Figure 5.

Transduction of MLL fragments with high menin-binding avidity inhibits cell growth. A, schematic of the experimental procedure (see Materials and Methods). B, immunoprecipitation analysis of MLL deletion mutants at 2 and 8 d after GFP sorting. Equal amounts of cell extracts from GFP-positive–sorted BM cells were immunoprecipitated with M2 anti-FLAG monoclonal antibody, and then immunoblotted with anti-FLAG polyclonal antibody. At 8 d postsorting, the expression levels of MLL2–167 were significantly reduced compared with other MLL dominant negative mutants. C, GC analysis on GFP-sorted cells shows that all MLL dominant negative cells profoundly inhibited cell growth, whereas all the other transduced cells proliferated rapidly. Points, means; bars, SD. D, results of methylcellulose colony-forming assay summarized as a percentage of GFP-positive CFUs obtained from 2 × 10⁴ transduced cells 4 d after plating. MLL-AF9–transformed cells transduced with MLL2–167, MLL2–62, or MLL Δ35–103 fragments with high menin-binding avidity show a highly reduced percentage of GFP-positive colonies, whereas for MLL2–44 dominant negative cells, the reduction was ~50% compared with MLL fragments with weak or no interaction with menin. A representative of three independent experiments. Bars, SD. E, flow cytometric analysis of GFP signal of MLL-AF9–transformed cells after MLL fragment transduction and GFP sorting. The percentage of GFP-positive cells at 4 and 11 d after cell sorting. Expression of GFP in MLL2–167, MLL2–62, or MLL Δ35–103 is rapidly down-regulated or outcompeted by GFP-negative cells. In contrast, equally high and stable percentages of GFP-positive cells are seen within all the remaining transductions.