RESUMO
It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations.
Assuntos
Caderinas/genética , Replicação do DNA/genética , Instabilidade Genômica , Antígenos CD , Caderinas/antagonistas & inibidores , Linhagem Celular Tumoral , Ciclina B/genética , Dano ao DNA , Fase G1/genética , Humanos , Mitose/genética , Quinolinas/farmacologia , Fase S/genética , Tiazóis/farmacologiaRESUMO
Structural centrosome aberrations are frequently observed in early stage carcinomas, but their role in malignant transformation is poorly understood. Here, we examined the impact of overexpression of Ninein-like protein (Nlp) on the architecture of polarized epithelia in three-dimensional mammospheres. When Nlp was overexpressed to levels resembling those seen in human tumors, it formed striking centrosome-related bodies (CRBs), which sequestered Ninein and affected the kinetics of microtubule (MT) nucleation and release. In turn, the profound reorganization of the MT cytoskeleton resulted in mislocalization of several adhesion and junction proteins as well as the tumor suppressor Scribble, resulting in the disruption of epithelial polarity, cell-cell interactions and mammosphere architecture. Remarkably, cells harboring Nlp-CRBs displayed an enhanced proliferative response to epidermal growth factor. These results demonstrate that structural centrosome aberrations cause not only the disruption of epithelial polarity but also favor overproliferation, two phenotypes typically associated with human carcinomas.
Assuntos
Mama/metabolismo , Centrossomo/fisiologia , Microtúbulos/fisiologia , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Proliferação de Células/fisiologia , Centrossomo/metabolismo , Feminino , Humanos , Proteínas Associadas aos Microtúbulos/biossíntese , Microtúbulos/metabolismo , Proteínas Nucleares/biossínteseRESUMO
Human Polo-like kinase 1 (Plk1) is an essential regulator of mitotic progression. Targeted inhibition of this kinase was effective in killing tumor cells in vitro and in vivo. The Plk1 inhibitor BI_2536 was well tolerated and showed antitumor activity in the first clinical trials enrolling patients with advanced solid tumors and refractory or relapsed acute myeloid leukemia.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Humanos , Pteridinas/farmacologia , Quinase 1 Polo-LikeRESUMO
Ensuring precise DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. Proteolytic control of cell cycle regulators by the anaphase-promoting complex, activated by Cdh1 (APC(Cdh1)), is responsible for a stable G1 phase after mitotic exit allowing accurate preparation for DNA replication in the following S phase. APC(Cdh1) target proteins are frequently upregulated in tumor cells and the inactivation of human Cdh1 might interfere with genome integrity by target stabilization. Here we show that APC(Cdh1) is required for maintaining genomic integrity in primary human cells. Lentiviral-delivered strong and stable suppression of Cdh1 by RNA interference (RNAi) causes aberrant accumulation of several APC(Cdh1) target proteins, such as cyclin A, B, Aurora A or Plk1, which control accurate and equal distribution of the genetic information to daughter cells. This induces a premature and prolonged S phase, mitotic-entry delay and defects in chromosome separation and cytokinesis. Cell cycle deregulation by stable knockdown of Cdh1 leads to activation of p53/p21 and genomic instability, which is further increased by codepletion of p53. Thus, stabilization of APC(Cdh1) targets may initiate aberrant DNA replication and chromosome separation, and trigger a p53 response by deregulating G1 in primary human cells.
Assuntos
Neoplasias Ósseas/genética , Instabilidade Genômica/fisiologia , Osteossarcoma/genética , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Aurora Quinases , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Ciclina A/genética , Ciclina A/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Fibroblastos/metabolismo , Fibroblastos/patologia , Citometria de Fluxo , Imunofluorescência , Fase G1/fisiologia , Humanos , Rim/metabolismo , Rim/patologia , Lentivirus/genética , Mitose/fisiologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/fisiologia , Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Quinase 1 Polo-LikeRESUMO
Genetic aberrations of cancer cells have a profound impact for prognosis in several malignant neoplasias. The understanding of their origin is the basis for the development of new therapeutic options. Aneuploidy is observed in a large variety of premalignancies and tumors. Aneuploid cells harbor less or more than 46 chromosomes. The exact role of aneuploidy in tumorigenesis is still not clear. It has long been debated, whether aneuploidy directly contributes to tumorigenesis or reflects nonspecific changes during tumor progression. Several mechanisms are thought to be responsible for the generation of aneuploid sets of chromosomes: these comprise failure in cell division, such as defective chromosome separation caused by compromised mitotic checkpoint signaling or centrosome aberrations. Moreover, telomere shortening and defective DNA-damage signaling appear to be powerful driving forces of genomic instability. The loss of telomere sequences at the end of each chromosome and DNA double-strand breakage accompanied by compromised damage signaling favor fusion of chromosomes and generation of aneuploidy. Furthermore, aneuploidy arises to a much higher degree from a tetraploid state when compared to diploid cells. The frequent observation of the described defects in pre- and malignant cells supports the hypothesis that aneuploidy contributes to tumorigenesis.