E2A-HLF-MEDIATED CELL-TRANSFORMATION REQUIRES BOTH THE TRANSACTIVATION DOMAINS OF E2A AND THE LEUCINE-ZIPPER DIMERIZATION DOMAIN OF HLF

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potentia l of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resist ance gene. Cells selected for resistance to the neomycin analog G418 f ormed aberrant colonies in monolayer cultures, marked by increased cel l density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF e xpression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to highe r plateau-phase cell densities in reduced-serum conditions than did pa rental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A -HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic pro perties, including anchorage-independent cell growth. Thus, both of th e E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2 A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemoge nesis.