RESUMO
Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off â¼40-fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAF-triggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.
Assuntos
Fatores Biológicos/metabolismo , Telomerase/antagonistas & inibidores , Catálise , Domínio Catalítico , Linhagem Celular , Ativação Enzimática , Humanos , Telomerase/metabolismoRESUMO
Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.
Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Leucemia Promielocítica Aguda/fisiopatologia , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/genética , Fenótipo , Transporte Proteico , Proteína SUMO-1/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle, and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A (TSA)-treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci, ALT-associated promyelocytic leukemia bodies, and telomere sister chromatid exchanges are activated upon TSA-induced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.
Assuntos
Ciclo Celular/fisiologia , Cromátides/metabolismo , Heterocromatina/metabolismo , Telômero/metabolismo , Cromátides/química , Eletroforese em Gel Bidimensional , Células HeLa , Heterocromatina/química , Humanos , Ácidos Hidroxâmicos/farmacologia , Telômero/químicaRESUMO
Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.
Assuntos
Envelhecimento/genética , Genômica/história , Neoplasias/genética , Telomerase/genética , Telômero/química , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Envelhecimento/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Genômica/métodos , História do Século XX , História do Século XXI , Humanos , Chaperonas Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Complexo Shelterina , Telomerase/metabolismo , Telômero/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Keloids are benign fibroproliferative skin tumors that can cause disfigurement and disability. Although they frequently recur after excision or medical management and can affect 6 to 16 percent of African Americans, there is no gold standard therapy. Keloids are challenging to study because there are no animal or in vitro models of this disorder. This makes it very difficult to validate data from treated tissue samples or cells and develop targeted therapies for this disease. In this study, the authors demonstrate that intralesional 5-fluorouracil injection after keloid excision prevents recurrence for 2 years, with no reported adverse events. The authors analyze the expression of treated and untreated biopsy specimens of the same keloids in their native context to capture insights that may be missed by in vitro cell culture models and correct for intrakeloid variability. Random forest analysis of the microarray data dramatically increased the statistical power of the authors' results, permitting hypothesis-free creation of a gene expression profile of 5-fluorouracil-treated keloids. Through this analysis, the authors found a set of genes, including YAP1 and CCL-2, whose expression changes predict 5-fluorouracil therapy status and include genes that have not previously been associated with keloid biology and are of unknown function. The authors further describe keloid heterogeneity for the first time using multidimensional analysis of their microarray results. The methods and tools the authors developed in this research may overcome some of the challenges in studying keloids and developing effective treatments for this disease. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, V.
Assuntos
Derme/patologia , Fluoruracila/administração & dosagem , Queloide/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biópsia , Quimiocina CCL2/metabolismo , Derme/cirurgia , Seguimentos , Perfilação da Expressão Gênica , Humanos , Injeções Intralesionais , Queloide/patologia , Queloide/cirurgia , Fosfoproteínas/metabolismo , Recidiva , Fatores de Transcrição , Resultado do Tratamento , Proteínas de Sinalização YAPRESUMO
Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Células A549 , Processamento Alternativo/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Antígeno Neuro-Oncológico Ventral , Splicing de RNA/genéticaRESUMO
Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.
Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Deleção de Genes , Inativação Gênica , Engenharia Genética , Genoma Humano , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Transplante de Neoplasias , Antígeno Neuro-Oncológico Ventral , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Telomerase/metabolismo , Telômero/ultraestruturaRESUMO
Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Telomerase/metabolismo , Animais , Linhagem Celular Tumoral , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Tionucleosídeos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Adenomatous polyposis coli (APC) is a key molecule to maintain cellular homeostasis in colonic epithelium by regulating cell-cell adhesion, cell polarity, and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (Asef). The APC-activated Asef stimulates the small GTPase, which leads to decreased cell-cell adherence and cell polarity, and enhanced cell migration. In colorectal cancers, while truncated APC constitutively activates Asef and promotes cancer initiation and progression, regulation of Asef by full-length APC is still unclear. Here, we report the autoinhibition mechanism of full-length APC. We found that the armadillo repeats in full-length APC interact with the APC residues 1362 to 1540 (APC-2,3 repeats), and this interaction competes off and inhibits Asef. Deletion of APC-2,3 repeats permits Asef interactions leading to downstream signaling events, including the induction of Golgi fragmentation through the activation of the Asef-ROCK-MLC2. Truncated APC also disrupts protein trafficking and cholesterol homeostasis by inhibition of SREBP2 activity in a Golgi fragmentation-dependent manner. Our study thus uncovers the autoinhibition mechanism of full-length APC and a novel gain of function of truncated APC in regulating Golgi structure, as well as cholesterol homeostasis, which provides a potential target for pharmaceutical intervention against colon cancers.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Mutação com Ganho de Função , Genes APC , Complexo de Golgi/metabolismo , Proteína da Polipose Adenomatosa do Colo/química , Sequência de Aminoácidos , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Colesterol/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Complexo de Golgi/patologia , Células HCT116 , Células HT29 , Homeostase , Humanos , Modelos Biológicos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Deleção de Sequência , Transdução de SinaisRESUMO
Telomerase is a cellular RNA template-dependent reverse transcriptase that adds telomere repeats to the 3' ends of chromosomes. Telomerase is expressed almost universally in tumor cells (>85%) to maintain telomere length, thus providing the ability of tumor cells to avoid senescence and to have unlimited replication ability, one of the key hallmarks of cancer. ddTRAP (droplet digital Telomere Repeat Amplification Protocol) is a two-step assay with whole cell lysates that utilizes a telomerase-mediated primer extension followed by droplet digital PCR (ddPCR) detection of extended products. The adoptation of the TRAP assay to ddPCR has resulted in improved throughput, increased sensitivity and better repeatability of the TRAP assay. The protocol described below details our procedures for ddTRAP.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , DNA de Neoplasias/isolamento & purificação , Neoplasias/diagnóstico , Reação em Cadeia da Polimerase/métodos , Telomerase/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Primers do DNA/genética , DNA de Neoplasias/genética , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patologia , Reação em Cadeia da Polimerase/instrumentação , Telômero/genética , Telômero/metabolismoRESUMO
We developed methods for conditionally reprogramming (CR) primary human bronchial epithelial cells (HBECs) to extend their functional lifespan and permit their differentiation into both upper and lower airway lung epithelium. We also developed a bioreactor to support vascular perfusion and rhythmic breathing of decellularized mouse lungs reconstituted with CR HBECs isolated from patients with and without cystic fibrosis (CF). While conditionally reprogrammed cells only differentiate into an upper airway epithelium after 35 days at the air-liquid interface, in reconstituted lungs these cells differentiate into upper airway bronchial epithelium and lower airway alveolar structures after 12 days. Rapid scale-up and the ability to obtain clonal derivatives of primary patient-derived HBECs without the need for genetic manipulation may permit rapid reconstitution of the lung epithelium; facilitating the study of lung disease in tissue-engineered models.
Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Pulmão/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Camundongos , Engenharia TecidualRESUMO
Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. We developed Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. TeSLA improves the specificity and efficiency of TL measurements that is facilitated by user friendly image-processing software to automatically detect and annotate band sizes, calculate average TL, as well as the percent of the shortest telomeres. Compared with other TL measurement methods, TeSLA provides more information about the shortest telomeres. The length of telomeres was measured longitudinally in peripheral blood mononuclear cells during human aging, in tissues during colon cancer progression, in telomere-related diseases such as idiopathic pulmonary fibrosis, as well as in mice and other organisms. The results indicate that TeSLA is a robust method that provides a better understanding of the shortest length of telomeres.
Assuntos
Envelhecimento/genética , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Telômero/genética , Adulto , Idoso , Animais , Southern Blotting , Feminino , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente/métodos , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3 , SoftwareRESUMO
Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.
Assuntos
Reparo do DNA/fisiologia , DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Telomerase/metabolismo , Homeostase do Telômero/fisiologia , Telômero/metabolismo , Linhagem Celular , Replicação do DNA/fisiologia , Humanos , Recombinação Genética/genéticaRESUMO
Telomerase is present in most human cancers, and proliferative stem cells including germline cells. Telomerase plays an essential role in tumorigenesis by maintaining/elongating telomeric DNA, and thus preventing the telomere shortening that results in replicative senescence. Understanding telomerase action in vivo has important implication for both cancer and aging, but there are not robust methods for monitoring telomerase action. By combining a series of cell biological and biochemical approaches, and taking advantage of the enzyme DSN that specifically cuts double-stranded DNA and releases the telomeric overhangs, we have developed a method to monitor telomerase action during one cell cycle. Here, we describe this method using Hela carcinoma cells as an example.
Assuntos
DNA/genética , Telômero/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Células HeLa , Humanos , Telomerase/genéticaRESUMO
Alternative lengthening of telomeres (ALT) is a telomerase independent telomere maintenance mechanism that occurs in â¼15% of cancers. The potential mechanism of ALT is homology-directed telomere synthesis, but molecular mechanisms of how ALT maintains telomere length in human cancer is poorly understood. Here, we generated TERC (telomerase RNA) gene knockouts in telomerase positive cell lines that resulted in long-term surviving clones acquiring the ALT pathway but at a very low frequency. By comparing these ALT cells with parental telomerase positive cells, we observed that ALT cells possess excessively long telomeric overhangs derived from telomere elongation processes that mostly occur during S phase. ALT cells exhibited preferential elongation of the telomeric lagging strands, whereas telomerase positive cells exhibited similar elongation between leading and lagging strands. We propose that the ALT pathway preferentially occurs at telomeric lagging strands leading to heterogeneous telomere lengths observed in most ALT cancers.
Assuntos
Neoplasias/genética , Homeostase do Telômero , Telômero/metabolismo , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , DNA Helicases/antagonistas & inibidores , Técnicas de Inativação de Genes , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , RNA/genética , Fase S/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/química , Proteína Nuclear Ligada ao XRESUMO
Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.
Assuntos
Envelhecimento/genética , Regulação Enzimológica da Expressão Gênica , Neoplasias/genética , Telomerase/genética , Telômero , Animais , Cromossomos Humanos Par 5 , Humanos , Primatas/genéticaRESUMO
Mutations in the adenomatous polyposis coli (APC) gene are common in colorectal cancer (CRC), and more than 90% of those mutations generate stable truncated gene products. We describe a chemical screen using normal human colonic epithelial cells (HCECs) and a series of oncogenically progressed HCECs containing a truncated APC protein. With this screen, we identified a small molecule, TASIN-1 (truncated APC selective inhibitor-1), that specifically kills cells with APC truncations but spares normal and cancer cells with wild-type APC. TASIN-1 exerts its cytotoxic effects through inhibition of cholesterol biosynthesis. In vivo administration of TASIN-1 inhibits tumor growth of CRC cells with truncated APC but not APC wild-type CRC cells in xenograft models and in a genetically engineered CRC mouse model with minimal toxicity. TASIN-1 represents a potential therapeutic strategy for prevention and intervention in CRC with mutant APC.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Terapia de Alvo Molecular , Piperidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Proliferação de Células , Colesterol/química , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Genes Supressores de Tumor , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Loss of function in genes required for telomere maintenance result in disorders known as telomeropathies, which are characterized by a pattern of symptoms including generalized and specific lymphocytopenias as well as very short telomere length and disease anticipation. METHODS: Because human LARP7 is the most likely ortholog of the Tetrahymena p65 protein, which is required for telomerase activity in that organism, we investigated the effects of LARP7 silencing in human cells as well as in two distinct families with Alazami syndrome (loss of function of LARP7). RESULTS: Depletion of LARP7 caused a reduction in telomerase enzymatic activity and progressively shorter telomeres in human cancer cell lines. Alazami syndrome patients from two separate cohorts exhibited very short lymphocyte telomeres. Further, wild-type offspring of LARP7 mutant individuals also had very short telomeres, comparable to what is observed in telomerase (hTERT) mutant cohorts. CONCLUSIONS: Together, these experiments demonstrate that in addition to the readily apparent developmental disorder associated with LARP7 deficiency, an underlying telomeropathy exists even in unaffected siblings of these individuals.
Assuntos
Estudos de Associação Genética , Ribonucleoproteínas/deficiência , Telômero/genética , Adulto , Linhagem Celular , Criança , Estudos de Coortes , Consanguinidade , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hibridização in Situ Fluorescente , Masculino , Modelos Genéticos , Mutação , Linhagem , Fenótipo , Homeostase do Telômero/genéticaRESUMO
Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.
Assuntos
Southern Blotting/métodos , DNA/análise , DNA/genética , Digoxigenina/química , Sondas de Oligonucleotídeos/genética , Homeostase do Telômero/genética , DNA/química , Técnicas de Sonda Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.