RESUMO
Nucleoli form interchromosomal contacts with genes controlling differentiation and carcinogenesis. DUX4 genes specify transcription factor possessing two homeodomains. Previously, using Circular Chromosome Conformation Capture (4С) approach on population of cells, it was demonstrated that DUX4 gene clusters form frequent contacts with nucleoli. It was found also that these contacts are almost completely abolished after heat shock treatment. 4C approach as all ligation-mediated methods is capable to detect rather close interactions between chromatin loops in nuclei. In order to independently confirm the formation and the frequency of the contacts in single cells we used FISH approach. Here, we show that DUX genes in single cells form stable contacts in all tested HEK293T cells. During heat shock, DUX4 genes reversibly move 1-3 µm away from the nuclei. We conclude that interchromosomal contacts formed by nucleoli are strong, dynamic, and reversible, providing both the initiation and maintenance of a differentiated state.
Assuntos
Nucléolo Celular , Proteínas de Homeodomínio , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células HEK293 , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Hibridização in Situ FluorescenteRESUMO
Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.
Assuntos
Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae , Humanos , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/genética , Sítios de Ligação , Replicação do DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromossomos Humanos/metabolismo , DNA/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Prostate cancer aggressiveness and metastatic potential are influenced by gene expression and genomic aberrations, features that can be influenced by the 3D structure of chromosomes inside the nucleus.â¯Using chromosome conformation capture (Hi-C), we conducted a systematic genome architecture comparison on a cohort of cell lines that model prostate cancer progression, from normal epithelium to bone metastasis. We describe spatial compartment identity (A-open versus B-closed) changes with progression in these cell lines and their relation to gene expression changes in both cell lines and patient samples. In particular, 48 gene clusters switch from the B to the A compartment, including androgen receptor, WNT5A, and CDK14. These switches are accompanied by changes in the structure, size, and boundaries of topologically associating domains (TADs). Further, compartment changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation.â¯These results suggest that discrete 3D genome structure changes play a deleterious role in prostate cancer progression.â¯.
Assuntos
Cromossomos Humanos/metabolismo , Progressão da Doença , Modelos Biológicos , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Estudos de Coortes , Genes Neoplásicos , Genoma Humano , Humanos , Masculino , Metástase Neoplásica , Neoplasias da Próstata/genética , Serina Endopeptidases/metabolismo , Ativação Transcricional/genéticaRESUMO
Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.
Assuntos
Autofagia , Capilares/citologia , Radiação Cósmica , Células Endoteliais/efeitos da radiação , Transdução de Sinais , Ausência de Peso , Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Sobrevivência Celular , Cromossomos Humanos/metabolismo , Citoesqueleto/metabolismo , Dano ao DNA , Fluorescência , Regulação da Expressão Gênica , Genoma Humano , Humanos , Masculino , Mecanotransdução Celular , Modelos Biológicos , Transdução de Sinais/efeitos da radiação , Voo Espacial , Estresse Fisiológico , Homeostase do Telômero , Transcriptoma/genéticaRESUMO
The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.
Assuntos
Cromossomos Humanos , Instabilidade Genômica , Proteínas de Neoplasias , Neoplasias , Proteínas Ligases SKP Culina F-Box , Transcrição Gênica , Animais , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismoRESUMO
Core binding factor acute myelogenous leukemia (CBF-AML), characterized by the presence of either t(8;21) (q22;q22) or inv(16) (p13q22)/t(16;16), is considered good-risk AML in the context of cytarabine based intensive chemotherapy. Still, outcome can be improved significantly through the effective implementation of available therapeutic measures and appropriate disease monitoring. The incorporation of gemtuzumab ozogamicin into frontline therapy should be standard. Cytarabine based induction/consolidation regimen may be combined with anthracycline (3 + 7 standard) or antimetabolite, fludarabine. Serial quantitative polymerase chain reaction (QPCR) monitoring of unique fusion transcripts allows monitoring for measurable residual disease clearance; this allows for better prognostication and well as treatment modifications.
Assuntos
Algoritmos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fatores de Ligação ao Core , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Antraciclinas/uso terapêutico , Inversão Cromossômica , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Fatores de Ligação ao Core/sangue , Fatores de Ligação ao Core/genética , Citarabina/uso terapêutico , Intervalo Livre de Doença , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Neoplasia Residual , Translocação GenéticaRESUMO
Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.
Assuntos
Cromossomos Humanos/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mitose , Cromossomos Humanos/genética , Fosfatases de Especificidade Dupla/genética , Células HCT116 , Células HeLa , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Mutação , Fosforilação/genéticaRESUMO
Well-differentiated liposarcoma/atypical lipomatous tumor (WDLS/ALT) and dedifferentiated liposarcoma (DDLS) have characteristic supernumerary ring and giant marker chromosomes involving the chromosomal region 12q13-15 which contains MDM2 (12q15), CDK4 (12q14.1), HMGA2 (12q14.3), YEATS4 (12q15), CPM (12q15), and FRS2 (12q15). Detecting MDM2 amplification by fluorescence in situ hybridization (FISH) is considered to be the gold standard for the diagnosis of WDLS/ALT and DDLS. In this study, formalin fixed paraffin embedded clinical specimens (16 liposarcomas and 19 benign lipomatous tumors) were used to detect MDM2 amplification and other chromosomal alterations in WDLS/ALT and DDLS by single nucleotide polymorphism-based chromosome microarray (CMA). All 16 liposarcomas showed MDM2 amplification with a MDM2/cep12 ratio from 2.4 to 8.4 by CMA. Ten (62.5%) of these cases had CDK4/cep12 ratio ≥2.0. All the cases without CDK4 amplification were from the thigh. The MDM2/cep12 ratio of all the benign lipomatous tumors (19/19) was within the normal limits. Twenty-one of the 35 benign lipomatous tumors and liposarcomas were also tested for MDM2 amplification by FISH. All the FISH results were consistent with the CMA results (100%). Along with MDM2 amplification, all 16 liposarcomas (100%) also showed amplification of YEATS4, CPM and FRS2. Only 11 of 16 (69%) cases showed HMGA2 amplification. In conclusion, this study demonstrated that CMA on routine formalin fixed paraffin embedded tissue is a sensitive and specific clinical test for detection of MDM2 gene amplification. Moreover, CMA allows simultaneous detection of genomic changes of interest including CDK4 and others, which provides enriched information for diagnosing lipomatous tumors.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos , Perfilação da Expressão Gênica , Lipossarcoma , Proteínas de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Feminino , Humanos , Hibridização in Situ Fluorescente , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Lipossarcoma/metabolismo , Lipossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genéticaRESUMO
Core-binding factor (CBF)-acute myeloid leukemia (AML) generally have a favorable prognosis. However, approximately 50% of patients experience disease relapse during or after post-remission therapy. Retrospective studies on autologous hematopoietic cell transplantation (AHCT) have shown improved survival with decreased relapse rate in CBF-AML. In this prospective study, we evaluate the outcomes of AHCT following high-dose cytarabine (HiDAC) consolidation in patients with CBF-AML in first complete remission (CR). Adult patients with CBF-AML achieving first CR after induction chemotherapy were eligible for the study. High-dose chemotherapy before AHCT included intravenous busulfan (3.2 mg/kg/day, days - 7 to - 5) and etoposide (400 mg/m2/day, days - 3 to - 2). Twenty-nine patients, 17 with t(8;21) and 12 with inv(16), underwent AHCT following 2 or 3 courses of HiDAC consolidation. The estimated 5-year overall and disease-free survival rates were between 89.0% and 82.5%, respectively. The cumulative incidences of relapse and non-relapse mortality were between 17.5% and 0%, respectively. Presence of measurable residual disease (MRD) before AHCT and KIT mutation were significantly associated with relapse after transplantation. In conclusion, the post-remission strategy of AHCT following HiDAC consolidation in CBF-AML was feasible and efficacious. Assays for MRD and KIT mutation may guide selection of patients who will benefit from AHCT in CBF-AML in first CR.
Assuntos
Quimioterapia de Consolidação , Fatores de Ligação ao Core , Citarabina/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Adulto , Autoenxertos , Aberrações Cromossômicas , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Fatores de Ligação ao Core/genética , Fatores de Ligação ao Core/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Indução de Remissão , Taxa de SobrevidaRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major component of the cancer stroma, and their response to therapeutic treatments likely impacts the outcome. We tested the hypothesis that CAFs develop unique characteristics that enhance their resistance to ionizing radiation. METHODS: CAFs were generated through intimate coculture of normal human fibroblasts of skin or lung origin with various human cancer cell types using permeable microporous membrane inserts. Fibroblasts and cancer cells are grown intimately, yet separately, on either side of the insert's membrane for extended times to generate activated fibroblast populations highly enriched in CAFs. RESULTS: The generated CAFs exhibited a decrease in Caveolin-1 protein expression levels, a CAF biomarker, which was further enhanced when the coculture was maintained under in-vivo-like oxygen tension conditions. The level of p21Waf1 was also attenuated, a characteristic also associated with accelerated tumor growth. Furthermore, the generated CAFs experienced perturbations in their redox environment as demonstrated by increases in protein carbonylation, mitochondrial superoxide anion levels, and modulation of the activity of the antioxidants, manganese superoxide dismutase and catalase. Propagation of the isolated CAFs for 25 population doublings was associated with enhanced genomic instability and a decrease in expression of the senescence markers ß-galactosidase and p16INK4a. With relevance to radiotherapeutic treatments, CAFs in coculture with cancer cells of diverse origins (breast, brain, lung, and prostate) were resistant to the clastogenic effects of 137Cs γ rays compared to naïve fibroblasts. Addition of repair inhibitors of single- or double-stranded DNA breaks attenuated the resistance of CAFs to the clastogenic effects of γ rays, supporting a role for increased ability to repair DNA damage in CAF radioresistance. CONCLUSIONS: This study reveals that CAFs are radioresistant and experience significant changes in indices of oxidative metabolism. The CAFs that survive radiation treatment likely modulate the fate of the associated cancer cells. Identifying them together with their mode of communication with cancer cells, and eradicating them, particularly when they may exist at the margin of the radiotherapy planning target volume, may improve the efficacy of cancer treatments. Video Abstract.
Assuntos
Antioxidantes/metabolismo , Fibroblastos Associados a Câncer/patologia , Reparo do DNA , Tolerância a Radiação , Fibroblastos Associados a Câncer/metabolismo , Caveolina 1/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Senescência Celular , Cromossomos Humanos/metabolismo , Técnicas de Cocultura , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Instabilidade Genômica , Humanos , Neoplasias/patologia , Oxirredução , Estresse Oxidativo , Superóxido Dismutase/metabolismoRESUMO
Genome instability in cancer drives tumor heterogeneity, undermines the success of therapies, and leads to metastasis and recurrence. Condensins are conserved chromatin-binding proteins that promote genomic stability, mainly by ensuring proper condensation of chromatin and mitotic chromosome segregation. Condensin mutations are found in human tumors, but it is not known how or even if such mutations promote cancer progression. In this study, we focus on condensin II subunit CAPH2 and specific CAPH2 mutations reported to be enriched in human cancer patients, and we test how CAPH2 cancer-specific mutations may lead to condensin II complex dysfunction and contribute to genome instability. We find that R551P, R551S, and S556F mutations in CAPH2 cause genomic instability by causing DNA damage, anaphase defects, micronuclei, and chromosomal instability. DNA damage and anaphase defects are caused primarily by ataxia telangiectasia and Rad3-related-dependent telomere dysfunction, as anaphase bridges are enriched for telomeric repeat sequences. We also show that these mutations decrease the binding of CAPH2 to the ATPase subunit SMC4 as well as the rest of the condensin II complex, and decrease the amount of CAPH2 protein bound to chromatin. Thus, in vivo the R551P, R551S, and S556F cancer-specific CAPH2 mutant proteins are likely to impair condensin II complex formation, impede condensin II activity during mitosis and interphase, and promote genetic heterogeneity in cell populations that can lead to clonal outgrowth of cancer cells with highly diverse genotypes.
Assuntos
Adenosina Trifosfatases/genética , Anáfase , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Complexos Multiproteicos/genética , Mutação/genética , Neoplasias/genética , Proteínas Nucleares/metabolismo , Telômero/patologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Micronúcleo Germinativo/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fase S , Telômero/metabolismoRESUMO
The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.
Assuntos
Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinases/metabolismo , Estresse Fisiológico , Anáfase , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Fase SRESUMO
Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = -0.35, p = 0.03 and r = -0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.
Assuntos
Carcinogênese/metabolismo , Cromossomos Humanos/metabolismo , Neoplasias do Endométrio/metabolismo , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Telômero/metabolismo , Transcrição Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Carcinogênese/patologia , Cromossomos Humanos/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Telômero/genética , Telômero/patologia , Homeostase do TelômeroRESUMO
The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.
Assuntos
Quebras de DNA de Cadeia Dupla , Expansão das Repetições de DNA/genética , Repetições de Dinucleotídeos/genética , Neoplasias/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Cromotripsia , Clivagem do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Instabilidade Genômica , Humanos , Recombinases/metabolismoRESUMO
We explain environmental and genetic factors determining male genetic conditions and infertility and evaluate the significance of environmental stressors in shaping defensive responses, which is used in the diagnosis and treatment of male infertility. This is done through the impact of external and internal stressors and their instability on sperm parameters and their contribution to immunogenetic disorders and hazardous DNA mutations. As chemical compounds and physical factors play an important role in the induction of immunogenetic disorders and affect the activity of enzymatic and non-enzymatic responses, causing oxidative stress, and leading to apoptosis, they downgrade semen quality. These factors are closely connected with male reproductive potential since genetic polymorphisms and mutations in chromosomes 7, X, and Y critically impact on spermatogenesis. Microdeletions in the Azoospermic Factor AZF region directly cause defective sperm production. Among mutations in chromosome 7, impairments in the cystic fibrosis transmembrane conductance regulator CFTR gene are destructive for fertility in cystic fibrosis, when spermatic ducts undergo complete obstruction. This problem was not previously analyzed in such a form. Alongside karyotype abnormalities AZF microdeletions are the reason of spermatogenic failure. Amongst AZF genes, the deleted in azoospermia DAZ gene family is reported as most frequently deleted AZF. Screening of AZF microdeletions is useful in explaining idiopathic cases of male infertility as well as in genetic consulting prior to assisted reproduction. Based on the current state of research we answer the following questions: (1) How do environmental stressors lessen the quality of sperm and reduce male fertility; (2) which chemical elements induce oxidative stress and immunogenetic changes in the male reproductive system; (3) how do polymorphisms correlate with changes in reproductive potential and pro-antioxidative mechanisms as markers of pathophysiological disturbances of the male reproductive condition; (4) how do environmental stressors of immunogenetic disorders accompany male infertility and responses; and (5) what is the distribution and prevalence of environmental and genetic risk factors.
Assuntos
Azoospermia , Exposição Ambiental/efeitos adversos , Estresse Oxidativo , Espermatogênese , Azoospermia/genética , Azoospermia/imunologia , Azoospermia/metabolismo , Azoospermia/patologia , Cromossomos Humanos/genética , Cromossomos Humanos/imunologia , Cromossomos Humanos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Masculino , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Polimorfismo Genético , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/imunologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogênese/genética , Espermatogênese/imunologiaAssuntos
Cromossomos Humanos , Amplificação de Genes , Neoplasias Hematológicas , Micronúcleos com Defeito Cromossômico , Transtornos Mieloproliferativos , Proteínas Proto-Oncogênicas c-myc , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Masculino , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
BACKGROUND: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. METHODS: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. RESULTS: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. CONCLUSIONS: These data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.
Assuntos
Benzodioxóis/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Quinolonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzodioxóis/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Microscopia Intravital , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/patologia , Quinolonas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.
Assuntos
Adenosina Trifosfatases , Aurora Quinase B , Aberrações Cromossômicas , Cromossomos Humanos , Proteínas de Ligação a DNA , Metáfase/genética , Complexos Multiproteicos , Proteínas de Neoplasias , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genéticaRESUMO
Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.
Assuntos
DNA Ribossômico/genética , Região Organizadora do Nucléolo/metabolismo , Região Organizadora do Nucléolo/fisiologia , Linhagem Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromossomos Humanos/metabolismo , DNA Ribossômico/metabolismo , Genoma Humano/genética , Genoma Humano/fisiologia , Humanos , Hibridização in Situ Fluorescente/métodos , Região Organizadora do Nucléolo/genética , Ribossomos/metabolismoRESUMO
Renal cell carcinoma (RCC) is characterized by a particularly broad metastatic swath, and, enigmatically, when the pancreas is a destination, the disease is associated with improved survival. Intrigued by this observation, we sought to characterize the clinical behavior, therapeutic implications, and underlying biology. While pancreatic metastases (PM) are infrequent, we identified 31 patients across 2 institutional cohorts and show that improved survival is independent of established prognostic variables, that these tumors are exquisitely sensitive to antiangiogenic agents and resistant to immune checkpoint inhibitors (ICIs), and that they are characterized by a distinctive biology. Primary tumors of patients with PM exhibited frequent PBRM1 mutations, 3p loss, and 5q amplification, along with a lower frequency of aggressive features such as BAP1 mutations and loss of 9p, 14q, and 4q. Gene expression analyses revealed constrained evolution with remarkable uniformity, reduced effector T cell gene signatures, and increased angiogenesis. Similar findings were observed histopathologically. Thus, RCC metastatic to the pancreas is characterized by indolent biology, heightened angiogenesis, and an uninflamed stroma, likely underlying its good prognosis, sensitivity to antiangiogenic therapies, and refractoriness to ICI. These data suggest that metastatic organotropism may be an indicator of a particular biology with prognostic and treatment implications for patients.