PMC

Expression of RFC3 and CREB1 is dependent on CDK activity. KG-1 cells were cultured with or without AT7519 (2 or 10 μM) for 16 hours. (a) RFC3 and CREB1 mRNA levels were determined by qRT-PCR. (b) CCNE1, CCNA2 and CCNB1 mRNA levels were measured to assess CDK inhibition. Expression of each gene was normalized against β-actin expression levels. Data are graphed as mean ± SEM (n = 3). **, p < 0.001.

CREB regulates loading of PCNA onto chromatin through RFC3. (a) Mitotic arrest KG-1 cells with a thymidine/nocodazole dual block were released and analyzed at the indicated times. Cells were extracted with NP-40 containing hypotonic buffer, fixed, then stained with anti-PCNA antibody and DAPI. Chromatin-bound PCNA and DNA content were determined by flow cytometry analysis. The region indicates chromatin-bound PCNA-positive populations. Plots are representative of three experiments with similar results. (b) Data represent the percentages of chromatin-bound PCNA compartments calculated using FlowJo software as mean ± SEM (n = 3).

Exogenous expression of RFC3 rescues the impaired G1/S progression in CREB knockdown KG-1 cells. Control or CREB knockdown (CREBshRNA-2) KG1 cells were transduced with lentiviral vectors expressing RFC3 and mCherry or mCherry alone. RFC3-high and low expressing cells were isolated based on mCherry levels. Expression levels of RFC3 and CREB were confirmed in mRNA levels by qRT-PCR (a) and protein levels by immunoblotting (b). mRNA expression levels of CREB1 and RFC3 were normalized against β-actin expression levels. Values are indicated as mean ± SEM (n = 3). **, p < 0.01. Cell lysates were analyzed for RFC3, CREB, and with β-tubulin as a loading control, by immunoblotting. A representative blot of at least three independent experiments is shown. (c) Exogenous RFC3 expression rescues the defective G1/S progression in CREB knockdown KG-1 cells. Cells were synchronized in mitosis by a thymidine/nocodazole dual block. Synchronized cells were released from the mitotic arrest and analyzed at the indicated times. DNA content was determined by flow cytometry analysis of propidium iodide stained cells. Cells started to enter S phase by 9 hours after release. Plots are representative of three experiments with similar results. (d) Data represent the percentages of cell populations residing at each cell cycle stage calculated using FlowJo software as mean ± SEM (n = 3).

RFC3 knockdown impairs the G1/S cell cycle progression. KG1 cells were transduced with pLKO.1 lentiviral vectors expressing RFC3 shRNA or luciferase shRNA, and then transduced cells were selected with puromycin. (a) Suppressed expression of RFC3 by corresponding specific shRNA was assessed at protein level. Total cell lysates were analyzed by immunoblotting for RFC3. β-tubulin was used as a loading control. (b) RFC3 knockdown inhibited proliferation of KG1 cells. A total of 1 × 10⁵ cells were seeded in 12-well plates and the number of viable cells was counted for 3 days. Values represent mean ± SEM (n = 3). **, p < .01. (c) Cell cycle profile of control or RFC3 knockdown KG1 cells released from thymidine block. Cells were synchronized at G1/S boundary with thymidine treatment (2 mM, 30 h), and then harvested at the indicated times after release into medium with nocodazole (300 nM). Cells were stained with PI following fixation with 70% cold ethanol, and then analyzed for DNA contents by flow cytometry. These data are representative plots from three experiments with similar results. (d) % cell populations at each cell cycle phase were calculated using FlowJo software and denoted as mean ± SEM (n = 3).

RFC3 as a direct target gene of CREB. (a) Protein expression levels of CREB and RFC3 were analyzed in CREB knockdown and control KG-1 cells by immunoblotting. Total lysates were immunoblotted for CREB, RFC3 and β-Tubulin (loading control). A representative blot of at least three different experiments is shown. (b) RFC3 mRNA expression levels were significantly decreased in CREB knockdown cells. RFC3 and CREB1 mRNA levels were determined by qRT-PCR and normalized against β-actin expression level. Relative expression levels are presented as mean ± SEM (n = 3). (c) Temporal expression of RFC3 and CREB1 mRNAs. Expression levels of RFC3 genes in synchronized cells were assessed by qRT-PCR. Values are indicated as mean ± SEM (n=3). **, p < 0.01. (d) Sequence of the human RFC3 and CREB1 promoter regions. Putative transcription factor binding sites are underlined. Sequences of PCR primers for CREB1 ChIP are shown in bold type. (e) CREB binds to the RFC3 promoter in vivo. ChIP assay was performed using normal rabbit IgG (negative control) or antibodies specific to Histone H3 (positive control), CREB, and E2F1 for demonstrating the in vivo binding of CREB and E2F1 to RFC3 and CREB1 promoters. RFC3 and CREB1 PCR primers were used to detect RFC3 and CREB1 promoter DNA fragments in chromatin immunoprecipitates, respectively. Two % of total in-put chromatin was used as a control. Relative PCR product levels are shown.

Impaired G1/S progression in CREB knockdown KG-1 cells. (a) Cell cycle profile of control and CREB knockdown cells by flow cytometry. KG-1 cells were infected by CREB shRNA-expressing or control lentiviruses, and then transduced GFP-positive cells were sorted. Cells synchronized using a thymidine plus nocodazole block. Synchronized cells were released from the nocodazole block (mitotic arrest) and collected at the indicated times. DNA content was analyzed using propidium iodide staining and flow cytometry analysis. Cells started to enter S phase by 8h after release. 2N indicates G1 DNA content. Plots are representative of three experiments with similar results. (b) Data represent the percentages of cell populations residing at each cell cycle stage calculated using FlowJo software and is expressed as mean ± SEM (n = 3). (c) Temporal expression patterns of CCNE1, PCNA, CCNA2 and CCNB1 genes in CREB knockdown KG-1 cells. CREB-knockdown and control KG1 cells were released from the mitotic arrest and harvested at indicated times. Relative mRNA expression of cyclin E1 (CCNE1), cyclin A2 (CCNA2), cyclin B1 (CCNB1) and PCNA genes were quantitated by qRT-PCR analysis. Expression of each gene was normalized against β-actin expression level. Relative expression levels are presented as fold induction above expression levels in control cells at 0 hours. Values are shown as mean ± SEM (n = 3). **, p < 0.01. (d) Cell extracts were prepared at the indicated times after release from mitotic arrest and protein expression levels of cyclin E1, cyclin A2, cyclin B1, PCNA and β-tubulin were analyzed by immunoblotting. β-tubulin was used as an internal control.

Correlation between the expression levels of RFC3 and CREB1 in AML. (a) CREB knockdown inhibits RFC3 expression in U937 and HL-60 AML cell lines. U937 and HL60 cells were transduced with lentiviral vector expressing CREBshRNA-2 or vector alone. Cells were sorted for GFP-positive cells, and then analyzed for mRNA expression levels of CREB1 and RFC3 by qRT-PCR. Expression of each gene was normalized against β-actin expression levels. Data are graphed as mean ± SEM (n = 3). **, p < 0.001. (b) Correlation between RFC3 and CREB1 mRNA expressions in diagnostic samples from AML patients (n = 19, Pearson r=0.6628, p = 0.002). The linear regression line is plotted and its slope is given. Relative mRNA expression levels of CREB1 and RFC3 were compared by qRT-PCR. Expression of genes was normalized against β-actin expression level. The values represent the ratio of fold change in gene expression from each sample relative to the average of three normal control samples. (c) Association of CREB with RFC3 promoter region in primary human AML cells. ChIP assays were undertaken using human AML cells and normal rabbit IgG (negative control) or antibodies specific to Histone H3 (positive control) and CREB. DNA fragments spanning CRE consensus motif in RFC3 promoter region were amplified from the immunoprecipitates by PCR using RFC3 primers and displayed by gel electrophoresis. Relative PCR product levels are shown as arbitrary numbers by setting the level of DNA from anti-Histone H3 antibody immunoprecipitate as 100.