A gaze at CDK9 isoforms in cell cycle and DNA damage response

INTRODUCTION AND OBJECTIVES. The DNA damage response (DDR) comprises an interwoven network that assures genomic integrity and constitutes a crucial barrier to cancer development and progression. The cell cycle checkpoint and DNA repair pathways play essential roles in the DDR because they allow a cell cycle-dependent and lesion-specific response. In a previous study, we identified CDK9 as a putative interactor of the DDR-related proteins BRCA1, BARD1, and PTIP. CDK9 acts in transcription elongation and presents two isoforms: CDK942k and CDK955k, which differ only by additional 117 amino acid residues at the N-terminal region of 55k. Previously, we demonstrated that BRCA1 recruitment to DNA damaged sites, and consequently the homologous recombination (HR) repair, depend on CDK942k. However, the literature suggests a possible role for CDK955k in the non-homologous end joining (NHEJ) repair. Here we investigate CDK9 regulation through cell cycle and explore functional differences between CDK942k and CDK955k, focusing on HR and NHEJ pathways. MATERIAL AND METHODS. We characterized CDK9 levels (both isoforms) throughout cell cycle, by real-time PCR and immunoblotting in synchronized MCF7 and hTERT-BJ cell lines. The impact of CDK9 isoforms overexpression (OE) on cell cycle dynamics and cell survival was evaluated in MCF7 cells using ionizing radiation (IR) and chemotherapeutic agents. To better understand the role of CDK9 in DNA repair, we evaluated the protein levels and subcellular localization of each isoform after IR treatment along with 53BP1 ionizing radiation-induced foci formation (IRIF) in MCF7 cells OE CDK942k or CDK955k. Subsequently, we also assessed the HR and NHEJ proficiency in MCF7 cells OE CDK942k or CDK955k. RESULTS AND CONCLUSION: Our results demonstrate that CDK955k levels (mRNA and protein) oscillate throughout the cell cycle, presenting a marked increase in G1/S transition, while CDK942k levels show no changes. Cell cycle dynamics and cell survival evaluation reinforced this correlation once CDK955k OE induces cell accumulation in G1 phase and confers resistance to chemotherapy treatment compared to control or cells OE CDK942k. Further, we demonstrate that CDK955k protein levels are increased upon IR treatment and only CDK942k presents IRIF. Although CDK955k do not form IRIF, cells OE CDK955k show an increase in 53BP1 IRIF and NHEJ efficiency, in contrast to cells OE CDK942k, that present no alterations in 53BP1 IRIF and an increase in HR efficiency. Taken together, our data suggest that CDK9 plays a dual role in DDR, possibly coordinating HR and NHEJ pathways through its two isoforms and that CDK955k levels are cell cycle regulated and response to DNA damage.