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1.
Nat Commun ; 12(1): 646, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510149

RESUMO

Polyploidy is a hallmark of cancer, and closely related to chromosomal instability involved in cancer progression. Importantly, polyploid cells also exist in some normal tissues. Polyploid hepatocytes proliferate and dynamically reduce their ploidy during liver regeneration. This raises the question whether proliferating polyploids are prone to cancer via chromosome missegregation during mitosis and/or ploidy reduction. Conversely polyploids could be resistant to tumor development due to their redundant genomes. Therefore, the tumor-initiation risk of physiologic polyploidy and ploidy reduction is still unclear. Using in vivo lineage tracing we here show that polyploid hepatocytes readily form liver tumors via frequent ploidy reduction. Polyploid hepatocytes give rise to regenerative nodules with chromosome aberrations, which are enhanced by ploidy reduction. Although polyploidy should theoretically prevent tumor suppressor loss, the high frequency of ploidy reduction negates this protection. Importantly, polyploid hepatocytes that undergo multiple rounds of cell division become predominantly mononucleated and are resistant to ploidy reduction. Our results suggest that ploidy reduction is an early step in the initiation of carcinogenesis from polyploid hepatocytes.


Assuntos
Transformação Celular Neoplásica/genética , Instabilidade Cromossômica/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Fígado/metabolismo , Poliploidia , Animais , Células Cultivadas , Hepatócitos/citologia , Humanos , Fígado/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitose/genética , Ploidias
2.
Mutat Res ; 786: 108342, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33339572

RESUMO

Micronuclei (MNi) are among the most widely studied biomarkers of DNA damage and chromosomal instability in humans. They originate from chromosome fragments or intact chromosomes that are not included in daughter nuclei during mitosis. The main reasons for their formation are a lack of functional centromere in the chromosome fragments or whole chromosomes or defects in one or more of the proteins of the mitotic system that, consequently, fails to segregate chromosomes properly. Assays have been developed to measure MNi in peripheral blood lymphocytes, red blood cells as well as various types of epithelial cells such as buccal, nasal, urothelial and cervical cells. Some of the assays have been further developed into micronucleus (MN) cytome assays to include additional nuclear anomalies, cell death and nuclear division biomarkers. In addition, the use of molecular probes has been adopted widely for the purpose of understanding the mechanistic origin of MNi. MN assays in humans are used for the purpose of investigating the genotoxic effects of adverse environmental, life-style and occupational factors, genetic susceptibility to DNA damage, and for determining risk of accelerated aging and diseases affected by genomic instability such as developmental defects and cancer. The emerging new knowledge showing that chromosomes trapped in MNi can undergo a high rate of fragmentation and become massively re-arranged have highlighted the possibility that MN formation is not only a biomarker of induced DNA damage but also a mechanism that drives hypermutation. Furthermore, another line of recent research showed that DNA and chromatin leaking from disrupted MNi triggers the innate immune cGAS-STING mechanism that promotes inflammation which can cause a wide-range of age-related diseases if left unresolved. For these reasons, MN assays in humans have become an increasingly important biomarker of disease initiation and progression across all life-stages.


Assuntos
Instabilidade Cromossômica/genética , Marcadores Genéticos/genética , Inflamação/genética , Micronúcleos com Defeito Cromossômico , Aneuploidia , Dano ao DNA , Humanos , Testes para Micronúcleos
3.
Nucleic Acids Res ; 48(16): 9161-9180, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797166

RESUMO

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.


Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , RNA Helicases/genética , Rad51 Recombinase/genética , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Humanos , Mitomicina/farmacologia , Reparo de DNA por Recombinação/genética
4.
Mutat Res ; 854-855: 503197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660821

RESUMO

Telomeres, specialized structures at the ends of linear chromosomes, protect chromosome ends from degradation, recombination, and mis-repair. Critically short telomere length (TL) may result in chromosome instability (CIN), causing tumor promotion and, at higher levels, cell death and tumor suppression. Homocysteine (Hcy) is a sulfur-containing amino acid involved in one-carbon metabolism. Elevated plasma Hcy is a cancer risk factor. Human SH-SY5Y neuroblastoma cells were treated with pathophysiological concentrations of Hcy (15-120 µM) for 14 and 28 days. The cytokinesis-block micronucleus cytome assay was used to determine cytostasis (nuclear division index, NDI), cell death (apoptosis and necrosis), and CIN (micronuclei, nucleoplasmic bridges, and nuclear buds in binucleated cells). Quantitative PCR was used to measure TL and the expression of hTERT, the gene encoding the catalytic subunit of telomerase for TL elongation. The results showed that Hcy induced elongation of TL and fluctuating changes in expression of hTERT. TL elongation was associated with increased CIN. Hcy decreased the NDI and increased cell death. This study shows that there is cross-talk between Hcy and TL in tumor cells and supports the concept that high Hcy inhibits cell division and promotes the death of tumor cells by abnormal elongation of TL and elevation of CIN.


Assuntos
Instabilidade Cromossômica/genética , Homocisteína/genética , Neuroblastoma/genética , Telômero/genética , Apoptose/genética , Morte Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Citocinese/genética , Dano ao DNA/genética , Humanos , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos/métodos , Necrose/genética , Telomerase/genética
5.
Tumour Biol ; 42(7): 1010428320938492, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32635826

RESUMO

Molecular classification of colorectal cancer is difficult to implement in clinical settings where hundreds of genes are involved, and resources are limited. This study aims to characterize the molecular subtypes of patients with sporadic colorectal cancer based on the three main carcinogenic pathways microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and chromosomal instability (CIN) in a Chilean population. Although several reports have characterized colorectal cancer, most do not represent Latin-American populations. Our study includes 103 colorectal cancer patients who underwent surgery, without neoadjuvant treatment, in a private hospital between 2008 and 2017. MSI, CIN, and CIMP status were assessed. Frequent mutations in KRAS, BRAF, and PIK3CA genes were analyzed by Sanger sequencing, and statistical analysis was performed by Fisher's exact and/or chi-square test. Survival curves were estimated with Kaplan-Meier and log-rank test. Based on our observations, we can classify the tumors in four subgroups, Group 1: MSI-high tumors (15%) are located in the right colon, occur at older age, and 60% show a BRAF mutation; Group 2: CIN-high tumors (38%) are in the left colon, and 26% have KRAS mutations. Group 3: [MSI/CIN/CIMP]-low/negative tumors (30%) are left-sided, and 39% have KRAS mutations; Group 4: CIMP-high tumors (15%) were more frequent in men and left side colon, with 27% KRAS and 7% presented BRAF mutations. Three percent of patients could not be classified. We found that CIMP-high was associated with a worse prognosis, both in MSI-high and MSI stable patients (p = 0.0452). Group 3 (Low/negative tumors) tend to have better overall survival compared with MSI-high, CIMP-high, and CIN-high tumors. This study contributes to understanding the heterogeneity of tumors in the Chilean population being one of the few characterizations performed in Latin-America. Given the limited resources of these countries, these results allow to improve molecular characterization in Latin-American colorectal cancer populations and confirm the possibility of using the three main carcinogenic pathways to define therapeutic strategies.


Assuntos
Carcinogênese/genética , Instabilidade Cromossômica/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Chile/epidemiologia , Neoplasias Colorretais/classificação , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
6.
Anticancer Res ; 40(7): 3759-3764, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620615

RESUMO

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy due to its increased ability for local metastases and distant lymph node metastases. Extensive cytogenetic analyses have detected chromosome instability (CI) patterns in OSCC including gross chromosome numerical alterations, such as polysomy and sporadically monosomy that negatively affect the biological behaviour of the malignancy. Our aim was to investigate the frequency and impact of chromosome 17 (Chr 17) numerical imbalances in OSCC. MATERIALS AND METHODS: Fifty (n=50) formalin-fixed, paraffin-embedded primary OSCCs tissue sections were used. Chromogenic in situ hybridization (CISH) was implemented for detecting Chr 17 centromeric numerical imbalances. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. RESULTS: Chr 17 multiple copies were observed in 12/50 (24%) of the examined cases. Polysomy was observed in 10/50 (20%) tissue sections, monosomy in 2/50 (4%), whereas the rest of them demonstrated a normal, diploid pattern (38/50-76%). Chr 17 numerical differences were associated with the grade of differentiation of the examined tumors (p=0.001). CONCLUSION: Chr 17 numerical imbalances (polysomy predominantly and monosomy) are observed in sub-groups of OSCCs correlating with a progressive dedifferentiation of malignant tissues. The proposed grid-based platform on CISH slides provides a novel, fast and accurate screening-mapping mechanism for detecting chromosome numerical aberrations.


Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 17/genética , Neoplasias Bucais/genética , Instabilidade Cromossômica/genética , Aberrações Cromossômicas , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Masculino
7.
Mutat Res ; 850-851: 503147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247562

RESUMO

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Fritillaria/química , Extratos Vegetais/farmacologia , Aurora Quinase B/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Instabilidade Cromossômica/genética , Colo/efeitos dos fármacos , Colo/patologia , Citocinese/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a Y-Box/genética
8.
Eur J Protistol ; 74: 125691, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32200034

RESUMO

We performed karyotyping of Amoeba sp. strain Cont. Based on the results of a cytological analysis, we concluded that the chromosome number of Amoeba sp. strain Cont in mitosis was unstable. In all cases they appeared to be hypergaploid (the basic chromosome number is 30), with monosomy of all chromosomes except four shortest ones. The presence of "extrachromosomes" in the nucleus could prolong until the beginning of the anaphase. It was only then that they were ejected from the nucleus and the euploidy (haploidy) was restored. The stage of endoprophase nucleus was revealed in the cell cycle of Amoeba sp. strain Cont. This stage has not yet been found in other amoebae from the "proteus-type" group that had been previously studied (A. proteus strain B and A. borokensis). The maximum number of endoreplication rounds in the strain Cont amoebae nuclear cycle was 4 or 5. The regular extrusion of chromosomes from the nucleus into the cytoplasm occurred in each of the endoreplication rounds. Comparative cytological analysis of A. proteus strain B, A. borokensis and Amoeba sp. strain Cont karyotypes indicated that strain Cont, though rather close to the former two amoebae, is actually a distinct species.


Assuntos
Amoeba/citologia , Amoeba/genética , Instabilidade Cromossômica/genética , Cariótipo , Mitose/genética , Especificidade da Espécie
9.
Proc Natl Acad Sci U S A ; 117(14): 7917-7928, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193338

RESUMO

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.


Assuntos
Centrômero/genética , Criptococose/genética , Cryptococcus neoformans/genética , Isolamento Reprodutivo , Sistemas CRISPR-Cas/genética , Instabilidade Cromossômica/genética , Cromossomos/genética , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Quebras de DNA de Cadeia Dupla , Genoma Fúngico/genética , Genômica , Humanos , Translocação Genética/genética
10.
DNA Repair (Amst) ; 88: 102801, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32032862

RESUMO

High fidelity of genetic transmission in neural stem and progenitor cells (NSPCs) has been long time considered to be crucial for brain development and homeostasis. However, recent studies have identified recurrent DSB clusters in dividing NSPCs, which may underlie the diversity of neuronal cell types. This raised the interest in understanding how NSPCs sense and repair DSBs and how this mechanism could be altered by environmental genotoxic stress caused by pollutants or ionizing radiation. Here, we show that embryonic mouse neural stem and progenitor cells (NSPCs) have significantly higher capacity than mouse embryonic fibroblasts (MEFs) to maintain their chromosome stability in response to acute (γ-radiation) and chronic (tritiated thymidine -3H-T- incorporation into DNA) genotoxic stress. Cells deficient for XLF/Cernunnos, which is involved in non-homologous end joining DNA (NHEJ) repair, highlighted important variations in fidelity of DNA repair pathways between the two cell types. Strikingly, a progressive and generalized chromosome instability was observed in MEFs cultured with 3H-T at long-term, whereas NSPCs cultured in the same conditions, preserved their chromosome stability thanks to higher DNA repair activity further enhanced by an adaptive response and also to the elimination of damaged cells by apoptosis. This specific DNA damage response of NSPCs may rely on the necessity for preservation of their genome stability together with their possible function in creating neuronal genetic diversity.


Assuntos
Instabilidade Cromossômica/genética , Dano ao DNA , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Células-Tronco Neurais/metabolismo , Animais , Reparo do DNA/genética , Camundongos , Fatores de Tempo
11.
Oncogene ; 39(16): 3245-3257, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32086441

RESUMO

ATR and CHK1 play key roles in the protection and recovery of the stalled replication forks. Claspin, an adaptor for CHK1 activation, is essential for DNA damage signaling and efficient replication fork progression. Here, we show that tristetraprolin (TTP), an mRNA-binding protein, can modulate the replication stress response via stabilization of Claspin mRNA. TTP depletion compromised specifically in the phosphorylation of CHK1, but not p53 or H2AX among other ATR substrates, and produced CHK1-defective replication phenotypes including accumulation of stalled replication forks. Importantly, the expression of siRNA-resistant TTP in TTP-deficient cells restored CHK1 phosphorylation and reduced the number of stalled replication forks as close to the control cells. Besides, we found that TTP was required for efficient replication fork progression even in the absence of exogenous DNA damage in a Claspin-dependent manner. Mechanistically, TTP was able to bind to the 3'-untranslated region of Claspin mRNA to increase the stability of Claspin mRNA which eventually contributed to the subsequent ATR-CHK1 activation upon DNA damage. Taken together, our results revealed an intimate link between TTP-dependent Claspin mRNA stability and ATR-CHK1-dependent replication fork stability to maintain replication fork integrity and chromosomal stability.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Replicação do DNA/genética , Estabilidade de RNA/genética , Tristetraprolina/genética , Regiões 3' não Traduzidas/genética , Células A549 , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase 1 do Ponto de Checagem/genética , Instabilidade Cromossômica/genética , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Células HCT116 , Histonas/genética , Humanos , RNA Mensageiro/genética , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/genética
12.
Oncogene ; 39(16): 3396-3410, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32103168

RESUMO

E3 ubiquitin ligases (E3s) play essential roles in the maintenance of tissue homeostasis under normal and stress conditions, as well as in disease states, particularly in cancer. However, the role of E3s in the initiation of human tumors is poorly understood. Previously, we reported that genetic ablation of the HECT-type E3 ubiquitin ligase Smurf2 induces carcinogenesis in mice; but whether and how these findings are pertinent to the inception of human cancer remain unknown. Here we show that SMURF2 is essential to protect human dermal fibroblasts (HDFs) from malignant transformation, and its depletion converts HDFs into tumorigenic entity. This phenomenon was associated with the radical changes in chromatin structural and epigenetic landscape, dysregulated gene expression and cell-cycle control, mesenchymal-to-epithelial transition and impaired DNA damage response. Furthermore, we show that SMURF2-mediated tumor suppression is interlinked with SMURF2's ability to regulate the expression of two central chromatin modifiers-an E3 ubiquitin ligase RNF20 and histone methyltransferase EZH2. Silencing these factors significantly reduced the growth and transformation capabilities of SMURF2-depleted cells. Finally, we demonstrate that SMURF2-compromised HDFs are highly tumorigenic in nude mice. These findings suggest the critical role that SMURF2 plays in preventing malignant alterations, chromosomal instability and cancer.


Assuntos
Carcinogênese/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Neoplasias/genética , Ubiquitina-Proteína Ligases/genética , Animais , Cromatina/genética , Instabilidade Cromossômica/genética , Derme/metabolismo , Derme/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Fibroblastos/metabolismo , Fibroblastos/patologia , Inativação Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitinação/genética
13.
Exp Cell Res ; 387(2): 111805, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31877307

RESUMO

Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.


Assuntos
Instabilidade Cromossômica/genética , Segregação de Cromossomos/genética , Cromossomos Artificiais Humanos/genética , Transgenes/genética , Humanos , Neoplasias/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genética
14.
Nat Med ; 25(11): 1699-1705, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31686035

RESUMO

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.


Assuntos
Instabilidade Cromossômica/genética , Variações do Número de Cópias de DNA/genética , Desenvolvimento Embrionário/genética , Fertilização In Vitro/efeitos adversos , Blastocisto/metabolismo , Linhagem da Célula/genética , Embrião de Mamíferos , Feminino , Feto , Genótipo , Humanos , Recém-Nascido , Masculino , Placenta/metabolismo , Placenta/patologia , Polimorfismo de Nucleotídeo Único/genética , Gravidez
15.
J Cell Sci ; 132(24)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31757888

RESUMO

Chromosomal instability, one of the most prominent features of tumour cells, causes aneuploidy. Tetraploidy is thought to be an intermediate on the path to aneuploidy, but the mechanistic relationship between the two states is poorly understood. Here, we show that spindle polarity (e.g. bipolarity or multipolarity) in tetraploid cells depends on the level of functional phosphorylated Eg5, a mitotic kinesin, localised to the spindle. Multipolar spindles are formed in cells with high levels of phosphorylated Eg5. This process is suppressed by inhibition of Eg5 or expression of a non-phosphorylatable Eg5 mutant, as well as by changing the balance between opposing forces required for centrosome separation. Tetraploid cells with high levels of functional Eg5 give rise to a heterogeneous aneuploid population through multipolar division, whereas cells with low levels of functional Eg5 continue to undergo bipolar division and remain tetraploid. Furthermore, Eg5 protein levels correlate with ploidy status in tumour specimens. We provide a novel explanation for the tetraploid intermediate model, i.e. spindle polarity and subsequent tetraploid cell behaviour are determined by the balance of forces generated by mitotic kinesins at the spindle.


Assuntos
Cinesina/metabolismo , Fuso Acromático/metabolismo , Tetraploidia , Instabilidade Cromossômica/genética , Instabilidade Cromossômica/fisiologia , Citometria de Fluxo , Células HCT116 , Células HeLa , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cinesina/genética , Fosforilação/genética , Fosforilação/fisiologia
16.
CRISPR J ; 2(6): 406-416, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31742432

RESUMO

CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.


Assuntos
Instabilidade Cromossômica/genética , Análise Citogenética/métodos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Instabilidade Cromossômica/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Rearranjo Gênico/genética , Humanos , Mutagênese/genética , Mutação , Neoplasias/genética , RNA Guia/genética , Deleção de Sequência/genética
17.
Nat Commun ; 10(1): 4834, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645568

RESUMO

Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.


Assuntos
Anáfase , Blastômeros/metabolismo , Instabilidade Cromossômica/genética , Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Tetraploidia , Animais , Centríolos , Embrião de Mamíferos/embriologia , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Mitose , Neoplasias/genética , Fuso Acromático
18.
Mol Cancer Res ; 17(12): 2480-2491, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31611308

RESUMO

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.


Assuntos
Neoplasias da Próstata/genética , RNA/genética , Telomerase/genética , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Masculino , Mutação , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Telômero/genética
19.
Cell Death Dis ; 10(10): 697, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541076

RESUMO

Gastric cancer is characterized by chromosomal instability. In this study, we investigated chromosomal instability quantified by copy number instability (CNI) score of circulating tumor DNA (ctDNA) during the drug treatment in advanced gastric cancer (AGC). A total of 55 pretherapeutic plasmas from 55 AGC patients and 75 plasmas during drug treatment of 26 AGC patients were collected. Plasma ctDNA was extracted and assessed by whole-genome sequencing (WGS) for somatic copy number alteration (SCNA), and according to which we calculated the CNI scores. We next assessed the correlations between chromosomal instability and therapeutic response. The cutoff value of chromosomal instability was defined as the mean + SD of the CNI scores (56.60) in cfDNA of plasmas from 100 healthy people. For 55 enrolled cases, chromosomal instability was observed in 27 (49%) prior to drug treatment, whose response rate (59%, 16/27) was higher than in 28 patients with stable chromosomes (32%, 9/28, P = 0.043). We also observed that CNI scores fluctuated during treatment in 26 patients. Specifically, the CNI scores in 93% (14/15) of patients sensitive to drug treatment reduced to the level of chromosomal stability and the CNI scores in 52% (13/25) of patients resistant to treatment elevated again. For ctDNA with developed resistance, the SCNA patterns were identical to those before treatment, whereas the CNI scores were lower than the pretherapeutic scores. We found that chromosomal instability based on ctDNA could predict and monitor therapeutic response in gastric cancer, although validation in a larger cohort will be necessary.


Assuntos
Instabilidade Cromossômica/genética , DNA Tumoral Circulante/metabolismo , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
20.
Cancer Res ; 79(21): 5536-5549, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530568

RESUMO

High-grade serous ovarian carcinoma commonly arises from fallopian tube secretory epithelium and is characterized by a high level of chromosomal instability. To model the acquisition of aneuploidy during early carcinogenesis, chromosome missegregation was induced in immortalized tubal epithelial cells, which proved acutely detrimental to cellular fitness. The phenotype was characterized by accumulation of misfolded proteins, activation of the unfolded protein response (UPR), decreased protein synthesis, and enhanced vulnerability to proteasome inhibition. However, chromosome missegregation also resulted in heightened transformation potential, assessed by colony formation in soft agar. Ovarian cancer cells retained intrinsic sensitivity to proteasome inhibitors under adherent culture conditions, but acquired resistance as spheroids (recapitulating their native configuration in ascites) by downregulating protein synthesis via mTORC1 suppression. Loss of PTEN drove constitutive mTORC1 activity, enhanced proteotoxic stress, as evidenced by UPR induction, and resensitized tumor spheroids to proteasome inhibition both in vitro and in vivo. In cohorts of primary ovarian carcinomas, mTORC1 and UPR signaling pathways were closely associated. These results implicate attenuation of protein synthesis as a protective mechanism in tumor spheroids, which may explain the overall poor response to bortezomib in clinical trials of patients with advanced ovarian cancer. However, patients with PTEN-deficient tumors may represent a subpopulation potentially amenable to treatment with proteasome inhibitors or other therapeutic agents that disrupt protein homeostasis. SIGNIFICANCE: Chromosome instability and protein synthesis are important factors that determine the efficacy of proteotoxic stress-inducing agents, such as proteasome inhibitors, in the treatment of ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5536/F1.large.jpg.


Assuntos
Instabilidade Cromossômica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Animais , Bortezomib/farmacologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Instabilidade Cromossômica/efeitos dos fármacos , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Epitélio/efeitos dos fármacos , Epitélio/patologia , Neoplasias das Tubas Uterinas/genética , Neoplasias das Tubas Uterinas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética
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