Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , RNA Helicases DEAD-box/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Pirazinas/farmacologia , Ribonuclease III/metabolismo , Western Blotting , Bortezomib , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mutação/genética , RNA Interferente Pequeno/genética , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaAssuntos
Compostos Alílicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/fisiologia , Mieloma Múltiplo/patologia , Quinazolinas/farmacologia , Medula Óssea/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
Small molecule inhibitors targeting CDK1/CDK2 have been clinically proven effective against a variety of tumors, albeit at the cost of profound off target toxicities. To separate potential therapeutic from toxic effects, we selectively knocked down CDK1 or CDK2 in p53 mutated HACAT cells by siRNA silencing. Using dynamic, cell cycle wide proteome arrays, we observed minor changes in overall abundance of proteins critically involved in cell cycle transition despite profound G2/M or G1/S arrest, respectively. Employing phospho site specific analyses, we identified uncoupled mitogenic, yet pro-apoptotic signaling from counter balancing anti-apoptotic activity in CDK2 disrupted cells. Moreover, a crucial role of CDK2 activity in early serum response was observed, extending well-established roles of CDKs outside their cell cycle regulating functions. In contrast, disruption of CDK1 only marginally affected phosphorylation events of crucial signaling nodes prior to G2/S transition. The data presented here suggest that the temporal separation of pro- and anti-apoptotic pathways by selective inhibition of CDK2 disrupts coherent signaling modules and may synergize with anti-proliferative drugs, averting toxic side effects from CDK1 inhibition.
Assuntos
Apoptose , Proteína Quinase CDC2/metabolismo , Inativação Gênica , Mitose , Transdução de Sinais , Mutações Sintéticas Letais/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Quinase CDC2/deficiência , Linhagem Celular , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Cinética , Mitose/genética , Fosforilação , RNA Interferente Pequeno/metabolismoRESUMO
miRNAs are critically implicated in the initiation process of and progression through cancerogenesis. The mechanisms, however, by which miRNAs interfere with the signalosomes of human cancer cells, are still obscure. We utilized the p53-mutated human keratinocyte cell line HACAT to investigate the biological significance and extent to which miRNAs regulate proliferation, cell growth, and apoptosis in transformed phenotypes. Silencing of the miRNA-processing enzyme Dicer1 resulted in cell cycle arrest at the G1/S border, along with restoration of CDK inhibitor p21(CIP)expression. Employing a cell cycle-wide phospho-proteomic approach, we detected neglectable changes in abundance and schedule of overall and cell cycle periodic protein expression despite cell cycle arrest of Dicer1-depleted cells. Instead, we found substantially delayed post-translational modifications of some, but not all, signaling nodes. Phospho-site-specific analyses revealed that pro-apoptotic information elicited by Myc, ß-catenin, and other mitotic pathways early in G1 are absorbed and balanced by anti-apoptotic signaling from AKT and NFκB in Dicer1-competent cells. The absence of regulatory miRNAs, however, led to a substantial delay of anti-apoptotic signaling, leaving pro-apoptotic stress unbalanced in Dicer1-deprived cells. We here show that this temporal separation of pro- and anti-apoptotic signaling induced by inhibition of Dicer1 is synergistic and synthetic lethal to low-dose 5-FU chemotherapy in p53-mutated HACAT cells. The findings reported here contribute to the understanding of the complex interactions of miRNAs with the signalosom of transformed phenotypes and may help to design novel strategies to fight cancer.