Isolation of growth factor-independent retrovirally-infected tf-i cells

TF-I is a factor-dependent cell line derived from a patient with erythroieukemia, Whereas GM-CSF is required for long-term growth, erythropoietin (Epo) can sustain growth for approximately 7 days. Low levels, of both factors act synergistically to maintain proliferation of TF-1 cells for more than 14 days. Previously we have reported thatTF-t cells infected with a retroviral vector, pBABE neo bearing the human Epo gene, survive for more than 14 days in a low concentration of GM-CSF, without the addition of exogenous Epo (Percy el al.. Leukemia 9: S66, 1995)- Epo was detectable ai low levels in TF-I cell culture supernatants, and Epo and neomycin (neo) gene expression were detected by RT-PCR. After prolonged culture, cell mortality increased and subsequently clones evolved which were growth factorindependent although they remained responsive to GM-CSF, Epo w/as undctectable in (he eel! culture supernatants by biological assay and by radioimmunoassay. RT-PCR showed a 10-fold decrease in Epo gene expression but neo gene expression was unchanged. Addition of Epo antisense oligonucleotides to the cultures did not produce any significant decrease in growth suggesting that the cells had become Epo-independent. The reduction of Epo gene expression may be caused by a gradual loss of function of (he retroviral 5' LTR promoter, resulting in selection for growth factor independence. It is unclear whe(her (he growth factor-independent TF-1 cell mutations occur spontaneously or are caused by random retroviral insertion.