RESUMEN
Background: Well characterized human cell lines are needed for preclinical treatment studies of anaplastic thyroid cancer (ATC).Aims/Objectives: The aim was to establish, verify and characterize a panel of ATC cell lines.Material and methods: Cell lines were established from ATC fine-needle aspiration biopsies and characterized genetically and functionally regarding treatment sensitivities.Results: Eight cell lines were established in vitro and the anaplastic thyroid origin was verified. Seven of the cell lines were also grown as xenografts. The cell lines harboured complex karyotypes with modal numbers in hyperdiploid to near-pentaploid range. Five were TP53 mutated and three carried the BRAFV600E mutation. None had rearrangements of RET. For doxorubicin, IC50 ranged from 0.42 to 46 nmol/L and for paclitaxel from 1.6 to 196 nmol/L. Radiation sensitivity varied between 2.6 and 6.3 Gy. Two of the BRAF mutated cell lines displayed high sensitivity to vemurafenib, while the third was similar to the wild-type ones.Conclusions and significance: We describe a series of new ATC cell lines demonstrating large heterogeneity in the response to cytostatic drugs and the BRAF inhibitor vemurafenib. The observations are relevant to future attempts to optimize treatment combinations for ATC.
Asunto(s)
Línea Celular Tumoral , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Anciano , Anciano de 80 o más Años , Animales , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Xenoinjertos , Humanos , Masculino , Ratones Desnudos , Tolerancia a RadiaciónRESUMEN
BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral/citología , Cisplatino/administración & dosificación , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/terapia , Neoplasias Tonsilares/virología , Animales , Línea Celular Tumoral/virología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cisplatino/uso terapéutico , Genoma Viral , Papillomavirus Humano 16/efectos de los fármacos , Papillomavirus Humano 16/efectos de la radiación , Humanos , Concentración 50 Inhibidora , Cariotipo , Masculino , Ratones , Persona de Mediana Edad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Radioterapia , Neoplasias Tonsilares/genética , Neoplasias Tonsilares/terapia , Carga Viral/efectos de los fármacos , Carga Viral/efectos de la radiación , Secuenciación Completa del Genoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Constitutional aneuploidy is typically caused by a single-event meiotic or early mitotic error. In contrast, somatic aneuploidy, found mainly in neoplastic tissue, is attributed to continuous chromosomal instability. More debated as a cause of aneuploidy is aneuploidy itself; that is, whether aneuploidy per se causes chromosomal instability, for example, in patients with inborn aneuploidy. We have addressed this issue by quantifying the level of somatic mosaicism, a proxy marker of chromosomal instability, in patients with constitutional aneuploidy by precise background-filtered dual-color FISH. In contrast to previous studies that used less precise methods, we find that constitutional trisomy, even for large chromosomes that are often trisomic in cancer, does not confer a significantly elevated rate of somatic chromosomal mosaicism in individual cases. Constitutional triploidy was associated with an increased level of somatic mosaicism, but this consisted mostly of reversion from trisomy to disomy and did not correspond to a proportionally elevated level of chromosome mis-segregation in triploids, indicating that the observed mosaicism resulted from a specific accumulation of cells with a hypotriploid chromosome number. In no case did the rate of somatic mosaicism in constitutional aneuploidy exceed that of "chromosomally stable" cancer cells. Our findings show that even though constitutional aneuploidy was in some cases associated with low-level somatic mosaicism, it was insufficient to generate the cancer-like levels expected if aneuploidy single-handedly triggered cancer-like chromosomal instability.
Asunto(s)
Aneuploidia , Inestabilidad Cromosómica/genética , Neoplasias/genética , Línea Celular Tumoral , Trastornos de los Cromosomas , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , MosaicismoRESUMEN
Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.
Asunto(s)
Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Cavidad Nasal/virología , Mucosa Nasal/virología , Faringe/virología , Carcinoma , Línea Celular Transformada , Células Epiteliales/metabolismo , Células Epiteliales/patología , Infecciones por Virus de Epstein-Barr/patología , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Cavidad Nasal/metabolismo , Cavidad Nasal/patología , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , Faringe/metabolismo , Faringe/patología , Complejo Represivo Polycomb 1/metabolismo , Latencia del VirusRESUMEN
An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (pâ=â0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.
Asunto(s)
Aneuploidia , Neoplasias Colorrectales/metabolismo , Células Cultivadas , Niño , Preescolar , Aberraciones Cromosómicas , Segregación Cromosómica/genética , Evolución Clonal/genética , Evolución Clonal/fisiología , Neoplasias Colorrectales/genética , Fibroblastos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Lactante , Cariotipo , MasculinoRESUMEN
Neuroblastomas (NBs) are tumours of the sympathetic nervous system accounting for 8-10% of paediatric cancers. NBs exhibit extensive intertumour genetic heterogeneity, but their extent of intratumour genetic diversity has remained unexplored. We aimed to assess intratumour genetic variation in NBs with a focus on whole chromosome changes and their underlying mechanism. Allelic ratios obtained by SNP-array data from 30 aneuploid primary NBs and NB cell lines were used to quantify the size of clones harbouring specific genomic imbalances. In 13 cases, this was supplemented by fluorescence in situ hybridisation to assess copy number diversity in detail. Computer simulations of different mitotic segregation errors, single cell cloning, analysis of mitotic figures, and time lapse imaging of dividing NB cells were used to infer the most likely mechanism behind intratumour variation in chromosome number. Combined SNP array and FISH analyses showed that all cases exhibited higher inter-cellular copy number variation than non-neoplastic control tissue, with up to 75% of tumour cells showing non-modal chromosome copy numbers. Comparisons of copy number profiles, resulting from simulations of different segregation errors to genomic profiles of 120 NBs indicated that loss of chromosomes from a tetraploid state was more likely than other mechanisms to explain numerical aberrations in NB. This was supported by a high frequency of lagging chromosomes at anaphase and polyploidisation events in growing NB cells. The dynamic nature of numerical aberrations was corroborated further by detecting substantial copy number diversity in cell populations grown from single NB cells. We conclude that aneuploid NBs typically show extensive intratumour chromosome copy number diversity, and that this phenomenon is most likely explained by continuous loss of chromosomes from a polyploid state.
Asunto(s)
Cromosomas Humanos , Variación Estructural del Genoma , Modelos Genéticos , Neoplasias del Sistema Nervioso/genética , Neuroblastoma/genética , Aneuploidia , Niño , Preescolar , Segregación Cromosómica , Simulación por Computador , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Recién Nacido , Masculino , Mitosis/genética , Neoplasias del Sistema Nervioso/patología , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Poliploidía , Cultivo Primario de Células , Imagen de Lapso de TiempoRESUMEN
Soft tissue angiofibroma is a recently delineated tumor type of unknown cellular origin. Cytogenetic analysis of four cases showed that they shared a t(5;8)(p15;q13). In three of them it was the sole change, underlining its pathogenetic significance. FISH mapping suggested the involvement of the aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2) genes in 5p15 and 8q13, respectively. RT-PCR revealed in-frame AHRR/NCOA2 and NCOA2/AHHR transcripts in all four cases. Interphase FISH on paraffin-embedded tissue from 10 further cases without cytogenetic data showed that three were positive for fusion of AHRR and NCOA2. While AHRR has never been implicated in gene fusions before, NCOA2 is the 3'-partner in fusions with MYST3 and ETV6 in leukemias and with PAX3 and HEY1 in sarcomas. As in the previously described fusion proteins, NCOA2 contributes with its two activation domains to the AHRR/NCOA2 chimera, substituting for the repressor domain of AHRR. Because the amino terminal part of the transcription factor AHRR, responsible for the recognition of xenobiotic response elements in target genes and for heterodimerization, shows extensive homology with the aryl hydrocarbon receptor (AHR), the fusion is predicted to upregulate the AHR/ARNT signaling pathway. Indeed, global gene expression analysis showed upregulation of CYP1A1 as well as other typical target genes of this pathway, such as those encoding toll-like receptors. Apart from providing a diagnostic marker for soft tissue angiofibroma, the results also suggest that this tumor constitutes an interesting model for evaluating the cellular effects of AHR signaling.
Asunto(s)
Angiofibroma/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación Neoplásica de la Expresión Génica , Coactivador 2 del Receptor Nuclear/genética , Fusión de Oncogenes , Proteínas Represoras/genética , Neoplasias de los Tejidos Blandos/genética , Translocación Genética , Cariotipo Anormal , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Bandeo Cromosómico , Puntos de Rotura del Cromosoma , Cromosomas Humanos Par 5 , Cromosomas Humanos Par 8 , Femenino , Perfilación de la Expresión Génica , Orden Génico , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Regulación hacia Arriba , Adulto JovenRESUMEN
The purpose of the study was to investigate the expression of Aurora-A in human laryngeal squamous cell carcinoma (LSCC) and to explore the effects of Aurora-A silencing on invasion and chromosomal instability in laryngeal cancer HEp-2 cells. The expression of Aurora-A mRNA and protein were studied using reverse transcription-PCR and Western blot in LSCC tissues and corresponding normal epithelium, respectively. In addition, the correlation between Aurora-A expression and clinicopathologic characteristics was analyzed in LSCC patients. Furthermore, HEp-2 cells were transfected with Aurora-A short hairpin RNA and the effects of knockdown of Aurora-A on tumor invasion and chromosomal instability were investigated. The results showed that expression of Aurora-A mRNA was significantly upregulated in laryngeal tumor tissue compared with that in normal tissue (P = 0.001), and overexpression of Aurora-A was found in 64.0% (16 of 25) of the patients by Western blotting. Upregulation of Aurora-A mRNA was significantly correlated with regional lymph node metastasis (P = 0.007) and clinical stage III/IV (P = 0.022). Overexpression of Aurora-A was significantly associated with lymph node metastasis (P = 0.027). Furthermore, disruption of Aurora-A using RNA interference technique suppressed invasive ability and chromosomal instability in HEp-2 cells. In conclusion, Aurora-A expression is elevated in human LSCC and associated with regional lymph node metastasis and late clinical stage. Overexpression of Aurora-A may contribute to LSCC carcinogenesis and progression partially due to enhancement of invasion ability and chromosomal instability.
Asunto(s)
Carcinoma de Células Escamosas/genética , Inestabilidad Cromosómica/genética , Neoplasias Laríngeas/genética , Proteínas Serina-Treonina Quinasas/genética , Anciano , Aurora Quinasas , Western Blotting , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Silenciador del Gen , Humanos , Neoplasias Laríngeas/patología , Laringe/patología , Metástasis Linfática/genética , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Estadificación de Neoplasias , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genéticaRESUMEN
One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.
Asunto(s)
Segregación Cromosómica/genética , Citocinesis/fisiología , Neoplasias Renales/genética , Mitosis/fisiología , Modelos Biológicos , Trisomía/patología , Tumor de Wilms/genética , Hibridación Genómica Comparativa , Citocinesis/genética , Técnica del Anticuerpo Fluorescente , Genes cdc/fisiología , Humanos , Hibridación Fluorescente in Situ , Neoplasias Renales/patología , Microscopía Fluorescente , Mitosis/genética , Huso Acromático/genética , Tumor de Wilms/patologíaRESUMEN
Cell immortalization is regarded as an early and pre-requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein-Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV-encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c-Myc, Bmi-1, and Id-1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT-immortalized nasopharyngeal epithelial cells. The LMP1/hTERT-immortalized NP446 cells were non-tumorigenic in immunosuppressed nude mice and retained anchorage-dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV-encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma.
Asunto(s)
Transformación Celular Neoplásica , Células Epiteliales/citología , Nasofaringe/citología , Telomerasa/biosíntesis , Proteínas de la Matriz Viral/biosíntesis , Adulto , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , MasculinoRESUMEN
BACKGROUND: Amplification of the oncogene MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development. PRINCIPAL FINDINGS: We show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers. CONCLUSION: Binomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.
Asunto(s)
Segregación Cromosómica , Amplificación de Genes , Genes myc , Mutación , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Variación Genética , Humanos , Hibridación Fluorescente in Situ , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Mitosis , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Distribución AleatoriaRESUMEN
Many solid tumors exhibit characteristic gene fusions, which are reflected by balanced translocations at the cytogenetic level. These changes might be useful diagnostic and prognostic tools. In Wilms tumor (WT, nephroblastoma) no fusions genes or recurrent balanced translocations have been described thus far. To screen for cryptic balanced translocations, we have analyzed 17 renal neoplasms, histopathologically classified as WT, by a combination of G-banding, multicolor FISH, and subtelomeric FISH. This approach revealed several submicroscopic chromosomal aberrations and three different seemingly balanced translocations, resulting in a heterozygous deletion of HACE1, an EWSR1/ERG fusion, and an EWSR1/FLI1 fusion, respectively. As EWSR1 rearrangements are known to be a characteristic of Ewing tumors (ET), our findings illustrate the diagnostic problems regarding small cell kidney tumors and strongly argue for the need of adjuvant diagnostic techniques in this group of neoplasms. In summary, our genomic screening approach proved efficient in finding structural chromosomal aberrations. The fact that no recurrent translocations were found in the WTs of this study argues against the presence of a frequent pathognomonic translocation in this disease entity.
Asunto(s)
Aberraciones Cromosómicas , Neoplasias Renales/genética , Translocación Genética , Tumor de Wilms/genética , Adulto , Niño , Preescolar , Bandeo Cromosómico , Cromosomas Humanos/genética , Diagnóstico Diferencial , Humanos , Hibridación Fluorescente in Situ , Lactante , Cariotipificación , Neoplasias Renales/patología , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1 , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telómero/genética , Factores de Transcripción/genética , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Tumor de Wilms/patologíaRESUMEN
BACKGROUND: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally. PRINCIPAL FINDINGS: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells. CONCLUSION: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.
Asunto(s)
Genoma , Mitosis , Evolución Biológica , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Cromátides/química , Cromátides/ultraestructura , Mapeo Cromosómico , Segregación Cromosómica , Fibroblastos/metabolismo , Variación Genética , Humanos , Hibridación Fluorescente in Situ , Modelos Genéticos , Factores de TiempoRESUMEN
Previous studies on lymphocytes have suggested that patients with head and neck squamous cell carcinoma (HNSCC) have an increased susceptibility for chromosomal damage induced by bleomycin, a known radiomimetic mutagen. However, it has so far not been possible to study whether this genetic instability is present also in the epithelial component of the upper aerodigestive tract mucosa, the tissue from which HNSCC originates. In the present study, we have successfully cultured epithelial cells and fibroblasts isolated from non-neoplastic mucosa samples of 30 HNSCC patients and 56 controls. All cell cultures were exposed to bleomycin and chromosome instability was assessed by analysis of chromosome breakage in cells harvested after 2h of exposure and subsequent removal of bleomycin. Furthermore, the status of the fragile histidine triad gene (FHIT) in chromosome band 3p14.2 was studied by fluorescence in situ hybridization (FISH) in epithelial cells that had been cultured after removal of bleomycin. Chromosomal damage, in the form of chromosomal breaks and gaps, was seen in all cell cultures harvested 2h after exposure to bleomycin. In epithelial cells, the frequency of chromosome breakage was significantly higher among HNSCC patients than among controls [mean breaks per cell (b/c) 1.02 vs. 0.77, p=0.02]. When subdivided according to smoking status, age, and sex, a significantly higher frequency of chromosome breakage was still found in HNSCC patients (smokers, p=0.01, age=70 group, p=0.03, male, p=0.02). However, no significant difference was found between fibroblasts from HNSCC patients and controls (b/c 1.21 vs. 1.23). In the cell cultures growing after termination of bleomycin exposure, the frequency of chromosome breakage was generally very low and no significant difference could be found between the HNSCC patients and controls when epithelial cells were examined. In subcultured fibroblasts, a higher frequency was found in HNSCC patients than in controls (b/c 0.59 vs. 0.19, p=0.03). Interphase FISH on cultured epithelial cells from HNSCC patients (n=10) and controls (n=12) showed that the frequency of FHIT deletion was significantly higher in HNSCC patients than in controls. Our results support the notion that HNSCC patients accumulate genetic damage more rapidly, possibly due to an inherent susceptibility, which could explain the high risk for multi-focal neoplastic cell transformation.
Asunto(s)
Bleomicina/efectos adversos , Carcinoma de Células Escamosas/patología , Mucosa Gástrica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/patología , Mucosa Bucal/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Ácido Anhídrido Hidrolasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Antibióticos Antineoplásicos/efectos adversos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Mucosa Gástrica/patología , Eliminación de Gen , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Mucosa Bucal/patología , Pruebas de Mutagenicidad , Proteínas de Neoplasias/genética , Mucosa Respiratoria/patologíaRESUMEN
DNA methylation is an important epigenetic modification that regulates several genes crucial for tumor development. To identify epigenetically regulated genes in bladder cancer, we performed genome wide expression analyses of eight-bladder cancer cell lines treated with the demethylating agents 5-aza-2'-cytidine and zebularine. To identify methylated C-residues, we sequenced cloned DNA fragments from bisulfite-treated genomic DNA. We identified a total of 1092 genes that showed > or =2-fold altered expression in at least one cell line; 710 showed up-regulation and 382 down-regulation. Extensive sequencing of promoters from 25 genes in eight cell lines showed an association between methylation pattern and expression in 13 genes, including both CpG island and non-CpG island genes. Overall, the methylation patterns showed a patchy appearance with short segments showing high level of methylation separated by larger segments with no methylation. This pattern was not associated with MeCP2 binding sites or with evolutionarily conserved sequences. The genes UBXD2, AQP11, and TIMP1 showed particular patchy methylation patterns. We found several high-scoring and evolutionarily conserved transcription factor binding sites affected by methylated C residues. Two of the genes, FGF18 and MMP11, that were down-regulated as response to 5-aza-2'-cytidine and zebularine treatment showed methylation at specific sites in the untreated cells indicating an activating result of methylation. Apart from identifying epigenetically regulated genes, including TGFBR1, NUPR1, FGF18, TIMP1, and MMP11, that may be of importance for bladder cancer development the presented data also highlight the organization of the modified segments in methylated promoters. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
Asunto(s)
Epigénesis Genética , Regiones Promotoras Genéticas , Neoplasias de la Vejiga Urinaria/genética , Secuencia de Bases , Línea Celular Tumoral , Islas de CpG , Metilación de ADN , Cartilla de ADN , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
PURPOSE: In many childhood neoplasms, prognostic subgroups have been defined based on specific chromosome changes. In Wilms' tumor (WT), such subclassification has been hampered by the diverse and relatively unspecific pattern of chromosomal imbalances present in these tumors. Unspecific patterns of cytogenetic imbalances in tumors are often caused by mitotic segregation errors due to short dysfunctional telomeres. As an alternative to cytogenetic classification, we therefore have evaluated whether the rate of telomere-dependent chromosomal instability could influence the clinical course in WT patients. EXPERIMENTAL DESIGN: Telomere function and mitotic segregation errors were assessed in 12 cultured tumors and in tumor tissue sections from 41 WT patients. RESULTS: Abnormal telomere shortening was found in cultured cells and in tissue sections from highly aggressive tumors. In vitro, dysfunctional telomeres were associated to specific cell division abnormalities, including anaphase bridges and multipolar mitoses. Assessment of mitotic figures in tissue sections revealed that anaphase bridges and multipolar mitoses were predominantly, but not exclusively, present in high-risk tumors and were predictors of poor event-free and overall survival. CONCLUSIONS: Telomere-dependent mitotic instability is present in a subgroup of WT, predominantly consisting of high-risk tumors.
Asunto(s)
Segregación Cromosómica , Neoplasias Renales/patología , Telómero/ultraestructura , Tumor de Wilms/patología , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Neoplasias Renales/mortalidad , Masculino , Mitosis , Pronóstico , Tumor de Wilms/mortalidadRESUMEN
PURPOSE: Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth. EXPERIMENTAL DESIGN: We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation. RESULTS: Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival. CONCLUSION: Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.
Asunto(s)
Inestabilidad Cromosómica , Segregación Cromosómica/genética , Neoplasias Urológicas/genética , Neoplasias Urológicas/patología , Anciano , Anafase , Línea Celular Tumoral , Humanos , Mitosis , Invasividad Neoplásica , Intercambio de Cromátides Hermanas , Análisis de Supervivencia , Telómero/química , Translocación Genética , Neoplasias Urológicas/mortalidad , Urotelio/patologíaRESUMEN
Infection with high-risk human papillomavirus (HPV) has been implicated in the pathogenesis of esophageal squamous cell carcinoma, and up-regulation of telomerase in esophageal adenocarcinoma. We immortalized normal esophageal epithelial cells by over-expression of the HPV16 E6/E7 and human telomerase reverse transcriptase (hTERT) genes. HPV16 E6/E7-induced immortalization was accompanied by reduced RB and p53, but increased p16 and p21, protein expression. hTERT-immortalized cells had unaffected RB and p53, but significantly decreased p16 and p21, protein expression. Aurora-A protein was also up-regulated in E6E7 immortalized cells, and to a less extent in hTERT immortalized cells. Fluorescence in situ hybridization showed that the Aurora-A gene locus was amplified in E6E7 immortalized cells, which might account in part for the Aurora-A over-expression. These molecular changes led to an abrogation of the G2 checkpoint. E6E7 and hTERT immortalized esophageal cells recapitulated many of the molecular changes observed in esophageal carcinomas, where RB and p53 are frequently down-regulated. However, down-regulation of p16 and p21 occurred frequently in esophageal cancer, owing to aberrant gene promoter methylation. We showed in the immortalized cells that aberrant methylation had not yet set in, suggesting that promoter methylation might not be necessary for cellular immortalization. In addition to supporting the role of HPV and telomerase in esophageal carcinogenesis, our cell lines may also be useful in vitro models for further studies of esophageal carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Represoras/genética , Telomerasa/genética , Aurora Quinasas , Western Blotting , Cadherinas/genética , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Esófago/citología , Esófago/metabolismo , Citometría de Flujo , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Nocodazol/farmacología , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Telomerasa/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
We characterized the molecular genetic consequences of a balanced chromosome translocation t(8;22)(p21;q12), which occurred as the sole cytogenetic aberration in short-term cultured cells from an intrathoracic mature teratoma in a 15-year-old girl. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction disclosed that t(8;22) resulted in the fusion of the genes PPP2R2A and CHEK2, with an inserted fragment belonging to class I endogenous retrovirus-related sequences at the junction. Sequencing of the two genes did not reveal any additional mutation. None of the three detected PPP2R2A/CHEK2 fusion transcripts resulted in an in-frame PPP2R2A/CHEK2 chimerical open reading frame; however, in all of them, the known open reading frame of CHEK2 was preserved. Thus, promoter swapping leading to deregulated CHEK2 expression would be the most likely oncogenic mechanism. Whereas inactivating mutations of CHEK2 previously have been described in a variety of sporadic tumors and in inherited cancer-predisposing syndromes, PPP2R2A, encoding a regulatory subunit of the multimeric enzyme phosphatase 2, has not been directly implicated in tumorigenesis. Our findings suggest that deregulation of CHEK2 and/or PPP2R2A is of pathogenetic importance in at least a subset of germ cell tumors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de Células Germinales y Embrionarias/genética , Proteínas de Fusión Oncogénica/genética , Fosfoproteínas Fosfatasas/genética , Proteínas Serina-Treonina Quinasas/genética , Teratoma/enzimología , Translocación Genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Quinasa de Punto de Control 2 , Aberraciones Cromosómicas , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 8 , Femenino , Humanos , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/fisiología , Regiones Promotoras Genéticas , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/fisiología , Teratoma/genéticaRESUMEN
To define the early cytogenetic events important in esophageal carcinogenesis, we immortalized normal esophageal epithelial cells by overexpression of human papillomavirus type 16 E6/E7 (HPV16E6/E7) and human telomerase reverse transcriptase (hTERT), and characterized the chromosomal abnormalities serially before and after cellular immortalizaiton. During crisis, most cells had simple nonclonal karyotypic changes with cytogenetic divergence. Mitotically unstable chromosomes (i.e., telomere association and dicentric chromosomes) were the most common aberrations. After crisis, the karyotypic patterns were more convergent with nonrandom clonal changes. A few clones dominated the culture. Gain of chromosome 20q was consistently observed in four HPVE6/E7 immortalized esophageal lines, whereas amplification of chromosome 5q was preferentially found in hTERT immortalized cells. In addition, chromosomal aberrations of immortalized cells, including del(3p) and centromere rearrangements, were similar to those observed in esophageal cancer. Furthermore, in E6/E7-expressing cells, the frequency of negative telomere termini and anaphase bridges were high during crisis and low after crisis. These findings suggested that telomere dysfunction might be an important cause of cellular crisis, and the resultant chromosomal aberrations, mainly amplification of chromosome 20q or 5q, might be early genetic events required in esophageal cell immortalization. These alterations might be valuable models for further study of molecular mechanisms contributing to esophageal carcinogenesis.