ZNF300 knockdown inhibits forced megakaryocytic differentiation by phorbol and erythrocytic differentiation by arabinofuranosyl cytidine in K562 cells

Abstract

Previously, we reported that ZNF300 might play a role in leukemogenesis. In this study, we further investigated the function of ZNF300 in K562 cells undergoing differentiation. We found that ZNF300 upregulation in K562 cells coincided with megakaryocytic differentiation induced by phorbol-12-myristate-13-acetate (PMA) or erythrocytic differentiation induced by cytosine arabinoside (Ara-C), respectively. To further test whether ZNF300 upregulation promoted differentiation, we knocked down ZNF300 and found that ZNF300 knockdown effectively abolished PMA-induced megakaryocytic differentiation, evidenced by decreased CD61 expression. Furthermore, Ara-C-induced erythrocytic differentiation was also suppressed in ZNF300 knockdown cells with decreased γ-globin expression and CD235a expression. These observations suggest that ZNF300 may be a critical factor controlling distinct aspects of K562 cells. Indeed, ZNF300 knockdown led to increased cell proliferation. Consistently, ZNF300 knockdown cells exhibited an increased percentage of cells at S phase accompanied by decreased percentage of cells at G0/G1 and G2/M phase. Increased cell proliferation was further supported by the increased expression of cell proliferation marker PCNA and the decreased expression of cell cycle regulator p15 and p27. In addition, MAPK/ERK signaling was significantly suppressed by ZNF300 knockdown. These findings suggest a potential mechanism by which ZNF300 knockdown may impair megakaryocytic and erythrocytic differentiation.

Figure 1. ZNF300 expression is upregulated in PMA-induced megakaryocytic differentiation in K562 cells.

(A) K562 cells were cultured with 10 nM phorbol myristate acetate (PMA) or vehicle control (Ctrl) for 72 hours and stained with Wright-Giemsa stains. The stained or un-stained cells were photographed under microscopy at the bright view of the microscope (magnification ×400 for stained cells and un-stained cells). (B) The resultant cells were also stained with PE-conjugated GPIIIa (CD61)-specific antibody. The samples were analyzed using flow cytometer. Data was analyzed with Flowjo and presented as histogram graph. (C, D) The mRNA level of ITGB3 (CD61) and ITGA2B (CD41α) in the resultant cells was measured by quantitative RT-PCR. Data was normalized to GAPDH and presented as bar graph. (E) The mRNA level of ZNF300 in the resultant cells was measured by quantitative RT-PCR and represented as the relative expression (mean±SD). Data were representative results of 3 independent experiments with similar results. *** indicates p<0.001. (F) The protein expression level of ZNF300 in resultant cells was measured by western blot and quantified by densitometry. Numbers indicate the densitometry of ZNF300 protein normalized by that of HSC70, which is further normalized to that of untreated cells. Result was the representative blot from 3 experiments with similar result.

Figure 2. ZNF300 expression is upregulated during the erythrocytic differentiation when K562 cells were induced by Ara-C.

(A) K562 cells were cultured in the absence (Ctrl) or presence of 1 µM Ara-C for 168 hours and were stained with Wright-Giemsa stains. Unstained cells were photographed under the dark field and the stained cells were photographed under the bright field (original magnification ×400). (B) The erythrocytic differentiation of resultant cells were determined by staining with PE-conjugated anti-CD235a antibody and analyzed by FACS. Histogram was the representative result from 3 independent experiments with similar results. (C) The erythrocytic differentiation of resultant cells was also determined by benzidine staining to measure the hemoglobin protein. The hemoglobin staining positive cells were counted under light microscope and data were presented as percentage of benzidine staining positive cells. Results were statistics of three independent experiments with similar results (mean±SD). *** indicates p<0.001. (D) The mRNA expression level of γ-hemoglobin (HBG1) in the resultant cells was measured by quantitative RT-PCR. (E) The mRNA level of ZNF300 in the resultant cells was measured by quantitative RT-PCR and represented as the relative expression (mean±SD). Results were representative data from 3 independent experiments with similar results. *** indicates p<0.001. (F) The protein expression level of ZNF300 in resultant cells was measured by western blot and quantified by densitometry. Numbers indicate the densitometry of ZNF300 protein normalized by that of HSC70, which is further normalized to that of untreated cells. Results were the representative blot from 3 experiments with similar results.

Figure 3. ZNF300 knockdown abolished megakaryocytic differentiation.

(A) Control and ZNF300 knockdown (shZNF300) cells were cultured in the presence of 10 nM PMA for 72 hours. The morphology of the treated cells was observed under the light microscope (×400 magnifications). (B) The megakaryocytic differentiation of the treated cells was measured by staining cells with PE-conjugated anti-CD61 antibody and analyzed by FACS. (C) The megakaryocyte differentiation of the treated cells was measured by detecting ITGB3 mRNA level (quantitative RT-PCR) and presented as relative expression level. (D) The megakaryocytic differentiation of the treated cells was also measured by detecting ITGA2B mRNA level (quantitative RT-PCR) and presented as relative expression level. Data were representatively results of 3 independent experiments with triplicates. *** indicates p<0.001

Figure 4. ZNF300 knockdown blocks Ara-C-induced erythrocytic differentiation.

(A) Control and ZNF300 knockdown cells (shZNF300) were cultured in the presence of Ara-C for 72 hours. The resultant cells were stained with benzidine to measure the hemoglobin protein. The stained cells were photographed under the bright field (original magnification ×200). (B) The hemoglobin staining positive cells were counted under microscope and data were presented as percentage of benzidine staining positive cells. The bar graph was the statistics of benzidine staining. (C) The erythrocyte differentiation of resultant cells was determined by staining cells with PE-conjugated CD235a antibody and measured by FACS. (D) The erythrocyte differentiation of resultant cells was also determined by detecting mRNA level of γ-hemoglobin (HBG1) through quantitative RT-PCR. *** indicates p<0.001. (E) Control and ZNF300 knockdown cells treated with (+) or without (−) Ara-C were collected for western blot with antibodies as indicated.

Figure 5. ZNF300 knockdown promotes proliferation in K562 cells.

(A) The same amount of control and ZNF300 knockdown cells (5×10³) were plated in triplicates in a 24-well plate and the cell number was counted for consecutive 6 days. Data were statistics (mean ±SD) of representative results from 3 independent experiments with similar results. (B) Cell proliferation assay was also performed by using Cell Counting Kit-8. The absorbance at 450 nm was measured for consecutive 3 days and normalized to that of the first day. The cell proliferation was presented as relative absorbance. (C) Control and ZNF300 knockdown cells were fixed, permeablized, and stained with DAPI. The DNA content was analyzed by FACS. The distribution of cells in G0/G1, S, and G2/M phases was further analyzed by ModFit LT. Data were the statistics of representative results from 3 independent experiments with similar results. Numbers indicate the percentage (mean ±SD). (D) Bar graph of the statistics of cell cycle profiling experiments. (E) Cell lysates were prepared from control or ZNF300 knockdown cells and the protein expression level was detected by western blot with antibodies as indicated. HSC70 served as a protein loading control. ** indicates p<0.01. (F) The cytosol fraction (Cyto) and nucleus (Nucl) fraction of K562 cells treated with (+) or without (−) PMA were used for western blot with antibodies as indicated.