The role of intracisternal A-type particles in the evolution of factor-independent murine hematopoietic cell lines.
Cocultivation of a clonal factor-dependent hematopoietic cell line (FDC-P1JL26) with an irradiated bone marrow stromal cell line (D2XRII) significantly increased the frequency of isolation of factor-independent subclones. Eight out of nine factor-independent subclonal lines showed expression of IL-3, GM-CSF or both cytokine mRNAs by reverse-transcription polymerase chain reaction (RT-PCR) and seven of these expressed biologically active GM-CSF or IL-3. In three cell lines that synthesized biologically active IL-3 (FIJ1, FIJ4D and FIJ10D) insertion of an IAP sequence into the IL-3 gene was detected by PCR analysis and the insertions were confirmed by DNA sequence analysis of PCR or RT-PCR fragments. In the four cell lines in which no IL-3 expression was detected no IAP insertions were detected. Rearrangements of the GM-CSF gene were detected in three factor-independent cell lines and an insertion of an IAP into the GM-CSF gene was confirmed by DNA sequence analysis of PCR fragments. In contrast to results with IL-3, insertion of an IAP into the GM-CSF gene did not correlate with GM-CSF expression. In one cell line that contained an IAP insertion into the GM-CSF gene, no GM-CSF was detected by biological assay nor by RT-PCR. Retrotransposition of IAPs may be responsible for the emergence of factor-independent cells in our cocultivation system and other IAP insertions may prove to be responsible for the factor-independent phenotype seen in the non-autocrine factor-independent cell line, FI7CL2.
Pogue-Geile
K L
KL
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, and University of Pittsburgh Medical Center, PA 15213, USA.
Gott
J A
JA
Greenberger
J S
JS
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002999:Clone Cells; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D017931:DNA Primers; D007426:Genes, Intracisternal A-Particle / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology* / Q000276:immunology; D007377:Interleukin-3 / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology
D017077:Culture Media, Conditioned; D017931:DNA Primers; D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor