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.

The amino terminus targets the mixed lineage leukemia (MLL) protein to the nucleolus, nuclear matrix and mitotic chromosomal scaffolds

“…The pUHD/MLL-AF4 and pUHD/MLL3AT vectors were constructed by subcloning the FLAG-tagged cDNAs into the BstXI and XhoI cloning sites of pUHD10B/X, a modified version of pUHD10S expression vector. 33 …”

“…9,10,13 MLL3AT encodes the amino terminal 410 amino acids of MLL and includes three AT-hook motifs that share homology with minor-groove DNA-binding proteins. 29,33 Both constructs contain an amino terminal FLAG epitope tag. SNL-1 and SNL-2, speckle nuclear localization domains 1 and 2; 53 trx, short regions of homology to Trithorax Drosophila protein; 53 MT, DNA methyltransferase homology region; 9,30,31 NLS, nuclear localization signal of AF4; 10 PHD, plant homeodomain zinc-fingers; [13][14][15][16][17][18] SET, domain of homology to regions of Su(var)3-9, E(z), and Trx Drosophila proteins.…”

“…The vector encodes the tTA, whose expression is autoregulated by tet operator sequences in the promoter (pTet). U937T clones conditionally expressing MLL3AT 33 were routinely maintained in RPMI 1640 medium supplemented with 10% FCS (RPMI-F), 0.5 g/ml puromycin, 0.4 mg/ml Hyg B, 1 g/ml tet. The acute mixed lineage cell lines RS(4;11) 36 and ALL-PO(4;11) 37 and the Jurkat T cell line were maintained in RPMI-F.…”

“…We found that MLL-AF9 is a potent inducer of macrophage differentiation and cell death and that small amino terminal portions of MLL containing two (MLL2AT) or three (MLL3AT) AT-hook motifs are sufficient for G0/G1 cell cycle arrest, p21 Cip1 and p27 Kip1 upregulation, and for promotion of macrophage differentiation. 33 In the current study, immunofluorescence, confocal laser scanning and immunoelectron microscopic techniques were employed to study the nuclear distribution of ectopically expressed epitope-tagged MLL3AT protein and compare it to ectopic MLL-AF4 and endogenous MLL.…”

Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia

“…For example, expression of MLL-AF9 promoted differentiation and cell-cycle arrest in the human U937 monoblastic cell line, and induced the downregulation of Hoxa7 in the murine myeloid 32Dcl3 cell lineFthe opposite to what has been observed in human leukemias, mouse models and in primary human hematopoietic cells. 7,[32][33][34][35] Also, as cell lines are already fully transformed and immortalized, they cannot be utilized to study the early events of leukemogenic initiation and progression that lead to the generation of leukemia stem cells. Together, these limitations underscore the need to study leukemia-associated oncogenes in a more relevant cellular context, namely primary hematopoietic stem and progenitor cell populations.…”