RESUMO
The last decade has come across an increasing demand for theranostic biocompatible nanodevices possessing the double ability of diagnosis and therapy. In this work, we report the design, synthesis and step-by-step characterization of rationally coated gold nanostars (GNSs) for the SERS imaging and photothermal therapy of HeLa cancer cells. The nanodevices were realized by synthesizing GNSs with a seed growth approach, coating them with a controlled mixture of thiols composed of a Raman reporter and a polyethylene glycol with a terminal amino group, and then reacting these amino groups with folic acid (FA), in order to impart selectivity towards cancer cells which overexpress folate receptors on their membranes. After a complete characterization, we demonstrate that these FA-functionalized GNSs (FA-GNSs) are able to bind selectively to the membranes of HeLa cells, acting as SERS tags and allowing SERS imaging. Moreover, we demonstrate that once bound to HeLa cell membranes, FA-GNSs exhibit photothermal effect which can be exploited to kill the same cells in vitro using laser irradiation in the NIR at a very low and safe irradiance. We thus demonstrate that the FA-GNSs designed following the described approach are an efficient prototype of theranostic nanodevices.
RESUMO
The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.
Assuntos
Bancos de Espécimes Biológicos , Genômica , Cavalos/genética , Animais , Feminino , FenótipoRESUMO
The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclear sequences of mitochondrial origin. In the horse reference genome, we identified 82 numts and showed that the entire horse mitochondrial DNA is represented as numts without gross bias. Numts were inserted in the horse nuclear genome at random sites and were probably generated during the repair of DNA double-strand breaks. We then analysed 12 numt loci in 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion, were present at six of these loci. At some loci, the null allele is prevalent in the sample analysed, suggesting that, in the horse population, the number of numt loci may be higher than 82 present in the reference genome. Contrary to humans, the insertion polymorphism of numts is extremely frequent in the horse population, supporting the hypothesis that the genome of this species is in a rapidly evolving state.
Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Cavalos/genética , Mutagênese Insercional , Animais , Humanos , Polimorfismo GenéticoRESUMO
Horses, asses and zebras belong to the genus Equus and are the only extant species of the family Equidae in the order Perissodactyla. In a previous work we demonstrated that a key factor in the rapid karyotypic evolution of this genus was evolutionary centromere repositioning, that is, the shift of the centromeric function to a new position without alteration of the order of markers along the chromosome. In search of previously undiscovered evolutionarily new centromeres, we traced the phylogeny of horse chromosome 5, analyzing the order of BAC markers, derived from a horse genomic library, in 7 Equus species (E. caballus, E. hemionus onager, E. kiang, E. asinus, E. grevyi, E. burchelli and E. zebra hartmannae). This analysis showed that repositioned centromeres are present in E. asinus (domestic donkey, EAS) chromosome 16 and in E. burchelli (Burchell's zebra, EBU) chromosome 17, confirming that centromere repositioning is a strikingly frequent phenomenon in this genus. The observation that the neocentromeres in EAS16 and EBU17 are in the same chromosomal position suggests that they may derive from the same event and therefore, E. asinus and E. burchelli may be more closely related than previously proposed; alternatively, 2 centromere repositioning events, involving the same chromosomal region, may have occurred independently in different lineages, pointing to the possible existence of hot spots for neocentromere formation. Our comparative analysis also showed that, while E. caballus chromosome 5 seems to represent the ancestral configuration, centric fission followed by independent fusion events gave rise to 3 different submetacentric chromosomes in other Equus lineages.
Assuntos
Centrômero , Mapeamento Cromossômico , Cavalos/genética , Filogenia , Animais , Cromossomos Artificiais Bacterianos , Sondas de DNA , Hibridização in Situ FluorescenteRESUMO
In addition to their location at terminal positions, telomeric-like repeats are also present at internal sites of the chromosomes (intrachromosomal or interstitial telomeric sequences, ITSs). According to their sequence organization and genomic location, two different kinds of ITSs can be identified: (1) heterochromatic ITSs (het-ITSs), large (up to hundreds of kb) stretches of telomeric-like DNA localized mainly at centromeres, and (2) short ITSs (s-ITSs), short stretches of telomeric hexamers distributed at internal sites of the chromosomes. Het-ITSs have been only described in some vertebrate species and they probably represent the remnants of evolutionary chromosomal rearrangements. On the contrary, s-ITSs are probably present in all mammalian genomes although they have been described in detail only in some completely sequenced genomes. Sequence database analysis revealed the presence of 83, 79, 244 and 250 such s-ITSs in the human, chimpanzee, mouse and rat genomes, respectively. Analysis of the flanking sequences suggested that s-ITSs were inserted during the repair of DNA double-strand breaks that occurred in the course of evolution. An extensive comparative analysis of the s-ITS loci and their orthologous 'empty' loci confirmed this hypothesis and suggested that the repair event involved the direct action of telomerase. Whereas het-ITSs seem to be intrinsically prone to breakage, the instability of s-ITSs is more controversial. This observation is consistent with the hypothesis that s-ITSs are probably not themselves prone to breakage but represent 'scars' of ancient breakage that may have occurred within fragile regions. This study will review the current knowledge on these two types of ITS, their molecular organization, how they arose during evolution, their implications for chromosomal instability and their potential applications as phylogenetic/forensic markers.
Assuntos
Evolução Molecular , Origem de Replicação/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Animais , Sequência de Bases , Centrômero/genética , Cromossomos/genética , Cromossomos Humanos/genética , Sequência Conservada , Cricetinae , Cricetulus , DNA/genética , Elementos de DNA Transponíveis/genética , Heterocromatina/genética , Humanos , Hibridização in Situ Fluorescente , Invertebrados/genética , Camundongos , Primatas/genética , Ratos , Vertebrados/genéticaRESUMO
We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.
Assuntos
Amidoidrolases/genética , Aspartato Carbamoiltransferase/genética , Evolução Biológica , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Cromossomos , DNA/genética , Di-Hidro-Orotase/genética , Amplificação de Genes , Complexos Multienzimáticos/genética , Animais , Ácido Aspártico/análogos & derivados , Southern Blotting , Linhagem Celular , Cricetinae , DNA/efeitos dos fármacos , Modelos Genéticos , Mutação , Ácido Fosfonoacéticos/análogos & derivadosRESUMO
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
Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.
Assuntos
Aspartato Carbamoiltransferase , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Di-Hidro-Orotase , Amplificação de Genes , Complexos Multienzimáticos , Mutação , Compostos Organofosforados/farmacologia , Ácido Fosfonoacéticos/farmacologia , Proteínas/genética , Animais , Ácido Aspártico/farmacologia , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Genes , Mesocricetus , Hibridização de Ácido Nucleico , Ácido Fosfonoacéticos/análogos & derivadosRESUMO
Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N-(phosphonacetyl)-L-aspartate (PALA) resistance is considered a measure of gene amplification because it is mainly attributable to amplification of the carbamyl-P-synthetase aspartate transcarbamylase dihydro-orotase (CAD) gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs gene, displays also an increased frequency of gene amplification. In these cells, we found that the amplification of the CAD gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs(-/-) MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that gene amplification occurs in a genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify.
Assuntos
Proteínas de Ligação a DNA , Amplificação de Genes , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Domínio Catalítico/genética , Linhagem Celular , Cricetinae , Cricetulus , Reparo do DNA , Proteína Quinase Ativada por DNA , Di-Hidro-Orotase/genética , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Camundongos , Complexos Multienzimáticos/genética , Ácido Fosfonoacéticos/análogos & derivados , Ácido Fosfonoacéticos/farmacologiaRESUMO
The concentration of evolutionary breakpoints in primate karyotypes in some particular regions or chromosome bands suggests that these chromosome regions are more prone to breakage. This is the first extensive comparative study which investigates a possible relationship of two genetic markers (intrachromosomal telomeric sequences [TTAGGG]n, [ITSs] and fragile sites [FSs]), which are implicated in the evolutionary process as well as in chromosome rearrangements. For this purpose, we have analyzed: (a) the cytogenetic expression of aphidicolin-induced FSs in Cebus apella and Cebus nigrivittatus (F. Cebidae, Platyrrhini) and Mandrillus sphinx (F. Cercopithecidae, Catarrhini), and (b) the intrachromosomal position of telomeric-like sequences by FISH with a synthetic (TTAGGG)n probe in C. apella chromosomes. The multinomial FSM statistical model allowed us to determinate 53 FSs in C. apella, 16 FSs in C. nigrivittatus and 50 FSs in M. sphinx. As expected, all telomeres hybridized with the probe, and 55 intrachromosomal loci were also detected in the Cebus apella karyotype. The chi(2) test indicates that the coincidence of the location of Cebus and Mandrillus FSs with the location of human FSs is significant (P < 0.005). Based on a comparative cytogenetic study among different primate species we have identified (or described) the chromosome bands in the karyotypes of Papionini and Cebus species implicated in evolutionary reorganizations. More than 80% of these evolutionary breakpoints are located in chromosome bands that express FSs and/or contain ITSs.
Assuntos
Cebus/genética , Quebra Cromossômica/genética , Sítios Frágeis do Cromossomo/genética , Cromossomos de Mamíferos/genética , Evolução Molecular , Telômero/genética , Animais , Afidicolina/farmacologia , Sítios Frágeis do Cromossomo/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , Feminino , Masculino , Mandrillus/genética , Metáfase/genéticaRESUMO
Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra- and inter-species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription-PCR-based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH-1, eCATH-2 and eCATH-3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C-terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin-conserved propiece revealed a polymorphic DNA region with several hybridization-positive fragments and suggested the presence of additional genes. A null eCATH-1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH-1-specific mRNA were found in myeloid cells of gene-positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH-2 and eCATH-3 propeptides, but not of eCATH-1-related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate-stimulated neutrophils. These results thus suggest that eCATH-2 and eCATH-3 are functional genes, whereas eCATH-1 is unable to encode a polypeptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Cavalos/fisiologia , Leucócitos/fisiologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/efeitos dos fármacos , Sequência de Bases , Southern Blotting , Western Blotting , Catelicidinas , Grânulos Citoplasmáticos/metabolismo , Dados de Sequência Molecular , Família Multigênica/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência , Homologia de Sequência de Aminoácidos , Elementos Nucleotídeos Curtos e Dispersos/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.
Assuntos
Instabilidade Cromossômica/efeitos da radiação , Cromossomos Humanos/efeitos da radiação , Sequências Repetitivas de Ácido Nucleico , Telômero/genética , Carcinoma de Células Escamosas/patologia , Aberrações Cromossômicas , Quebra Cromossômica , Coloração Cromossômica , Cromossomos Humanos/genética , Cromossomos Humanos/ultraestrutura , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 2/efeitos da radiação , Cromossomos Humanos Par 2/ultraestrutura , Raios gama/efeitos adversos , Humanos , Recém-Nascido , Tolerância a Radiação/genética , Sequências Repetitivas de Ácido Nucleico/genética , Telômero/fisiologia , TransfecçãoRESUMO
In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has always been found associated with amplification of the CAD gene (carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase). We describe two PALA resistant Chinese hamster mutant cell lines in which amplification of the CAD gene was not present. The PALA resistant phenotype was stable when the cells were grown in non-selective medium. However, after prolonged growth in the presence of the same drug concentration used for selection, cells with increased CAD gene copy number and higher levels of resistance overrode the original population. In these cell populations, a heterogeneous organization of the CAD genes was revealed by fluorescence in situ hybridization on mitotic chromosomes indicating that the additional copies of the gene were generated in several ways, such as non-disjunction and breakage-fusion-bridge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberrations in the cells grown in the presence of the drug, could have favored the late onset of the amplified mutants. It is tempting to speculate that, during the expansion of tumor populations, different drug resistance mechanisms, including gene amplification, could occur in succession and lead to the generation of cells highly resistant to chemotherapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Di-Hidro-Orotase/genética , Amplificação de Genes , Complexos Multienzimáticos/genética , Ácido Fosfonoacéticos/análogos & derivados , Animais , Ácido Aspártico/farmacologia , Células CHO , Cricetinae , Resistencia a Medicamentos Antineoplásicos , Dosagem de Genes , Mutação , Ácido Fosfonoacéticos/farmacologiaRESUMO
A mutation assay in cultured mammalian cells was developed based on direct analysis of minisatellite DNA. Chinese hamster cells (V79) were mutagenized with nitrosoguanidine and independent colonies were isolated and expanded. DNA fingerprints were then obtained after digestion with HinfI or HaeIII and hybridization with 33.15 and 33.6 probes (Jeffreys et al., 1985). 12 colonies from untreated cells were also analyzed. Digestion with HaeIII and hybridization with 33.15 probe detected the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic effect of chemical agents. We have also analyzed the DNA fingerprints of 17 independent Chinese hamster (CHO) cell lines carrying amplification of the CAD gene. The DNA fingerprint analysis showed a variation in minisatellite regions in 3 lines while no variation was observed in independent colonies from the CHO parental cell line. The results suggest that these sequences may be hot spots for recombination during gene amplification.
Assuntos
DNA Satélite/genética , Variação Genética , Sequências Repetitivas de Ácido Nucleico , Animais , Células CHO , Linhagem Celular , Cricetinae , Impressões Digitais de DNA , Amplificação de Genes , Mutagênese , NitrosoguanidinasRESUMO
Two gastric cancer cell lines, AKG and GK2, were established from a pleural and an ascitic effusion, respectively. GK2 cells have a pseudodiploid karyotype with an add(6)(q27) chromosome in all metaphases examined. The karyotype of AKG cells is highly rearranged: FISH analysis with painting probes has shown that DNA sequences derived from single chromosomes are scattered on several (as many as eight) markers. In this cell line, the C-MYC and the K-RAS oncogenes are amplified. The organization and the copy number of the C-MYC-amplified units are different from the K-RAS units, suggesting that the two oncogenes were amplified independently. The presence of a few marker chromosomes carrying both C-MYC and K-RAS could be due to translocation events that followed the amplification.
Assuntos
Carcinoma/genética , Carcinoma/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Animais , Testes de Carcinogenicidade , Carcinoma/tratamento farmacológico , Divisão Celular , Aberrações Cromossômicas , Resistencia a Medicamentos Antineoplásicos , Feminino , Dosagem de Genes , Rearranjo Gênico , Genes myc , Genes ras , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Gástricas/tratamento farmacológicoRESUMO
In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.
Assuntos
Ácido Aspártico/análogos & derivados , Dipiridamol/farmacologia , Resistência a Medicamentos , Ácido Fosfonoacéticos/análogos & derivados , Uridina/metabolismo , Animais , Aspartato Carbamoiltransferase/biossíntese , Ácido Aspártico/farmacologia , Transporte Biológico/efeitos dos fármacos , Células CHO , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Di-Hidro-Orotase/biossíntese , Relação Dose-Resposta a Droga , Amplificação de Genes , Cinética , Complexos Multienzimáticos/biossíntese , Mutagênese , Proteínas de Neoplasias/biossíntese , Ácido Fosfonoacéticos/farmacologia , Uridina/farmacologiaRESUMO
(TTAGGG)n sequences have been localized on the chromosomes of the Chinese hamster V79 cell line. A correlation between telomeric-like repeats and chromosome breakage has been found. Moreover, the analysis of the truncated chromosomes, typical of this cell line, has suggested that intrachromosomal (TTAGGG)n DNA may be important in the stabilization of the new telomeres.
Assuntos
Aberrações Cromossômicas , Telômero , Animais , Linhagem Celular , Mapeamento Cromossômico , Cricetinae , Cricetulus , Hibridização in Situ Fluorescente , MetáfaseRESUMO
The frequency of sister-chromatid exchanges (SCE) was determined on second-division metaphases of lymphocytes stimulated by phytohaemagglutinin (PHA) during 9 days of culture. By using either a continuous or a pulsed bromodeoxyuridine (BUdR) treatment, cells were selected that had divided only twice, or at least twice, after different culture periods. No significant differences were observed in the SCE frequencies among the various samples. The incidence of SCE appears to be independent of the proliferation properties of cultured lymphocytes, such as length of cell cycle, fast or delayed response to PHA and number of divisions performed in vitro.
Assuntos
Divisão Celular , Troca Genética , Linfócitos/ultraestrutura , Troca de Cromátide Irmã , Adulto , Bromodesoxiuridina , Células Cultivadas , Feminino , Humanos , Cinética , Coloração e Rotulagem , Fatores de TempoRESUMO
In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2-5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.