Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells.

Low expression levels of CREB‑binding protein (CREBBP) have been demonstrated to be associated with high minimal residual disease at the end of induction therapy and adverse long‑term outcomes in pediatric patients with acute lymphoblastic leukemia (ALL). However, the effect of low CREBBP expression on the prognosis of ALL has not yet been investigated. In the present study, CREBBP was downregulated and overexpressed in ALL cell lines (Jurkat and Reh). Sensitivity to chemotherapy and cell proliferation activity was determined via a Cell Counting Kit‑8 assay. Cell cycle analysis was performed using flow cytometry. Immunofluorescence confocal microscopy and co‑immunoprecipitation (Co‑IP) assays were performed to determine the interaction between CREBBP and E2F transcription factor 3a (E2F3a). The binding of CREBBP to downstream gene caspase 8 associated protein 2 (CASP8AP2) promoters was assessed using a chromatin immunoprecipitation assay, and mRNA expression levels were detected via reverse transcription‑quantitative PCR. Western blot analysis was performed to detect protein expression of CREBBP, E2F3a and CASP8AP2. Downregulation of CREBBP increased the IC50 value of daunorubicin; however, no significant affects were observed on the IC50 values of vincristine and L‑asparaginase. Furthermore, downregulation of CREBBP notably inhibited leukemia cell proliferation, accumulated cells in the G0/G1 phase and decreased cell proportions in the S and G2/M phases. Co‑IP analysis demonstrated that CREBBP interacted with E2F3a, a transcription factor involved in G1/S transition. Immunofluorescence confocal microscopy indicated co‑localization of CREBBP and E2F3a at the cell nucleus. Furthermore, E2F3a protein expression decreased in CREBBP RNA interference treated Jurkat and Reh cells. CASP8AP2, a target gene of E2F3a, was also identified to be a downstream gene of CREBBP. In addition, decreased IC50 value and cell proportions in the G0/G1 phase, accelerated cell proliferation and upregulated E2F3a and CASP8AP2 expression were exhibited in CREBBP overexpressed cells. Taken together, the results of the present study suggested that CREBBP downregulation affects proliferation and cell cycle progression in leukemia cells, potentially via the interaction and regulation of E2F3a, resulting in chemotherapy resistance. Thus, targeting CREBBP may be a therapeutic strategy for treating pediatric patients with ALL.

Citation

Chao Gao, Shu-Guang Liu, Wen-Ting Lu, Zhi-Xia Yue, Xiao-Xi Zhao, Tian-Yu Xing, Zhen-Ping Chen, Hu-Yong Zheng, Zhi-Gang Li. Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells. Molecular medicine reports. 2020 Oct;22(4):2905-2915

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PMID: 32945392

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