The amino terminus of the mixed lineage leukemia protein (MLL) promotes cell cycle arrest and monocytic differentiation

Abstract

Several lines of evidence suggest that the mixed lineage leukemia protein (MLL, ALL-1, HRX) plays a role in regulating myelomonocytic differentiation. In this study we examined the effect of expression of MLL-AF9 on differentiation of the monoblastic U937 cell line by using a tetracycline-inducible expression system. MLL-AF9 arrested growth of U937 cells and induced these cells to differentiate into macrophages; induction was accompanied by expression of CD11b and CD14 and ultimately cell death. Deletion mutants of MLL-AF9 were used to map the sequences responsible for this effect. The amino-terminal half of MLL was sufficient for both cell cycle arrest and macrophage differentiation, whereas the carboxyl terminus of MLL or AF9 was found to be dispensable for this effect. Further deletions showed that a 35-kDa amino-terminal fragment spanning two AT hook motifs was sufficient for cell cycle arrest, up-regulation of p21(Cip1) and p27(Kip1), and partial differentiation toward macrophages. These findings suggest a possible role for the MLL AT hook-containing region in regulating myelomonocytic differentiation.

Figure 1

Schematic of MLL-AF9 and deletion mutants. All cDNAs were cloned into pUHD10B/X, a modified form of the pUHD10S expression vector (29). MLLΔT contains MLL truncated in the region where translocation breakpoints in MLL commonly occur. MLL3AT encodes the amino-terminal 410 amino acids of MLL and includes all three AT hook motifs of MLL. MLL2AT encodes the amino-terminal 312 amino acids of MLL and contains the two amino-terminal AT hook motifs. All constructs contain an amino-terminal FLAG epitope tag. AT, AT hook motifs; trx, short regions of homology to Trithorax; SNL-1 and SNL-2, subnuclear localization domains 1 and 2 as defined by Yano et al. (33); DMT, DNA methyltransferase homology region.

Figure 2

Immunofluorescent staining of U937T cells expressing MLL-AF9 and deletion mutants MLL3AT and MLL2AT, as well as the U937T parent. Cells were induced by tetracycline withdrawal for 48 hr and were stained with M2 FLAG monoclonal antibody followed by goat anti-mouse Alexa 488 conjugate. MLL-AF9 and MLL3AT have a predominantly punctate subnuclear distribution (green background with bright green/white spots), whereas MLL2AT is uniformly distributed in the nucleus (uniform green nuclear staining). (×1,000.)

Figure 3

Western blot analysis of expression of MLL fusion genes and deletion mutants in U937T cells. Cells were induced for 48 hr in tetracycline-free medium. Cell extracts were prepared, run on 6% (MLLΔT, MLL-AF9; A) or 12% (MLL2AT, MLL3AT; B) polyacrylamide/Tris–glycine gels, and blotted to nitrocellulose, and proteins were detected with M2 anti-FLAG monoclonal antibody.

Figure 4

Inhibition of cell growth by MLL-AF9 and deletion mutants as assessed by counting cells that exclude trypan blue. In the presence of tetracycline (+TET), the clones have roughly comparable growth rates. When tetracycline is removed (−TET), the growth of MLL-AF9-, MLL3AT-, and MLL2AT-expressing clones is markedly inhibited.

Figure 5

Cell cycle arrest and cell death in MLL-AF9-expressing cells. Propidium iodide cell cycle analysis was performed on cells cultured at the same initial density (3 × 10⁵ cells per ml) for 72 hr in the presence (Left) or absence (Right) of tetracycline. Cell cycle progression was unaffected by tetracycline withdrawal in parental U937T cells. Expression of MLL-AF9 was associated with marked cell death as evidenced by the large sub-2N population. The vast majority of cells in these cultures were morphologically degenerated and not viable as assessed by trypan blue exclusion. The viable population in these cultures was composed of small numbers of macrophages (see Fig. 6). Expression of the amino-terminal portion of MLL containing either 3 or 2 AT hook motifs (MLL3AT or MLL2AT, respectively) results in cell cycle arrest in G₀/G₁ without appreciable cell death. Morphologically and immunophenotypically these cells showed evidence of partial differentiation toward macrophages (Figs. 6 and 7).

Figure 6

Induction of macrophage differentiation in U937T cells. Wright–Giemsa-stained Cytospin preparation of U937T clones cultured for 72 hr in the presence (Left) or absence (Right) of tetracycline. (×400.) The morphology of the parental cell line U937T (Top) is unaffected by tetracycline withdrawal. Induction of MLL-AF9 expression in representative clone B37 results in differentiation into macrophages accompanied by apoptotic cell death. Similar results were seen with MLLΔT. Induction of expression of the amino-terminal portion of MLL containing 2 AT hook motifs (MLL2AT; Bottom) results in partial differentiation into macrophages as evidenced by increased amount of cytoplasm and less conspicuous nucleoli (clone C15).

Figure 7

The two left columns show induction of CD11b expression by MLL-AF9 and deletion mutants in U937T cells. The two right columns show induction of CD14 expression by MLL-AF9 but not MLL3AT or MLL2AT in U937T cells.

Figure 8

Western blot analysis of time course of p21^Cip1 and p27^Kip1 induction in MLL3AT clone. Equal quantities of cells were grown in the presence (+TET) or absence (−TET) of tetracycline for the indicated times, and whole-cell lysates were fractionated on SDS/15% polyacrylamide gels. Similar results were seen in three independent clones.