THE ROLE OF INTRACISTERNAL A-TYPE PARTICLES IN THE EVOLUTION OF FACTOR-INDEPENDENT MURINE HEMATOPOIETIC-CELL LINES

Cocultivation of a clonal factor-dependent hematopoletic cell line (FD C-P1JL26) with an irradiated bone marrow stromal cell line (D2XRII) si gnificantly increased the frequency of isolation of factor-independent subclones. Eight out of nine factor-independent subclonal lines showe d expression of IL-3, GM-CSF or bath cytokine mRNAs by reverse-transcr iption polymerase chain reaction (RT-PCR) and seven of these expressed biologically active GM-CSF or IL-3. In three cell lines that synthesi zed 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 i nsertions were confirmed by DNA sequence analysis of PCR or RT-PCR fra gments. In the four cell lines in which no IL-3 expression was detecte d no IAP Insertions were detected. Rearrangements of the GM-CSF gene w ere 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 IA P into the GM-CSF gene did not correlate with GM-CSF expression. In on e cell line that contained an IAP insertion into the GM-CSF gene, no G M-CSF was detected by biological assay nor by RT-PCR. Retrotranspositi on at IAPs may be responsible for the emergence of factor-independent cells in our cocultivation system and other lap insertions may prove t o be responsible for the factor-independent phenotype seen in the nona utocrine factor-independent cell line, FI7C12.