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 disru...

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Bibliographic Details
Published in:	Oncogene Vol. 25; no. 50; pp. 6595 - 6603
Main Authors:	KISER, K. F, COLOMBI, M, MORONI, C
Format:	Journal Article
Language:	English
Published:	Basingstoke Nature Publishing 26-10-2006 Nature Publishing Group
Subjects:	Aminacrine - analogs & derivatives Aminacrine - pharmacology Autonomy Benzenesulfonates - pharmacology Biological and medical sciences Butadienes - pharmacology Cell differentiation, maturation, development, hematopoiesis Cell fusion Cell physiology Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes Cell Transformation, Neoplastic - genetics Clone Cells - cytology Cloning Cytokines Extracellular signal-regulated kinase Frameshift Mutation - drug effects Fundamental and applied biological sciences. Psychology Gene expression Genes, Dominant Genes, Recessive H-Ras protein Hematopoiesis - genetics Humans Hybrids Insertion Integration Interleukin 3 Interleukin-3 - genetics Interleukin-3 - physiology Kinases MAP Kinase Kinase Kinases - metabolism Medical treatment Molecular and cellular biology Mutagenesis Mutagenesis, Insertional Mutation Neoplastic Stem Cells - cytology Niacinamide - analogs & derivatives Nitriles - pharmacology Nitrogen Mustard Compounds - pharmacology Phenylurea Compounds Phosphorylation Post-transcription Protein kinase Proto-Oncogene Proteins c-raf - metabolism Pyridines - pharmacology Raf-1 gene Retroviridae - genetics Stat5 protein STAT5 Transcription Factor - metabolism Transfection Viruses Characterization stat Hematopoiesis Interleukin 3 ICR191 Cytokine Isolation Raf-1 mast cells Carcinogenesis mutagenesis
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0950-9232 1476-5594
DOI:	10.1038/sj.onc.1209673