Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants.
Retroviral integration mutagenesis and treatment with the frameshift mutagen ICR191 were used to transform v-H-ras expressing PB-3c cells to interleukin-3 (IL-3) independence. Six clones displayed viral integrations into the 3' region of the IL-3 gene thus acting post-transcriptionally by disrupting the AU-rich instability element. Two clones contained reverse orientation integration into the raf-1 gene revealing an enhancer insertion mechanism. Growth by this mechanism was sensitive to the Raf-1 inhibitor BAY 43-9006 and the Mek inhibitor U0126. Following treatment with ICR191, IL-3-independent clones were recovered and studied by cell fusion. With 21/22 clones, IL-3 independence resulted from a recessive mechanism as cellular hybrids with parental cells reverted to IL-3 dependence. Recessive clone D2c displayed increased phospho-Erk1/2 levels and was growth sensitive to U0126, but not to BAY43-9006. The single dominant clone, D5a, showed no signs of mitogen-activated protein kinases pathway activation but displayed constitutive phosphorylation of Stat5. We conclude that PB-3c has several options to acquire IL-3 growth autonomy involving transcriptional or post-transcriptional mechanisms affecting the distal regulators Erk or Stat5. The reported panel of independent dominant and recessive transformants should provide a useful tool for inhibitor profiling.
Kiser
K F
KF
Institute for Medical Microbiology, Department of Clinical-Biological Sciences, University of Basel, Basel, Switzerland.
Colombi
M
M
Moroni
C
C
D000585:Aminacrine / Q000031:analogs & derivatives / Q000494:pharmacology; D001557:Benzenesulfonates / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002999:Clone Cells / Q000166:cytology*; D016368:Frameshift Mutation / Q000187:drug effects; D005799:Genes, Dominant; D005808:Genes, Recessive; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics* / Q000502:physiology*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D016254:Mutagenesis, Insertional; D014411:Neoplastic Stem Cells / Q000166:cytology; D009536:Niacinamide / Q000031:analogs & derivatives; D009570:Nitriles / Q000494:pharmacology; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology; D010671:Phenylurea Compounds; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000378:metabolism; D000077157:Sorafenib; D014162:Transfection
D001557:Benzenesulfonates; D002070:Butadienes; D007377:Interleukin-3; D009570:Nitriles; D009588:Nitrogen Mustard Compounds; D010671:Phenylurea Compounds; D011725:Pyridines; D050799:STAT5 Transcription Factor; C113580:U 0126; D009536:Niacinamide; D000585:Aminacrine; C004294:acridine half-mustard; D000077157:Sorafenib; D019908:Proto-Oncogene Proteins c-raf; D020930:MAP Kinase Kinase Kinases