FUNCTIONAL CHARACTERIZATION OF CDK9 55K ISOFORM IN DNA DAMAGE RESPONSE

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  • Presentation type: Pós-Doutorado
  • Track:
  • Keywords: CDK9; DNA damage; 53BP1; BRCA1;
  • 1 Pesquisa Clínica / INCA / Instituto Nacional de Câncer
  • 2 INCA / Pesquisa Clínica / Rio de Janeiro - Brasil
  • 3 H. Lee Moffitt Cancer Center & Research Institute
  • 4 Instituto Federal do Rio de Janeiro

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Abstract

INTRODUCTION AND OBJECTIVES. DNA damage response (DDR) is crucial to maintain genomic stability. Dysfunction in proteins involved in this pathway may lead to carcinogenesis. To better understand this network, our group performed a systematic study to identify new proteins involved in DDR. In a previous work we identified the cyclin-dependent kinase 9 (CDK9) as a putative interactor of BRCA1, BARD1 and PTIP. CDK9 has two isoforms, the 42k isoform plays a role in transcription elongation and homologous recombination control (through interaction with BRCA1 and BARD1). However, CDK9 55k isoform role remains unclear. In this work, we investigated functional differences between CDK9 42k and 55k, exploring CDK9 55k putative role in non-homologous end joining (NHEJ) repair pathway. MATERIAL AND METHODS. To inquire a possible cell cycle dependent behavior, CDK9 expression levels were evaluated by real-time PCR and immunoblotting in synchronized human cells - hTERT-BJ were synchronized in G0/G1 and MCF7 cells in G1/S. To investigate CDK9 isoforms impact in cell cycle dynamics, CDK9 42k or 55k were overexpressed in MCF7 cells (CDK9 proficient and deficient) after ionizing radiation (IR) treatment and evaluated by flow cytometry. We also assessed the effect of chemotherapeutic agents (cisplatin, etoposide and irinotecan) in cell viability using MCF7 CDK9 deficient cells reconstituted with CDK9 42k or 55k isoforms. To evaluate the putative interaction between CDK9 and PTIP, GST-pulldown and co-immunoprecipitation assays were performed. Also, CDK9 isoforms role in DDR was investigated by 53BP1 ionizing radiation-induced foci formation (IRIF) in MCF7 cells overexpressing the isoforms. RESULTS AND CONCLUSION: Data from synchronized cell analysis suggested that CDK9 55k levels (mRNA and protein) are cell cycle dependent, markedly increased in G1 phase. Cell cycle dynamics evaluation reinforced the correlation between CDK9 55k and G1 phase - 55k isoform overexpression leads to cell accumulation in G1 phase. Furthermore, cells reconstituted with CDK9 55k showed a significant increase in cellular viability upon damage when compared to cells reconstituted with 42k isoform. Interestingly, MCF7 cells overexpressing CDK9 42k exhibited a characteristic IRIF upon IR treatment; on the other hand, 55k overexpression sustained a nuclear diffuse localization. Unlike CDK9 42k that had no impact in 53BP1 IRIF, 55k isoform overexpression showed a significant increase in foci formation – indicating NHEJ repair pathway activation. Preliminary data also indicated a selective interaction between CDK9 55k and PTIP (a known player in NHEJ). Taken together, our data suggest that CDK9 55k plays a role in cell cycle control and in NHEJ repair pathway.

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Author

Vanessa Fernandes

Olá Anderson, não entendi ao certo qual seria sua questão. Nesse ensaio nós plaqueamos as células e coletamos amostras após um dois ou três dias (cada amostra foi analisada por citometria e immunoblotting), assim esses resultados indicam uma correlação entre a confluência da monocamada, o perfil de ciclo celular e os níveis proteicos de CDK9. Células em cultura (mesmo que sejam transformadas) tendem a diminuir a velocidade de replicação devido à confluência ou ao esgotamento do meio de cultura. Nós utilizamos esse racional para realizar essa avaliação, como podemos observar as amostras obtidas em 48 e 72 horas apresentam um aumento progressivo do número de células em G1, quando comparadas com as células coletadas 24 horas após o plaqueamento.  Nós então avaliamos os níveis proteicos de CDK9 dessas amostras e percebemos que, paralelamente ao acúmulo de células em G1, ocorre um aumento dos níveis proteicos da isoforma 55k. Segurindo uma possível correlação entre a isoforma 55k e o controle do ciclo celular, mais precisamente da fase G1. Espero ter ajudado, caso não tenha conseguido, por favor sinalize para que eu possa responder melhor sua pergunta. Vanessa