RESUMEN
Aneuploidy, a state of chromosome imbalance frequently found in cancer, results in convoluted cancer genomes. Here, Kuzmin and colleagues1 identify how the aneuploid genome in triple-negative breast cancer is being shaped by unique genome network interactions.
Asunto(s)
Aneuploidia , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Femenino , Genoma Humano , Genómica/métodos , Neoplasias/genéticaRESUMEN
Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.
Asunto(s)
Aneuploidia , Inestabilidad Cromosómica , Animales , Transformación Celular Neoplásica/genética , Centrosoma , Inestabilidad Cromosómica/genética , Evolución Clonal , Inestabilidad Genómica/genética , RatonesRESUMEN
Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.
Asunto(s)
Cromotripsis , Evolución Molecular , Amplificación de Genes/genética , Neoplasias/genética , Oncogenes/genética , Daño del ADN , Reparación del ADN por Unión de Extremidades , ADN Circular/química , ADN Circular/metabolismo , ADN de Neoplasias/química , ADN de Neoplasias/metabolismo , Proteína Quinasa Activada por ADN , Resistencia a Antineoplásicos , Células HEK293 , Células HeLa , Humanos , Micronúcleos con Defecto Cromosómico , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Selección Genética , Secuenciación Completa del GenomaRESUMEN
Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.
Asunto(s)
Proteína A Centromérica/genética , Centrómero/genética , Cromosomas Humanos/genética , Replicación del ADN/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Fase G1/genética , Células HeLa , Histonas/genética , Humanos , Nucleosomas/genéticaRESUMEN
Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.
Asunto(s)
Segregación Cromosómica/genética , Reordenamiento Génico/genética , Animales , Células Cultivadas , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , Genoma Humano/genética , Genómica/métodos , Células HEK293 , Humanos , Neoplasias/genética , Translocación Genética/genética , Secuenciación Completa del Genoma/métodos , Xenopus laevis/genéticaRESUMEN
Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements known as chromothripsis, but the underlying mechanisms remain unresolved. Here we developed an inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimaera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated following centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harbouring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJ-mediated reassembly in the subsequent interphase.
Asunto(s)
Centrómero/metabolismo , Cromosomas Humanos Y/metabolismo , Cromotripsis , Reparación del ADN por Unión de Extremidades , Micronúcleos con Defecto Cromosómico , Autoantígenos/metabolismo , Línea Celular Tumoral , Proteína A Centromérica , Proteína B del Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Humanos , Hibridación Fluorescente in Situ , MitosisRESUMEN
INTRODUCTION: Multiple myeloma is still incurable in most cases. Polyclonal anti T lymphocyte globulins (ATG) have been reported to kill human myeloma cells in vitro and in mouse models. METHODS: Anti-human-myeloma globulins (AMG) were produced by immunizing rabbits with human myeloma cell lines RPMI-8226 (AMG-8226) or KMS-12-BM (AMG-12-BM). Cytotoxicity of the polyclonal antibodies was analyzed in vitro and in a xenograft NOD-SCID mouse model. RESULTS: Both AMG had stronger cytotoxicity against myeloma cells compared to ATG. In primary T cells, AMG-8226 showed greater complement-dependent cytotoxicity (CDC) than ATG, whereas complement-independent cytotoxicity did not differ. Effects on non-hematopoietic cell lines were also similar. Competitive blocking assays revealed fourfold more antibodies against CD38 in AMG-8226 compared to ATG. Low concentrations of AMG-8226 and ATG increased ADCC. At higher concentrations, ATG inhibited ADCC more potently than AMG-8226. Combinations of ATG and AMG-8226 with melphalan or bortezomib showed additive to synergistic cytotoxicity on myeloma cells. The cytotoxic effects of AMG and ATG were confirmed in the xenograft NOD-SCID mouse model. CONCLUSION: Our data show more potent antimyeloma effects of AMG compared to ATG. These results lay the ground for the development of polyclonal antibodies for the treatment of multiple myeloma.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Suero Antilinfocítico/farmacología , Antineoplásicos/farmacología , Mieloma Múltiple , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase-mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.
Asunto(s)
División Celular Asimétrica/fisiología , Centrosoma/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Regulación de la Expresión Génica , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias del Timo/genética , Neoplasias del Timo/metabolismo , Neoplasias del Timo/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Over a decade of intensive investigation of the possible plasticity of mammalian cells has eventually substantiated that mammalian species are endowed with a remarkable capacity to change mature cell fates. We review below the evidence for the occurrence of processes such as dedifferentiation and transdifferentiation within mammalian tissues in vivo, and in cells removed from their protective microenvironment and seeded in culture under conditions poorly resembling their physiological state in situ. Overall, these studies point to one major conclusion: stressful conditions, whether due to in vivo tissue damage or otherwise to isolation of cells from their in vivo restrictive niches, lead to extreme fate changes. Some examples of dedifferentiation are discussed in detail showing that rare cells within the population tend to turn back into less mature ones due to severe cell damage. It is proposed that cell stress, mechanistically sensed by isolation from neighboring cells, leads to dedifferentiation, in an attempt to build a new stem cell reservoir for subsequent regeneration of the damaged tissue. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
Asunto(s)
Desdiferenciación Celular , Fenómenos Fisiológicos Celulares , Estrés Fisiológico/fisiología , Animales , Reprogramación Celular , Humanos , MamíferosRESUMEN
INTRODUCTION: Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state. METHODS: MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA). RESULTS: We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs. CONCLUSIONS: The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.
Asunto(s)
Tejido Adiposo/citología , Inestabilidad Genómica , Células Madre Mesenquimatosas/metabolismo , ARN Largo no Codificante/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Cultivadas , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Ploidias , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mesenchymal stromal cell populations include a fraction, termed mesenchymal stem cells, exhibiting multipotency. Other cells within this population possess a lesser differentiation range. This was assumed to be due to a mesenchymal cellular cascade topped by a multipotent cell, which gives rise to progeny with diminishing differentiation potentials. Here, we show that mesenchymal cells, a priori exhibiting a limited differentiation potential, may gain new capacities and become multipotent following single-cell isolation. These fate changes were accompanied by upregulation of differentiation promoting genes, many of which also became H4K20me1 methylated. Early events in the process included TGFß and Wnt modulation, and downregulation of hypoxia signaling. Indeed, hypoxic conditions inhibited the observed cell changes. Overall, cell isolation from neighboring partners caused major molecular changes and particularly, a newly established epigenetic state, ultimately leading to the acquisition of new differentiation potentials and an altered cell fate.
Asunto(s)
Diferenciación Celular/fisiología , Separación Celular , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Linaje de la Célula , Inmunoprecipitación de Cromatina , Células Clonales/citología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.
Asunto(s)
Transformación Celular Neoplásica/genética , Silenciador del Gen/fisiología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Poliploidía , ARN Largo no Codificante/genética , Animales , Transformación Celular Neoplásica/patología , Células Cultivadas , Inestabilidad Genómica , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neoplasias/genética , ARN Largo no Codificante/antagonistas & inhibidoresRESUMEN
Differentiation cascades are arranged hierarchically; stem cells positioned at the top of the hierarchy generate committed progenitors that, in turn, proliferate and further differentiate stepwise into mature progeny. This rigid, irreversible structure ensures the phenotypic stability of adult tissues. However, such rigidity may be problematic under conditions of tissue damage when reconstitution is required. Although it may seem unlikely that the restrictions on changes in cell phenotypes would be lifted to enable tissue reconstitution, it is nevertheless possible that mammalian tissues are endowed with sufficient flexibility to enable their adaptation to extreme conditions.
Asunto(s)
Desdiferenciación Celular/fisiología , Mamíferos , Estrés Fisiológico , Animales , Diferenciación Celular , Homeostasis , Humanos , Neoplasias/etiología , Células Madre/fisiologíaRESUMEN
Mesenchymal stromal cells (MSCs) are capable of differentiating into bone-forming osteoblasts. A recent Nature Medicine study (Medici et al., 2010) shows that the mislocalized bone in the human disease fibrodisplasia ossificans progressiva (FOP) originates from vascular endothelium that gives rise to MSCs.