title: Synergy between AUUUA motif disruption and enhancer insertion results in autocrine transformation of interleukin-3-dependent hematopoietic cells.
abstract: Previously, we characterized the transposition of an intracisternal type A particle (IAP) to the 3' untranslated region (UTR) of the interleukin-3 (IL-3) gene, which displaced two of the six AUUUA motifs associated with mRNA stability in an IL-3-secreting clone. To determine whether this rearrangement was involved in the autocrine transformation of the parental IL-3-dependent FL5.12 cell line, the germline (gIL-3) and rearranged IL-3 (rIL-3) genes were isolated and subcloned into a gene transfer vector. Moreover, the IAP-long terminal repeat (LTR) and the 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 transformation 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 transformation. 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 greater 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, which was equal to rIL-3 + delta LTR, which was greater than gIL-3. The half-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 tumorigenic after injection into BALB/c nude mice. These results indicated that two events could synergize in the autocrine transformation of hematopoietic cells: (1) addition of a transcriptional enhancer present in a retroviral LTR, and (2) disruption of an mRNA stability region.
authors.affiliation: Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Mayo
authors.forename: X Y
authors.initials: XY
authors.lastname: Wang
authors.forename: P A
authors.initials: PA
authors.lastname: Algate
authors.forename: G F
authors.initials: GF
authors.lastname: Arana
authors.forename: P E
authors.initials: PE
authors.lastname: Hoyle
authors.forename: L S
authors.initials: LS
authors.lastname: Steelman
authors.forename: J A
authors.initials: JA
authors.lastname: McCubrey
mesh_terms: D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D003043:Cocarcinogenesis; D004742:Enhancer Elements, Genetic; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D007377:Interleukin-3; D011994:Recombinant Proteins