SYNERGY BETWEEN AUUUA MOTIF DISRUPTION AND ENHANCER INSERTION RESULTSIN AUTOCRINE TRANSFORMATION OF INTERLEUKIN-3-DEPENDENT HEMATOPOIETIC-CELLS

Previously, we characterized the transposition of an intracisternal ty pe A particle (IAP) to the 3' untranslated region (UTR) of the interle ukin-3 (IL-3) gene, which displaced two of the six AUUUA motifs associ ated with mRNA stability in an IL-3-secreting clone. To determine whet her this rearrangement was involved in the autocrine transformation of the parental IL-3-dependent FL5.12 cell line, the germline (gIL-3) an d rearranged IL-3 (rIL-3) genes were isolated and subcloned into a gen e transfer vector. Moreover, the IAP-long terminal repeat (LTR) and th e IL-3 3'UTR AUUUA motifs were deleted (rIL-3 + Delta LTR and gIL-3 Delta AUUUA) in some IL-3 constructs to ascertain their role in the tr ansformation process. The IAP-LTR was also added to these constructs ( rIL-3 + Delta LTR + IAP-LTR, gIL-3 + Delta AUUUA + IAP-LTR, and gIL-3 + IAP-LTR), to determine whether it was necessary for autocrine transf ormation. The ability of the modified IL-3 genes to abrogate the IL-3 dependency of FL5.12 cells had the following rank order: rIL-3 was gre ater than rIL-3 + Delta LTR + IAP-LTR, which was greater than gIL-3 Delta AUUUA + IAP-LTR, which was greater than gIL-3 + Delta AUUUA, whi ch was equal to rIL-3 + Delta LTR, which was greater than gIL-3. The h alf-life of IL-3 mRNA was 20-fold longer in cells containing a mutated as opposed to a wild-type AUUUA region. All of the factor-independent cells that expressed the IL-3 transgenes secreted IL-3 and were tumor igenic after injection into BALB/c nude mice. These results indicated that two events could synergize in the autocrine transformation of hem atopoietic cells: (1) addition of a transcriptional enhancer present i n a retroviral LTR, and (2) disruption of an mRNA stability region. (C ) 1995 by The American Society of Hematology.