Ikaros induces quiescence and T-cell differentiation in a leukemia cell line

Abstract

Ikaros is a hematopoietic cell-specific zinc finger DNA binding protein that plays an important role in lymphocyte development. Genetic disruption of Ikaros results in T-cell transformation. Ikaros null mice develop leukemia with 100% penetrance. It has been hypothesized that Ikaros controls gene expression through its association with chromatin remodeling complexes. The development of leukemia in Ikaros null mice suggests that Ikaros has the characteristics of a tumor suppressor gene. In this report, we show that the introduction of Ikaros into an established mouse Ikaros null T leukemia cell line leads to growth arrest at the G0/G1 stage of the cell cycle. This arrest is associated with up-regulation of the cell cycle-dependent kinase inhibitor p27kip1, the induction of expression of T-cell differentiation markers, and a global and specific increase in histone H3 acetylation status. These studies provide strong evidence that Ikaros possesses the properties of a bona fide tumor suppressor gene for the T-cell lineage and offer insight into the mechanism of Ikaros's tumor suppressive activity.

FIG. 1.

Retroviral expression levels of Ik-1 are comparable to expression levels of DNA-binding Ikaros isoforms in wild-type thymocytes. Whole-cell extracts of wild-type thymocytes and purified JE131 cells successfully transduced with the MSCV-(Ik-1)-IRES-H-2Kk (Ik-1) or the MSCV-(Ik-7)-IRES-H-2Kk (Ik-7) retrovirus were prepared. A total of 10 μg of protein was subjected to SDS-PAGE, and Western blotting was performed with an anti-Ikaros monoclonal antibody. The two Ikaros bands visible in the wild-type thymocytes correspond to the predominantly expressed Ikaros isoforms Ik-1 (top band) and Ik-2 and Ik-3, which comigrate (bottom band). The Ik-1 band in the retrovirally transduced cells is of slightly larger molecular weight due to the Flag epitope tag. Densitometry readings are shown under each band. This figure was generated by using Adobe Photoshop software. Thy, thymocytes; Ab, antibody.

FIG. 2.

Reintroduction of Ikaros into the JE131 cells has a profound effect on their growth properties. (A) JE131 cells were infected with the MSCV-IRES-GFP or MSCV-(Ik-1)-IRES-GFP retrovirus. Successfully transduced cells were purified and stained with propidium iodide to measure DNA content as a measure of cell cycle status. Shown are percentages of cells that fall into the G₀/G₁, S, and G₂/M phases of the cell cycle for successfully transduced (GFP⁺) and nontransduced (GFP⁻) cells from each culture. (B) JE131 cells were infected with the MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-7)-IRES-H-2Kk (Ik-7), or the MSCV-(Ik-1)-IRES-H-2Kk (Ik-1) retrovirus. Successfully transduced cells were purified and plated at 10⁶ cells/well in a 24-well plate. Counts of viable cells were performed every 24 h. (C) H-2Kk expression as monitored every 24 h in the purified cell populations by flow cytometry. (D) After 8 days in culture, cells were resorted for H-2Kk expression, and whole-cell protein extracts were prepared from H-2Kk⁺ and H2-Kk⁻ fractions. A total of 10 μg of protein was loaded onto an SDS-PAGE gel, and Western blotting was performed with an anti-Ikaros monoclonal antibody. This figure was generated by using CellQuest, Microsoft Excel, and Adobe Photoshop software. Ab, antibody.

FIG. 3.

Appearance of a small cell population in Ik-1-expressing JE131 cells. At 48 h after infection, JE131 cells infected with the MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-7)-IRES-H-2Kk (Ik-7), or MSCV-(Ik-1)-IRES-H-2Kk (Ik-1) retrovirus were stained with fluorochrome-conjugated anti-H-2Kk, and flow cytometric analysis was performed. (A) A unique population of cells with low forward and side scatter (R2) are observed in the Ik-1-transduced culture. (B) This effect is confined to successfully transduced cells, since the small cell population is almost uniformly H-2Kk⁺ and, therefore, Ik-1⁺. The larger cell population (R1) is already beginning to lose H-2Kk and, therefore, Ik-1 expression. This figure was generated by using CellQuest and Adobe Photoshop software. FSC, forward scatter; SSC, side scatter.

FIG. 4.

Small population of Ik-1-expressing cells are arrested at G₀/G₁. (A) Three days after infection of the JE131 cells with MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-7)-IRES-H-2Kk (Ik-7), or MSCV-(Ik-1)-IRES-H-2Kk (Ik-1) retrovirus, DNA content was analyzed by propidium iodide staining as a measure of cell cycle status. Cells that fall into the R1 gate show similar cell cycle profiles in all three transduced cultures. However, the unique small cell population (R2) observed in the Ik-1-transduced culture is blocked at the G₀/G₁ phase of the cell cycle. (B) At day 6 postinfection, successfully transduced cells were sorted from the cultures, and Pyronin Y staining was performed to measure cytoplasmic RNA content. Pyronin Y-negative cells are in G₀, whereas Pyronin Y-positive cells are in G₁, S, or G₂/M phase. The majority of Ik-1-transduced cells fell into the small cell population and failed to stain with Pyronin Y. This figure was generated by using CellQuest and Adobe Photoshop software.

FIG. 5.

Ik-1-transduced JE131 cells display an increase in p27^kip1 expression and can be slowed in their growth by retroviral transduction of p27^kip1. JE131 cells with and without Ik-1 activity were analyzed for RNA and protein expression of p27^kip1. (A) At 24 h after infection with the MSCV-IRES-GFP (Ik−) or MSCV-(Ik-1)-IRES-GFP (Ik+) retrovirus, successfully transduced JE131 cells were purified and cDNA was prepared. RT-PCR was performed by using a range of cDNA amounts. Shown here are the results of twofold dilutions (from 1:256 to 1:1024 for each sample). (B) At 24 h after infection with MSCV-(Ik-1)-IRES-GFP (Ik-1), MSCV-(Ik-7)-IRES-GFP (Ik-7), or MSCV-IRES-GFP (Ik−) retrovirus, successfully transduced JE131 cells were purified, and protein extracts were prepared. A total of 10 μg of protein was subjected to SDS-PAGE. Blots were probed with antibodies against p27^kip1 or actin (as a loading control). Results from two representative experiments are shown. (C) JE131 cells were infected with the MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-7)-IRES-H-2Kk (Ik-7), MSCV-(Ik-1)-IRES-H-2Kk (Ik-1), or MSCV-(p27^kip1)-IRES-H-2Kk (p27) retrovirus. Successfully transduced cells were purified and plated at 10⁶ cells/well in a 24-well plate. Counts of viable cells were performed every 24 h. This figure was generated by using CellQuest, Microsoft Excel, and Adobe Photoshop software.

FIG. 6.

Reintroduction of Ik-1 into JE131 cells induces a T-cell differentiation program. (A) Model depicting developmental progression of thymocytes. DN thymocytes can be broken down into four developmental stages based on cell surface expression of CD44 and CD25 as depicted here. (B and C) JE131 cells were infected with the MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-7)-IRES-H-2Kk (Ik-7), MSCV-(Ik-1)-IRES-H-2Kk (Ik-1), or the MSCV-(p27^kip1)-IRES-GFP (p27) retrovirus. Two to three days postinfection, cells were stained with fluorochrome-conjugated anti-H-2Kk antibody to identify successfully transduced cells, together with antibodies against defined T-cell differentiation markers. This figure was generated by using CellQuest and Adobe Photoshop software.

FIG. 7.

Expression of Ik-1 in JE131 cells induces widespread changes in histone acetylation. JE131 cells were infected with the MSCV-IRES-H-2Kk (Ik−), MSCV-(Ik-1)-IRES-H-2Kk (Ik-1), or the MSCV-(p27^kip1)-IRES-GFP (p27) retrovirus. At 3 days postinfection, successfully transduced cells were purified. Histones were prepared by using an acid extraction procedure. A total of 10 μg of protein extract was subjected to SDS-PAGE. The blots in panels A and B are representative of six and two independent experiments, respectively. This figure was generated by using Adobe Photoshop software.