RESUMO
Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.
RESUMO
BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.
Assuntos
Lipossarcoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipossarcoma/patologia , Invasividade Neoplásica/fisiopatologia , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: This study investigated the effect of comorbidity, age, health insurance payer status, and race on the risk of patient nonadherence to NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Colon and Rectal Cancers. In addition, the prognostic impact of NCCN treatment nonadherence on overall survival was assessed. PATIENTS AND METHODS: Patients with CRC who received primary treatment at Memorial University Medical Center from 2003 to 2010 were eligible for this study. Modified Poisson regression was used to obtain risk ratios for the outcome of nonadherence with NCCN Guidelines. Hazard ratios (HRs) for the relative risk of death from all causes were obtained through Cox regression. RESULTS: Guideline-adherent treatment was received by 82.7% of patients. Moderate/severe comorbidity, being uninsured, having rectal cancer, older age, and increasing tumor stage were associated with increased risks of receiving nonadherent treatment. Treatment nonadherence was associated with 3.6 times the risk of death (HR, 3.55; 95% CI, 2.16-5.85) in the first year after diagnosis and an 80% increased risk of death (HR, 1.80; 95% CI, 1.14-2.83) in years 2 to 5. The detrimental effect of nonadherence declined with increasing comorbidity and varied according to age. CONCLUSIONS: Although medically justifiable reasons exist for deviating from NCCN Guidelines when treating patients with colorectal cancer (CRC), those who received nonadherent treatment had much higher risks of death, especially in the first year after diagnosis. This study's results highlight the importance of cancer health services research to drive quality improvement efforts in cancer care for patients with CRC.
Assuntos
Neoplasias Colorretais/epidemiologia , Fidelidade a Diretrizes , Adulto , Idoso , Idoso de 80 Anos ou mais , Causas de Morte , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Comorbidade , Feminino , República da Geórgia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Razão de Chances , Prognóstico , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Resultado do TratamentoRESUMO
Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P < 0.001). Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation.
RESUMO
Telomere maintenance is an essential characteristic of cancer cells, most commonly achieved by activation of telomerase. Telomeres can also be maintained by a recombination-based mechanism, alternative lengthening of telomeres (ALT). Cells using ALT are characterized by the presence of ALT-associated promyelocytic leukemia (PML) bodies (APB), long, heterogeneously sized telomeres, extrachromosomal telomeric circular DNA, and elevated telomeric recombination. Consistent with other reports, we found that liposarcomas containing APBs, but lacking telomerase expression, always contained C-rich circles (C-circles), and these C-circles were never present in the absence of APBs, indicating a tight link between these features in ALT cells. However, a rare subgroup of tumors showing evidence of telomere maintenance by both telomerase and ALT did not contain C-circles. To test the hypothesis that telomerase expression disrupts the tight link between APBs and C-circles, we used ALT cell lines that were engineered to express telomerase. Introduction of telomerase activity in these ALT cells resulted in, on average, shorter telomeres with retention of APBs. However, at high passage, the level of C-circles was significantly reduced, which was paralleled by a switch from C-strand overhangs to G-strand overhangs. We propose that by extending critically short telomeres in these cells, telomerase is disrupting a key step in the ALT pathway necessary for production and/or maintenance of C-circles.
Assuntos
Conformação de Ácido Nucleico , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Humanos , Corpos de Inclusão/metabolismo , Modelos Biológicos , Proteína 1 de Ligação a Repetições Teloméricas/metabolismoRESUMO
BACKGROUND: Pleomorphic liposarcoma (PLS) is a rare high-grade sarcoma that has lipoblastic differentiation. In this study, the authors evaluated PLS natural history, patient outcomes, and commonly deregulated protein biomarkers. METHODS: Medical records from patients (n = 155) who had PLS from 1993 to 2010 were reviewed. Univariate and multivariate analyses were conducted to identify independent prognosticators. A PLS tissue microarray (TMA) (n = 56 patient specimens) was constructed for immunohistochemical analysis of molecular markers, and p53 gene sequencing (exons 5-9) was conducted. RESULTS: The average patient age was 57 years, and the patients presented with primary disease (n = 102), recurrent disease (n = 16), and metastatic disease (n = 37). Lower extremity was the most common disease site (40%), and the average tumor size was 11 cm. Complete follow-up data were available for 83 patients, and their median follow-up was 22.6 months. The 5-year disease-specific survival rate was 53%; and recurrent disease, unresectability, and microscopic positive margins were identified as predictors of a poor prognosis. Systemic relapse (the strongest poor prognostic determinant) developed in 35% of patients with localized PLS. Immunohistochemical analysis revealed increased expression of peroxisome proliferator-activated receptor gamma (an adipogenic marker), B-cell leukemia 2 and survivin (survival factors), vascular endothelial growth factor (an angiogenic factor), matrix metalloproteinase 2, and other biomarkers. Frequent loss of retinoblastoma protein expression and high p53 mutation rates (approximately 60%) were observed. CONCLUSIONS: PLS is an aggressive, metastasizing sarcoma. Identifying ubiquitous molecular events underlying PLS progression is crucial for progress in patient management and outcomes.
Assuntos
Lipossarcoma/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Genes p53 , Humanos , Lipossarcoma/química , Lipossarcoma/mortalidade , Lipossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Mutação , PPAR gama/análise , PPAR gama/fisiologia , Prognóstico , Modelos de Riscos Proporcionais , Análise Serial de TecidosRESUMO
Soft tissue sarcomas are a diverse set of fatal human tumors where few agents have demonstrable clinical efficacy, with the standard therapeutic combination of doxorubicin and ifosfamide showing only a 25% to 30% response rate in large multi-institutional trials. Although liposarcomas are the most common histologic form of adult soft tissue sarcomas, research in this area is severely hampered by the lack of experimentally tractable in vitro model systems. To this end, here we describe a novel in vitro model for human pleomorphic liposarcoma. The cell line (LS2) is derived from a pleomorphic liposarcoma that uses the alternative lengthening of telomeres (ALT) mechanism of telomere maintenance, which may be important in modulating the response of this tumor type to DNA-damaging agents. We present detailed baseline molecular and genomic data, including genome-wide copy number and transcriptome profiles, for this model compared with its parental tumor and a panel of liposarcomas covering multiple histologies. The model has retained essentially all of the detectable alterations in copy number that are seen in the parental tumor, and shows molecular karyotypic and expression profiles consistent with pleomorphic liposarcomas. We also show the utility of this model, together with two additional human liposarcoma cell lines, to investigate the relationship between topoisomerase 2A expression and the sensitivity of ALT-positive liposarcomas to doxorubicin. This model, together with its associated baseline data, provides a powerful new tool to develop treatments for this clinically poorly tractable tumor and to investigate the contribution that ALT makes to modulating sensitivity to doxorubicin.
Assuntos
Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Lipossarcoma/genética , Neoplasias de Tecidos Moles/genética , Telômero/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Aberrações Cromossômicas/induzido quimicamente , Análise Citogenética , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Instabilidade Genômica/efeitos dos fármacos , Células HeLa , Humanos , Lipossarcoma/tratamento farmacológico , Lipossarcoma/patologia , Modelos Teóricos , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/patologia , Telômero/metabolismoRESUMO
Mammalian telomeres consist of TTAGGG repeats organized in nucleosomes and associated with a six-protein complex known as shelterin, which preserves telomere structure and protects chromosome ends from the cellular DNA damage response. Recent studies have found that telomeres are transcribed into telomeric UUAGGG repeat-containing RNA (TERRA) starting from subtelomeric regions. TERRA binding at telomeres appears to be involved in cis-based mechanisms of telomeric chromatin organization and maintenance. A number of histone methyltransferases (HMTs) are known to influence telomeric chromatin status; however, the regulatory mechanisms of telomere transcription are poorly understood. Here, we show that the histone 3/lysine 4 (H3/K4) HMT and the transcriptional regulator MLL associate with telomeres and contribute to their H3/K4 methylation and transcription in a telomere length-dependent manner. In human diploid fibroblasts, RNA interference-mediated MLL depletion affects telomere chromatin modification and transcription and induces the telomere damage response. Telomere uncapping through either TRF2 shelterin protein knockdown or exposure to telomere G-strand DNA oligonucleotides significantly increases the transcription of TERRA, an effect mediated by the functional cooperation between MLL and the tumor suppressor p53. In total, our findings identify a previously unrecognized role of MLL in modifying telomeric chromatin and provide evidence for the functional interaction between MLL, p53, and the shelterin complex in the regulation of telomeric transcription and stability.
Assuntos
Proteína de Leucina Linfoide-Mieloide/metabolismo , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Telômero , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Senescência Celular/fisiologia , Centrômero/metabolismo , Técnicas de Silenciamento de Genes , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo Shelterina , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Chromosome ends are maintained by telomere-repeat-binding factors (TRFs) that coordinate DNA end protection with telomere replication. The origin recognition complex (ORC) coordinates bidirectional DNA replication at most chromosomal sites, but it is also known to function in transcriptional silencing, heterochromatin formation, and sister-chromatid cohesion. We now show that ORC localizes to telomere repeats and contributes to telomere maintenance. We found that ORC subunits can be affinity purified with telomere-repeat DNA along with other components of the known "shelterin" complex. ORC subunits colocalized with telomere-repeat foci and coimmunoprecipitated with TRF2 but not TRF2 lacking its amino-terminal basic domain (TRF2DeltaB). ORC2 depletion or hypomorphic cell lines caused a loss of telomere-repeat signal intensity and the appearance of dysfunctional telomeres, including telomere-signal-free ends and telomere-repeat-containing double minutes. Two-dimensional agarose gel electrophoresis revealed that ORC2 depletion increased telomere circle formation, comparable to the overexpression of TRF2DeltaB. A similar increase in telomere circle formation was induced by hydroxyurea treatment, providing evidence that replication stress produces telomere circles. These findings suggest that ORC recruitment by TRF2 contributes to telomere integrity by facilitating efficient telomere DNA replication and preventing the generation of telomere-repeat-containing circles.
Assuntos
Aberrações Cromossômicas , Complexo de Reconhecimento de Origem/metabolismo , Sequências Repetitivas de Ácido Nucleico/fisiologia , Telômero/genética , Células HCT116 , Humanos , Complexo de Reconhecimento de Origem/genética , Telômero/metabolismoRESUMO
Telomere attrition ultimately leads to the activation of protective cellular responses, such as apoptosis or senescence. Impairment of such mechanisms can allow continued proliferation despite the presence of dysfunctional telomeres. Under such conditions, high levels of genome instability are often engendered. Data from both mouse and human model systems indicate that a period of genome instability might facilitate tumorigenesis. Here, we use a liposarcoma model system to assay telomere maintenance mechanism (TMM)-specific genetic alterations. A multiassay approach was used to assess the TMMs active in tumors. Genomic DNA from these samples was then analyzed by high-resolution DNA mapping array to identify genetic alterations. Our data reveal a higher level of genome instability in alternative lengthening of telomere (ALT)-positive tumors compared with telomerase-positive tumors, whereas tumors lacking both mechanisms have relatively low levels of genome instability. The bulk of the genetic changes are amplifications, regardless of the mode of telomere maintenance used. We also identified genetic changes specific to the ALT mechanism (e.g., deletion of chromosome 1q32.2-q44) as well as changes that are underrepresented among ALT-positive tumors, such as amplification of chromosome 12q14.3-q21.2. Taken together, these studies provide insight into the molecular pathways involved in the regulation of ALT and reveal several loci that might be exploited either as prognostic markers or targets of chemotherapeutic intervention.
Assuntos
Lipossarcoma/genética , Telômero/genética , Idoso , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Genoma Humano , Instabilidade Genômica , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE OF REVIEW: To examine the activation of telomere maintenance in a variety of sarcoma subtypes, and to review the consequences of telomere maintenance with respect to genome stability and tumor progression. RECENT FINDINGS: A hallmark of tumor cells is replicative immortality, which can be achieved, in part, by the activation of a telomere maintenance mechanism. A significant proportion of tumors show activation of telomerase, a specialized enzyme that adds telomeric repeats to pre-existing telomeres. Recent work has demonstrated, however, that a telomerase-independent mechanism called ALT (alternative lengthening of telomeres) is activated as frequently as telomerase in a variety of tumor types, particularly those of mesenchymal origin. Accordingly, panels of mesenchymal tumors have been interrogated for telomere maintenance mechanism, as well as characteristics such as tumor grade and patient survival. SUMMARY: These studies indicate a strong positive correlation between the activation of a telomere maintenance mechanism and tumor progression in sarcomas. In addition, the activation of either ALT or telomerase is correlated with poorer patient prognosis as compared with a lack of telomere maintenance. Ongoing studies aimed at elucidating the roles of ALT and telomerase in tumorigenesis should ultimately allow for the development of strategies to improve treatment of these malignancies.
Assuntos
Sarcoma/patologia , Telomerase , Telômero , Condrossarcoma Mesenquimal , Instabilidade Cromossômica , Progressão da Doença , Humanos , Sarcoma/mortalidade , SobrevidaRESUMO
We have previously reported the identification and characterization of a novel BRCA1/2 interacting protein complex, BRCC (BRCA1/2-containing complex). BRCC36, one of the proteins in BRCC, directly interacts with BRCA1, and regulates the ubiquitin E3 ligase activity of BRCC. Importantly, BRCC36 is aberrantly expressed in the vast majority of breast tumors, indicating a potential role in the pathogenesis of this disease. To further elucidate the functional consequence of abnormal BRCC36 expression in breast cancer, we have done in vivo silencing studies using small interfering RNAs targeting BRCC36 in breast cancer cell lines, i.e., MCF-7, ZR-75-1, and T47D. Knock-down of BRCC36 alone does not affect cell growth, but when combined with ionizing radiation (IR) exposure, it leads to an increase in the percentage of cells undergoing apoptosis when compared with the small interfering RNA control group in breast cancer cells. Immunoblot analysis shows that inhibition of BRCC36 has no effect on the activation of ATM, expression of p21 and p53, or BRCA1-BARD1 interaction following IR exposure. Importantly, BRCC36 depletion disrupts IR-induced phosphorylation of BRCA1. Immunofluorescent staining of BRCA1 and gamma-H2AX indicates that BRCC36 depletion prevents the formation of BRCA1 nuclear foci in response to DNA damage in breast cancer cells. These results show that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR by inhibiting BRCA1 activation, thereby sensitizing breast cancer cells to IR-induced apoptosis.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose/fisiologia , Apoptose/efeitos da radiação , Proteína BRCA1/antagonistas & inibidores , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Reparo do DNA/fisiologia , Regulação para Baixo , Expressão Gênica , Humanos , Fosforilação/efeitos da radiação , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
PURPOSE: Telomeres are specialized nucleoprotein complexes that protect and confer stability upon chromosome ends. Loss of telomere function as a consequence of proliferation-associated sequence attrition results in genome instability, which may facilitate carcinogenesis by generating growth-promoting mutations. However, unlimited cellular proliferation requires the maintenance of telomeric DNA; thus, the majority of tumor cells maintain their telomeres either through the activity of telomerase or via a mechanism known as alternative lengthening of telomeres (ALT). Recent data suggest that constitutive telomere maintenance may not be required in all tumor types. Here we assess the role and requirement of telomere maintenance in liposarcoma. EXPERIMENTAL DESIGN: Tumor samples were analyzed with respect to telomerase activity, telomere length, and the presence of ALT-specific subcellular structures, ALT-associated promyelocytic leukemia nuclear bodies. This multi-assay assessment improved the accuracy of categorization. RESULTS: Our data reveal a significant incidence (24%) of ALT-positive liposarcomas, whereas telomerase is used at a similar frequency (27%). A large number of tumors (49%) do not show characteristics of telomerase or ALT. In addition, telomere length was always shorter in recurrent disease, regardless of the telomere maintenance mechanism. CONCLUSIONS: These results suggest that approximately one half of liposarcomas either employ a novel constitutively active telomere maintenance mechanism or lack such a mechanism. Analysis of recurrent tumors suggests that liposarcomas can develop despite limiting or undetectable activity of a constitutively active telomere maintenance mechanism.
Assuntos
Lipossarcoma/ultraestrutura , Telômero/ultraestrutura , Adulto , Idoso , Southern Blotting , Proliferação de Células , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Genoma , Humanos , Processamento de Imagem Assistida por Computador , Lipossarcoma/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Nucleoproteínas/metabolismo , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/ultraestrutura , RNA Mensageiro/metabolismo , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismoRESUMO
Telomere stabilization is critical for tumorigenesis. A number of tumors and cell lines use a recombination-based mechanism, alternative lengthening of telomeres (ALT), to maintain telomere repeat arrays. Current data suggest that the mutation of p53 facilitates the activation of this pathway. In addition to its functions in response to DNA damage, p53 also acts to suppress recombination, independent of transactivation activity, raising the possibility that p53 might regulate the ALT mechanism via its role as a regulator of recombination. To test the role of p53 in ALT we utilized inducible alleles of human p53. We show that expression of transactivation-incompetent p53 inhibits DNA synthesis in ALT cell lines but does not affect telomerase-positive cell lines. The expression of temperature-sensitive p53 in clonal cell lines results in ALT-specific, transactivation-independent growth inhibition, due in part to the perturbation of S phase. Utilizing chromatin immunoprecipitation assays, we demonstrate that p53 is associated with the telomeric complex in ALT cells. Furthermore, the inhibition of DNA synthesis in ALT cells by p53 requires intact specific DNA binding and suppression of recombination functions. We propose that p53 causes transactivation-independent growth inhibition of ALT cells by perturbing telomeric recombination.
Assuntos
Divisão Celular/genética , Telômero/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Alelos , Linhagem Celular , Replicação do DNA , Humanos , Mutação/fisiologia , Recombinação Genética , Fase S , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Transformation of the human breast epithelial cells (HBEC) MCF-10F with the carcinogen benz(a)pyrene (BP) into BP1-E cells resulted in the loss of the chromosome 17 p13.2 locus (D17S796 marker) and formation of colonies in agar-methocel (colony efficiency (CE)), loss of ductulogenic capacity in collagen matrix, and resistance to anti-Fas monoclonal antibody (Mab)-induced apoptosis. For testing the role of that specific region of chromosome 17 in the expression of transformation phenotypes, we transferred chromosome 17 from mouse fibroblast donors to BP1-E cells. Chromosome 11 was used as negative control. After G418 selection, nine clones each were randomly selected from BP1-E-11neo and BP1-E-17neo hybrids, respectively, and tested for the presence of the donor chromosomes by fluorescent in situ hybridization and polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analyses. Sensitivity to Fas Mab-induced apoptosis and evaluation of transformation phenotype expression were tested in MCF-10F, BP1-E, and nine BP1-E-11neo and BP1-E-17neo clones each. Six BP1-E-17neo clones exhibited a reversion of transformation phenotypes and a dose dependent sensitivity to Fas Mab-induced apoptosis, behaving similarly to MCF-10F cells. All BP1-E-11neo, and three BP1-E-17neo cell clones, like BP1-E cells, retained a high CE, loss of ductulogenic capacity, and were resistant to all Fas Mab doses tested. Genomic analysis revealed that those six BP1-E-17neo clones that were Fas-sensitive and reverted their transformed phenotypes had retained the 17p13.2 (D17S796 marker) region, whereas it was absent in all resistant clones, indicating that the expression of transformation phenotypes and the sensitivity of the cells to Fas-mediated apoptosis were under the control of genes located in this region.
Assuntos
Apoptose , Mama/citologia , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 17/genética , Células Epiteliais/citologia , Receptor fas/metabolismo , Animais , Benzo(a)pireno/toxicidade , Mama/enzimologia , Mama/patologia , Divisão Celular/genética , Células Cultivadas , Cromossomos Humanos Par 11/genética , Células Clonais/citologia , Células Clonais/enzimologia , Colágeno/metabolismo , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Células Híbridas/citologia , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Repetições de Microssatélites/genética , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Telomerase/metabolismo , Transfecção , Receptor fas/genética , Receptor fas/imunologiaRESUMO
Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for the complete replication and stability of naturally occurring chromosome ends. Telomere stabilization is critical for the unlimited cellular proliferation that is necessary for tumor formation. While most tumors achieve telomere stabilization through activation of telomerase, a subset of tumors utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain chromosome termini. Tumors utilizing ALT for telomere preservation will likely be refractory to treatment with telomerase inhibitors. Furthermore, tumors carrying mutations that predispose a cell to utilize ALT may activate this pathway when challenged by telomerase inhibition. Mutation of the mismatch repair (MMR) pathway enhances telomerase independent survival in yeast, with the survivors using recombination-based pathways for telomere maintenance. One possibility is that mutation of the MMR pathways alleviates suppression of recombination, thereby abrogating the need for telomerase activation. If true, one might predict an increased frequency of tumors harboring MMR mutation to use ALT for telomere maintenance. Here we characterized tumors with and without MMR mutation for the presence of telomerase activity versus ALT. We found similarly frequent activation of telomerase in tumors with and without MMR mutation, suggesting that human tumors with MMR mutation may respond favorably to treatment with telomerase inhibitors.
Assuntos
Pareamento Incorreto de Bases/genética , Carcinoma de Células de Transição/genética , Neoplasias Colorretais/genética , Dano ao DNA , Repetições de Microssatélites , Neoplasias Ovarianas/genética , Telomerase/farmacologia , Telômero/metabolismo , Análise Mutacional de DNA , Reparo do DNA , Feminino , HumanosRESUMO
Telomeres are specialized structures at the ends of linear chromosomes that were originally defined functionally based on observations first by Muller (1938) and subsequently by McClintock (1941) that naturally occurring chromosome ends do not behave as double-stranded DNA breaks, in spite of the fact that they are the physical end of a linear, duplex DNA molecule. Double-stranded DNA breaks are highly unstable entities, being susceptible to nucleolytic attack and giving rise to chromosome rearrangements through end-to-end fusions and recombination events. In contrast, telomeres confer stability upon chromosome termini, as evidenced by the fact that chromosomes are extraordinarily stable through multiple cell divisions and even across evolutionary time. This protective function of telomeres is due to the formation of a nucleoprotein complex that sequesters the end of the DNA molecule, rendering it inaccessible to nucleases and recombinases as well as preventing the telomere from activating the DNA damage checkpoint pathways. The capacity of a functional end-protective complex to form is dependent upon maintenance of sufficient telomeric DNA. We have learned a great deal about telomere structure and how this specialized nucleoprotein complex confers stability on chromosome ends since the original observations that defined telomeres were made. This review summarizes our current understanding of mammalian telomere replication, structure and function.
RESUMO
Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5- to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G(1)/S while TRF1 binding peaks in G(2)/M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.
Assuntos
Replicação do DNA , Plasmídeos , Proteínas de Ligação a Telômeros/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Replicação Viral , Ciclo Celular , DNA/metabolismo , Células HeLa , Humanos , Complexo Shelterina , TelômeroRESUMO
BACKGROUND: We investigated the effects of telomerase inhibition by using the reverse transcriptase inhibitor azidothymidine (AZT) in the human colorectal cancer cell line HT-29 in the presence and absence of 5-fluorouracil (5-FU). METHODS: HT-29 cells were cultured in the presence of AZT. Telomerase activity was measured by using the telomerase repeat amplification protocol. Telomere length was determined by Southern analysis. The colorimetric microtiter assay was performed to determine the cytotoxic effects of AZT, alone and in combination with 5-FU. RESULTS: The presence of 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) effectively inhibited telomerase extracted from HT-29 cells. HT-29 cells cultured with 125 microM of AZT underwent fewer total population doublings over 91 days. Southern analysis revealed that telomere attrition occurred within this period. Exposure to 125 microM of AZT resulted in slightly reduced viability (10%) of HT-29 cells. However, the presence of AZT increased 5-FU cytotoxicity, suggesting that the effects of these two drugs are synergistic. CONCLUSIONS: The data are consistent with telomerase inhibition having growth-inhibitory effects in addition to those predicted to accompany loss of telomere function. Further studies using specific small-molecule inhibitors will confirm whether the growth-inhibitory and 5-FU-sensitivity effects seen here are a direct result of telomerase inhibition.