학술논문
Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells
Document Type
Academic Journal
Author
Source
Molecular Medicine Reports. October 2020, Vol. 22 Issue 4, p2905, 13 p.
Subject
Language
English
ISSN
1791-2997
Abstract
Introduction Acute lymphoblastic leukemia (ALL) is a common malignancy in children worldwide. Genome-wide profiling studies, including DNA microarray and next generation sequencing have identified several genetic alterations associated with childhood [...]
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 I[C.sub.50] value of daunorubicin; however, no significant affects were observed on the I[C.sub.50] values of vincristine and L-asparaginase. Furthermore, downregulation of CREBBP notably inhibited leukemia cell proliferation, accumulated cells in the [G.sub.0]/[G.sub.1] phase and decreased cell proportions in the S and [G.sub.2]/M phases. Co-IP analysis demonstrated that CREBBP interacted with E2F3a, a transcription factor involved in [G.sub.1]/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 I[C.sub.50] value and cell proportions in the [G.sub.0]/[G.sub.1] 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. Key words: acute lymphoblastic leukemia, CREB-binding protein, E2F transcription factor 3, cell cycle
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 I[C.sub.50] value of daunorubicin; however, no significant affects were observed on the I[C.sub.50] values of vincristine and L-asparaginase. Furthermore, downregulation of CREBBP notably inhibited leukemia cell proliferation, accumulated cells in the [G.sub.0]/[G.sub.1] phase and decreased cell proportions in the S and [G.sub.2]/M phases. Co-IP analysis demonstrated that CREBBP interacted with E2F3a, a transcription factor involved in [G.sub.1]/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 I[C.sub.50] value and cell proportions in the [G.sub.0]/[G.sub.1] 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. Key words: acute lymphoblastic leukemia, CREB-binding protein, E2F transcription factor 3, cell cycle