IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

Abstract

In the accompanying paper (1) in situ hybridization of globin DNA copy (cDNA) to fetal liver was used to elucidate the process of globin messenger RNA (mRNA) formation in erythroid precursor cells. The essential conclusions were also confirmed by conventional hybridization studies. In the present paper, the same dual approach is applied to a different experimental system in which hemoglobin synthesis in Friend virus-transformed mouse cells is induced by treatment with dimethylsulfoxide (DMSO) (2). The results obtained by in situ and conventional hybridization agree in showing that there is a minimum period of treatment with DMSO required in order that globin mRNA and hemoglobin may accumulate. M A T E R I A L S AND METHODS Cell CultureFriend cells (clone 707) and lymphoma cells (L 5178Y) were grown in Ham's FI2 medium (Flow Laboratories, Irvine, Scotland), supplemented with minimal essential medium amino acids, penicillin (60 U/ml), and 10-15% horse serum and buffered with HEPES or bicarbonate. LS cells were grown in Eagle's minimal essential medium plus 5% calf serum.All other methods are fully described in the accompanying paper (1). RESULTS

BUDR inhibition of post-DMSO-induced erythroleukaemia cell differentiation in vitro

“…It has previously been demonstrated that BUdR interferes with the DMSOinduced differentiation of Friend leukaemia cells (Ostertag et al, 1973; and that this inhibition results from interference with the accumulation of globin mRNA (Conkie et al, 1974;Preisler et al, 1973). These observations, taken together with those reported here, strongly suggest that these leukaemic cells continue to synthesize and accumulate globin mRNA after removal of DMSO from the culture medium and that iinterference with this process inhibits differentiation during secondary culture in DMSO-free medium.…”

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

“…Interestingly, some other proteins implicated in oncogenesis can also damage or kill specific cells (21,64,66,72,77 (25). Our preliminary studies suggest that the SFF-PU.1 virus differs from a Gag-Myb-Ets-encoding virus (2,62) 13,1993 stages of leukemic progression could be recapitulated in their cultures. As they reported, we have also observed rapid proliferation and terminal differentiation of Epo-independent CFU-E in cultures infected with polycythemic strains of SFFV (31a).…”

“…VOL. 13,1993 5672 SCHUETZE ET AL. [44,48]), the encoded helper-free viruses amplify by a back-and-forth (ping-pong) process of infection (6,38).…”

Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism.

“…The cell lines used were Hep3B and HepG2 (human hepatoma), MRC5 (human fetal lung fibroblast), 1BR3 (human skin fibroblast), U937 (human monocyte/macrophage), (monkey renal fibroblast), LLC-PK1 (pig renal epithelium), RAEC (rat aortic endothelium), a23 (Chinese hamster lung fibroblast), Kl Cell Cultures and cultured as specified by the supplier. Cells from other sources were a23 (11), RAEC (29), and MEL (30). The only nonadherent cells were MEL and U937.…”