and contain amplifications of the CAD gene as ladder-like structures on one of the two B9 chromosomes, where CAD is normally located. We now find that BHK cells resistant to high concentrations of PALA (approximately 15 x ID50) appear only after prior exposure to selective concentrations of PALA for approximately 72 h. Furthermore, in contrast to untreated cells, BHK cells pretreated with selective concentrations of MTX give colonies in high concentrations of PALA, and cells pretreated with selective concentrations of PALA give colonies in high concentrations of MTX or 5-fluorouracil. As judged by measuring numbers of cells and metaphase cell pairs, BHK cells do not arrest completely when starved for pyrimidine nucleotides by treatment with selective concentrations of PALA for up to 72 h. We propose that DNA damage, caused when cells fail to stop DNA synthesis promptly under conditions of dNTP starvation, stimulates amplification throughout the genome by mechanisms--such as bridge-breakage-fusion cycles--that are triggered by broken DNA. Amplified CAD genes were analyzed by fluorescence in situ hybridization both in cells where amplification was induced by PALA pretreatment and in cells in which the amplification occurred spontaneously, before selection with PALA. The ladder-like structures that result from bridge-breakage-fusion cycles were observed in both cases.

Asunto(s)

Aspartato Carbamoiltransferasa/genética , Ácido Aspártico/análogos & derivados , Amplificación de Genes/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Ácido Fosfonoacético/análogos & derivados , Animales , Ácido Aspártico/farmacología , División Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , Cricetinae , Daño del ADN , Fluorouracilo/farmacología , Humanos , Hibridación Fluorescente in Situ , Metotrexato/farmacología , Ácido Fosfonoacético/farmacología , Nucleótidos de Pirimidina/metabolismo , Células Tumorales Cultivadas

RESUMEN

In addition to its induction by DNA damage, p53 is induced by drugs that starve cells for DNA and RNA precursors, or by inhibitors of DNA or RNA polymerase. In normal cells, the induction of p53 by dNTP starvation serves a protective role, mediating rapid, reversible cell-cycle arrest without DNA damage. In most cell lines, this first line of defense is missing, so that starvation for dNTPs causes DNA to break, thus increasing the probability of genomic instability, chromosome deletions and gene amplification. The mechanism of how p53 is induced remains unclear.

Asunto(s)

ADN/biosíntesis , Genes p53 , ARN/biosíntesis , Animales , Ciclo Celular , Daño del ADN , Desoxirribonucleótidos/análisis , Desoxirribonucleótidos/genética , Amplificación de Genes , Regulación de la Expresión Génica , Humanos , Modelos Genéticos

RESUMEN

Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.

Asunto(s)

Amplificación de Genes , Regulación de la Expresión Génica , Animales , Humanos , Ratones

RESUMEN

A set of multidrug resistant (MDR) murine leukemia P388 sublines processing 30-50-fold mdr1 gene amplification was obtained as a result of experimental chemotherapy with rubomycin, ruboxyl, vinblastine, vincristine, or combination of rubomycin and vincristine. Significant differences of developed MDR sublines in response to treatment with cisplatin, tiophosphamide, sarcolysin, and dopad were found. Strong correlation between drug sensitivity and a copy number of gene coding for 19-22 kDa calcium-binding sorcin gene co-amplification were hypersensitive to cisplatin and alkylating agents, the cell sublines showing amplification of sorcin DNA sequences did not possess such collateral sensitivity and even acquired cross-resistance. The dependence of sensitivity to cisplatin on sorcin gene co-amplification was confirmed by analysis of Djungarian hamster DM15 cell sublines that selected for MDR in vitro by colchicine.

Asunto(s)

Alquilantes/farmacología , Proteínas de Unión al Calcio/genética , Cisplatino/farmacología , Resistencia a Múltiples Medicamentos/genética , Amplificación de Genes/genética , Leucemia P388/genética , Proteínas de Neoplasias/genética , Animales , Línea Celular Transformada , Cricetinae , Fibroblastos , Leucemia P388/tratamiento farmacológico

RESUMEN

Mouse leukemia P388 sublines that acquired the resistance to multiple drugs as a result of treatment in vivo with anthracyclines (rubomycin, ruboxyl) and/or vincristine were studied. The mdr gene amplification was found in all tested cell lines: in four of five sublines all three members of the mdr gene family showed increased copy numbers, and in one cell line, developed after treatment with ruboxyl, mdr1a and mdr1b genes were amplified to the same degree, whereas the mdr2 gene was not amplified at all. The levels of amplification of mdr genes varied in different cell lines from 30-fold to 50-fold. Unusual cytological manifestations--relatively large newly formed chromosomelike structures, were revealed in four of five long-term independent sublines. Some of these structures did not contained C blocks; the others, in contrast, were enriched by C-heterochromatin. In situ hybridization showed the presence of mdr genes in newly formed bodies. In the majority of cases, the formation of chromosomelike structures was preceded by the appearance of other, smaller size, structures: the so-called "small chromatin bodies" (minichromosomes) and/or homogeneously G-positive small ring chromosomes.

Asunto(s)

Antineoplásicos/farmacología , Cromosomas , Resistencia a Medicamentos/genética , Amplificación de Genes/efectos de los fármacos , Leucemia P388/genética , Glicoproteínas de Membrana/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Bandeo Cromosómico , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacología , Cariotipificación , Ratones , Células Tumorales Cultivadas , Vincristina/farmacología

RESUMEN

Sublines of the canine kidney epithelium cells (MDCK) resistant to various colchicine doses (0.3-5.0 mcg.ml-1) were obtained by multistep selection. The new lines were shown to possess multidrug-resistance (MDR) by means of Rhodamine 123 staining. The initial steps of resistance (2 mcg.ml-1, 300-fold increase in resistance) are evidently due to enhancement of mdr gene transcription. Approximately 4-fold gene amplification was revealed in the cells resistant to 3 mcg.ml-1 of colchicine. All the drug-resistant lines tested were found to be more prone to differentiation than the wild type cells (both spontaneous and induced by several drugs such as DMSO, cAMP and others). The data suggest that the selection for overexpression of mdr gene results in selection of variants that are more capable of differentiation than the rest of the population.

Asunto(s)

Diferenciación Celular , Resistencia a Medicamentos/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Colchicina/farmacología , ADN/genética , Dimetilsulfóxido/farmacología , Perros , Expresión Génica , Riñón , Fenotipo

RESUMEN

Five recombinant phages containing different parts of genomic regions amplified in multidrug-resistant (MDR) cells have been isolated from genomic libraries of colchicine- and actinomycin D-resistant Djungarian hamster cells. Fragments of these clones together with a part of Chinese hamster mdr gene (plasmid pDR4.7 a gift of Dr. I. Roninson) were used as hybridization probes to study composition and variations of amplified DNA in a large number of MDR cell lines. Two of the six probes used (pC52 and pDR4.7) showed DNA amplification in a large number of MDR cell lines tested (commonly amplified clones) regardless of their origin (Djungarian hamster or mouse), type of selective agent used (colchicine, actinomycin D, or anthracyclines), and mode of selection (in vitro or in vivo). These clones hybridized with two different RNA transcripts (pDR4.7, 5kb; pC52, 3.5 kb) that were overproduced in MDR cells. Degrees of amplification of both commonly amplified sequences correlated with levels of resistance in all but one of the cell lines. Other cloned sequences (sporadically amplified clones) were amplified to different extents (but never greater than the commonly amplified sequences) in some of the Djungarian hamster MDR cell lines. Such differential amplification is not the result of heterogeneity of cell population since 20 cell clones tested showed identical ratios of amplification of different amplified sequences. Sporadically amplified sequences usually coamplified with the commonly amplified ones at first steps of selection, but then they would cease to amplify and, at the later stages of selection, they could even be completely deamplified. It seems that disappearance of unnecessary parts of amplicons is a regular process accompanying stepwise gene amplification.

Asunto(s)

ADN/genética , Resistencia a Medicamentos/genética , Amplificación de Genes , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Enzimas de Restricción del ADN , Ratones

RESUMEN

The P388rm and P388rx cell lines resistant to antracycline antibiotics were obtained as a result of chemotherapy of mice bearing P388 leukemia, by means of increasing dosages of rubomycin and ruboxyl, respectively. These cell lines possessed cross-resistance to vinblastine, vincristine, colchicine, actinomycin D and some other drugs. Multidrug resistance (MDR) of P388rm and P388rx is due to decreased uptake of different cytotoxic compounds by the cells. Development of resistance to rubomycin and ruboxyl was accompanied by the appearance of additional chromosomal structures--long homogeneously staining regions (HSRs), double minute chromosomes and others usually containing amplified DNA sequences. Southern blot-hybridization with cloned DNA fragments amplified in Djungarian and Chinese hamster cell lines having MDR has revealed in P388rm and P388rx cells approximately 50-fold amplification of mdr and pC52 genes. Thus, in mouse leukemia cells which acquired MDR in vivo, as a result of chemotherapy, amplification is observed of the same genes that undergo amplification during selection of cell cultures for MDR in vitro.

Asunto(s)

Antineoplásicos/antagonistas & inhibidores , Amplificación de Genes , Leucemia P388/genética , Leucemia Experimental/genética , Selección Genética , Animales , ADN de Neoplasias/genética , Daunorrubicina/análogos & derivados , Daunorrubicina/antagonistas & inhibidores , Resistencia a Medicamentos/genética , Amplificación de Genes/efectos de los fármacos , Genotipo , Cariotipificación , Leucemia P388/tratamiento farmacológico , Ratones , Fenotipo , Células Tumorales Cultivadas

RESUMEN

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.

Asunto(s)

Resistencia a Medicamentos/genética , Amplificación de Genes , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , ADN/genética , Marcadores Genéticos , Hibridación de Ácido Nucleico

RESUMEN

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.

Asunto(s)

Clonación Molecular , Resistencia a Medicamentos/genética , Amplificación de Genes , Animales , Secuencia de Bases , Línea Celular , Cricetinae , ADN/genética , Hibridación de Ácido Nucleico

RESUMEN

Earlier we have found that the development of resistance to colchicine in mammalian cells in vitro is due to gene amplification leading to decreased plasma membrane permeability to the selective agent and some other unrelated drugs. By a stepwise self-renaturation procedure followed by chromatography on hydroxyapatite we isolated the fraction of middle-repeated sequences (DNAc0t = 10-250) enriched in amplified DNA from the DNA of colchicine-resistant Djungarian hamster cell line. Blotting-hybridization with [32P]DNAc0t = 10-250 performed in the presence of the excess of unlabelled DNA from wild type cells reveals amplified sequences in resistant cell lines. The comparison of DNAs from cell lines resistant to colchicine, adriablastin and actinomycin D showed that common but not identical DNA sequences are amplified in these cases. In situ hybridization with [3H]DNAc0t = 10-250 indicates that amplified sequences are located in the long homogeneously staining regions (HSRs) of the marker chromosomes. These results suggest that DNAc0t = 10-250 may be used for screening of recombinant molecules containing amplified sequences.

Asunto(s)

Colchicina/farmacología , ADN/genética , Amplificación de Genes , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Línea Celular , Cricetinae , Resistencia a Medicamentos , Marcadores Genéticos , Hibridación de Ácido Nucleico

RESUMEN

By multistep selection a set of clones and sublines possessing different levels of resistance to colchicine or adriablastin was obtained from the SV40-transformed Djungarian hamster cell lines, DM-15 and DMcap. Resistance to both colchicine and adriablastin is associated with an alteration of plasma membrane permeability leading to a decreased uptake of various drugs (3H-colchicine, 3H-cytochalasin B, 3H-actinomycin D, 3H-puromycin, 3H-vinblastine, 14C-chloramphenicol). The DNA of cells highly resistant to cholchicine can transmit resistance only to low dosages of the drug. Comparison of DNAs from wild-type and resistant cells digested by restriction endonucleases revealed new classes of repeated DNA sequences in resistant cell lines. The degree of DNA repetition was correlated with the level of drug resistance. The repeated DNA sequences evidently represent parts of the genome that are amplified in resistant cells. The size of the amplified sequences is 200-250 kilobase pairs (kb). Cell lines highly resistant to colchicine contain amplified DNA, which like mitochondrial DNA replicate asynchronously with the main portion of the cellular DNA and related but not identical DNA sequences are amplified in independent cell lines selected for resistance to colchicine, adriablastin, and actinomycin D. These cell lines display similar patterns of alterations of plasma membrane permeability. The amplified DNA sequences may contain a gene or genes the overexpression of which leads to change in plasma membrane permeability and a development of resistance to various drugs.

Asunto(s)

Permeabilidad de la Membrana Celular , Colchicina/metabolismo , Resistencia a Medicamentos , Amplificación de Genes , Mutación , Animales , Línea Celular , Cloranfenicol/metabolismo , Células Clonales , Cricetinae , Citocalasina B/metabolismo , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Dactinomicina/metabolismo , Cinética , Hibridación de Ácido Nucleico , Vinblastina/metabolismo