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. 2016 Jul:46:10-7.
doi: 10.1016/j.leukres.2016.03.013. Epub 2016 Apr 9.

Zfp521 promotes B-cell viability and cyclin D1 gene expression in a B cell culture system

Salma Al Dallal  1 , Kathryn Wolton  1 , Kathryn E Hentges  2
Affiliations

Zfp521 promotes B-cell viability and cyclin D1 gene expression in a B cell culture system

Salma Al Dallal et al. Leuk Res. 2016 Jul.

Abstract

Leukemia arises due to the dysregulated proliferation of hematopoietic progenitor cells. Errors in the multi-step commitment process result in excessive numbers of immature lymphocytes, causing malignant disease. Genes involved in the differentiation of lymphocytes are often associated with leukemia. One such gene, Zfp521, has been found to cause B-cell leukemia in mice when over-expressed. The role of Zfp521 in B-cell differentiation, and the mechanisms by which it leads to leukemic transformation, are unclear. In this study we report that Zfp521 knockdown causes apoptosis in a B-cell culture system and promotes down-regulation of genes acting at late stages of B-cell differentiation. We identify Pax5 and cyclin D1 as Zfp521 target genes, and suggest that excessive B-cell proliferation observed in mice with retroviral insertions near the Zfp521 gene is due to an up-regulation of cyclin D1 in B-cells. Overall, these results suggest links between dysregulated Zfp521 and B-cell survival.

Keywords: Cyclin D1; Ebf1; Evi3; Pax5; Zfp423; Zfp521.

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Figures

Fig. 1
Fig. 1
The Zfp521 BCL1 B-cell knockdown system. A. Zfp521 expression is present in the Pro-B cell line Ba/F3. Ladder = Bioline Hyperladder I, neg = no template control. B. Zfp521 expression is significantly reduced in BCL1 cells transfected with a specific shRNA as compared to mock-transfected cells at both 24 h (*p = 0.03; t test) and 48 h (*p = 0.006; t test). C. Zfp423 expression is unaffected in cells transfected with Zfp521 shRNA at either 24 h (p = 0.19; t test) or 48 h (p = 0.69; t test). D. The presence of viable cells is significantly reduced in cultures transfected with Zfp521 shRNA when compared to cells undergoing mock transfection at 3 days and 7 days post-transfection (*p < 0.05; t test), or transfection with vector containing no shRNA (negative) or scrambled shRNA sequence. E. The percentage of dead cells at days 3 and 7 post-transfection as identified by trypan blue staining is significantly increased in cells transfected with Zfp521 shRNA as compared to cells undergoing mock transfection (*p < 0.05; t test), or transfection with vector containing no shRNA (negative) or scrambled shRNA sequence. F. The fold-change in the levels of Caspase 3/7 activity in cells transfected with Zfp521 shRNA or control plasmids as compared to mock-transfected cells. A significant increase in Caspase 3/7 activity is detected in Zfp521 shRNA transfected cells compared to mock-transfected cells at day 3 and day 7 (*p < 0.05; t test) post-transfection.
Fig. 2
Fig. 2
Expression of B-cell genes in Zfp521 knock-down cells. A. The expression of early B-cell genes was assayed by qPCR at 24 and 48 h post-transfection in BCL1 cells transfected with Zfp521 shRNA or scrambled control plasmid. B. The expression of the recombination gene Rag1 in scrambled control or Zfp521 shRNA transfections. C. The expression of late B-cell marker genes at 24 and 48 h post-transfection. Expression was significantly reduced in Zfp521 shRNA transfected cells as compared to cells transfected with scrambled plasmid for all genes reported in Panel D (*p < 0.05; t test). All panels show expression normalized to 18S RNA control.
Fig. 3
Fig. 3
Viability and apoptosis levels in rescue assay cell cultures. A. The ratio of cell viability measured on day 7 post-transfection for cells with Zfp521 wild type rescue plasmid added (day 7 + ) to cells without rescue plasmid added (day 7). A significant increase in viability was found in shRNA cells with Zfp521 wild type rescue plasmid added as compared to mock-transfected cells with rescue (*p < 0.05; t test). B. The fold-change in Caspase 3/7 activity in each condition as compared to mock-transfected cells. Cells without rescue are shown on the left, and cells with rescue plasmid added are shown in the right. A significant different was found between caspase activity levels in cells without Zfp521 rescue plasmid transfection to cells with rescue (*p < 0.05; t test). C. The ratio of cell viability measured on day 7 post-transfection for cells with ΔZfp521 plasmid added. No significant difference was detected in shRNA cells with ΔZfp521 plasmid added as compared to mock-transfected cells (p = 0.67; t test). D. The ratio of cell viability measured on day 7 post-transfection for cells with Zfp521Stop plasmid added. No significant difference was detected in shRNA cells with Zfp521Stop plasmid added as compared to mock-transfected cells (p = 0.15; t test). For all panels, the original transfection condition is shown in the legend.
Fig. 4
Fig. 4
Cell viability measurements in rescue assays. A. The ratio of viable cells detected in cultures at day 7 post-transfection with wild type Pax5 rescue plasmid (7+) compared to cells mock transfected on day 7. A significant increase in viable cell number is seen in cells originally transfected with Zfp521 shRNA when compared to other transfection conditions (*p < 0.05; t test). B. The ratio of viable cells at day 7 post-transfection with Pax5 V26G mutant construct compared to cells without rescue. No significant differences were detected between any transfection groups. C. The ratio of viable cells in rescue assays with the addition of cyclin D1 plasmid as compared to cells with no rescue. A significant increase in viable cell number is seen in cells originally transfected with Zfp521 shRNA when compared to other transfection conditions (*p < 0.05; t test). D. The ratio of viable cells at day 7 post-transfection with Ebf1 plasmid compared to cells without rescue. No significant differences were detected between any transfection groups. For all panels, the original transfection condition is shown in the legend.
Fig. 5
Fig. 5
Expression analysis in AKXD27 tumors and leukemia cell lines. A. cyclin D1 expression measured by qPCR in tumors from mice with retroviral insertions at Evi3 (27-222 and 27-172) is increased as compared to a control tumor (27-165). B. Zfp521 expression levels in the same AKXD27 tumors. C. cyclin D1 expression levels in leukemia cell lines. The NFS-467 cell line has a retroviral insertion at the Evi3 locus, and shows a significant increase in cyclin D1 expression as compared to BCL1 cells transfected with the Zfp521 scrambled shRNA (*p < 0.05; t test). D. The expression levels of cyclin D1 in BCL1 cells transfected with either the Zfp521 shRNA or scrambled shRNA at 24 and 48 h post-transfection. A decrease in cyclin D1 expression is shown in Zfp521 shRNA transfections compared to cells transfected with scrambled shRNA sequence (*p < 0.1; t test).
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