The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials

Abstract

The t(1;19) translocation that characterizes 25% of pediatric pre-B cell acute lymphoblastic leukemias (pre-B ALL) produces a chimeric gene, joining 5' sequences that encode a transcriptional activator domain of E2A with 3' sequences that, in part, encode a homeo box domain of a new gene called pbx1. Two E2A-pbx1 transcripts have been cloned. They encode the putative fusion proteins, p85^(E2A-Pbx1) and p77^(E2A-Pbx1), which differ in Pbx1 sequences alone, containing unique carboxyl termini whose sequences diverge after the Pbx1 homeo box. In this study, an antiserum to Pbx1 was used to investigate the identity and abundance of E2A-Pbx1 fusion proteins in both the pre-B ALL cell line, 697, and in cryopreserved leukemic bone marrow cells, obtained from six children with t(1;19)-positive pre-B ALL. Five species of E2A-Pbx1 proteins were identified in all cells containing t(1;19), two of which were indistinguishable from in vitro-translated p85^(E2A-Pbx1) and p77^(E2A-Pbx1). To assess the biological properties of p85^(E2A-Pbx1) and p77^(E2A-Pbx1) in fibroblasts, the cDNAs encoding these proteins were cloned into retroviral vectors, and each was introduced into NIH-3T3 cells. Both p85^(E2A-Pbx1) and p77^(E2A-Pbx1) are localized in the nucleus, and expression of either resulted in malignant conversion of NIH-3T3 cells as assayed by tumor formation in nude mice. When scored by focus formation, density-independent growth, and growth in agar assays, p77^(E2A-Pbx1) was a much more potent transforming protein than was p85^(E2A-Pbx1). Because subtle mutations in p85^(E2A-Pbx1) converted its transforming activity into that of p77^(E2A-Pbx1), we suggest that a sequence within the unique carboxyl terminus of p85^(E2A-Pbx1) serves to negatively regulate its biochemical activity.

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