A new group of potent inducers of differentiation in murine erythroleukemia cells.

Abstract

This report identifies a group of compounds, polymethylene bisacetamides (acetylated diamines), which are potent inducers of erythroid differentiation in murine erythroleukemia cells. A known inducing agent, N-methylacetamide, was dimerized through varying numbers of methylenes in an attempt to increase the local effective concentration at adjacent target sites. The simple dimer was no more effective than N-methylacetamide alone; introduction of five to eight methylenes between acetamide groups substantially increased the effectiveness of these compounds. The hexamethylene bisacetamide was active between 0.5 mM and 5 mM; the percentage of cells induced and the rate at which they were recruited to differentiation was dependent upon the concentration of inducer within this range. At 5 mM hexamethylene bisacetamide essentially the entire population (>99%) was induced to a pathway of erythroid differentiation which was greater differentiation of the cultured cells than with any inducer yet tested.The murine erythroleukemia cell line established by Friend (1) shows a low percentage (<1%) of spontaneous erythroid differentiation; inclusion of 280 mM dimethylsulfoxide (Me2SO) in the culture medium results in an increased number of cells synthesizing hemoglobin (2). The program of erythroid differentiation induced by Me2SO includes characteristic changes in morphology (2), the appearance of globin mRNA (3, 4), an increase in iron uptake and heme synthesis (2), and the appearance of erythroid membrane antigens (5).Dimethylsulfoxide is not unique in this action in that several other compounds, having in common a hydrophilic or polar group covalently joined to a hydrophobic group, also induce differentiation in these cells (6, 7). With the knowledge that several types of organic compounds are active as inducing agents, we attempted to find a compound that would be better than those already investigated in terms of both lower effective concentrations and the percentage of cells that differentiate. One approach to this problem was to test dimers of a known inducing agent in order to increase the local effective concentration of inducer at adjacent target sites. The inducing agent N-methylacetamide was dimerized by linkage at nitrogen through a varying number of methylene groups. We have identified a group of compounds, polymethylene bisacetamides (acetylated diamines), that are potent inducers, in that, at concentrations well below the optimal for Me2SO, essentially the entire culture (>99%) of murine erythroleukemia cells differentiate. These compounds are structurally related to certain naturally occurring diamines, which are shown to be weak inducers. MATERIALS AND METHODSStrain 745A, a murine erythroleukemia cell line, was provided by Dr. Charlotte Friend, and maintained in continuous culture in our laboratory for 3 years as previously described (8). The extent of differentiation in a culture was determined by either of two methods. The percentage of hemoglobin-containing cells was determined by the benzidine rea...

Inhibition of Friend erythroleukaemia-cell tumours in vivo by a synthetic analogue of prostaglandin E2

“…Murine erythroleukaemia cells infected with the Friend virus (FLC) differentiate in vitro from a proerythroblast-like to a normoblast-like stage producing haemoglobin when they are treated with dimethylsulfoxide (DMSO) or other inducers (Friend et al, 1971;Leder & Leder, 1975;Reuben et al, 1976;Ebert et al, 1976). Chemically induced differentiation is promptly and spontaneously reversed when the inducing agents are removed from the culture medium (Friend et al, 1971).…”

Heterogeneous response to differentiation induction with different polar compounds in a clonal rat rhabdomyosarcoma cell line (BA-HAN-1C)

“…Therefore, we were looking for other effective inducers of differentiation, possibly having modes of action other than those of retinoic acid. Polar compounds such as dimethylsulphoxide (DMSO) (Friend et al, 1971;Collins et al, 1978;Tarella et al, 1982;Tsao et al, 1982), hexamethylene bisacetamide (HMBA) (Reuben et al, 1976;Ramsay et al, 1986;Mansfield et al, 1988;Snyder et al, 1988), sodium butyrate (NaBut) (Prasad & Sinha, 1976;Dexter et al, 1981;Augeron & Laboisse, 1984;Nordenberg et al, 1987;Lee et al, 1988), and N-monomethylformamide (NMF) (Dexter et al, 1982;Dibner et al, 1985;Cordeiro & Savarese, 1986;Iwakawa et al, 1987) have only been sporadically described (Dexter 1977;Garvin et al, 1986). Furthermore, DMSO, HMBA and NaBut have been reported to inhibit terminal differentiation and myotube-formation in non-tumorigenic myoblast cell lines (Miranda et al, 1978;Blau & Epstein, 1979;Fiszman et al, 1980;Endo & Nadal-Ginard, 1987).…”

The effect of hexamethylene‐bis‐acetamide on multiparameter analysis of friend leukemia cells

“…The induction of a differentiated state was associated with a decrease in cell volume and an increase of fluorescence anFriend leukemia cells (FLC) have been used as a model for the study of erythroid differentiation (7). They can be induced to differentiate and synthesize hemoglobin in vitro when grown in the presence of a variety of structurally unrelated agents (2, 3, 11,14,18,19,22). Among the agents particularly active, hexamethylene-bis-acetamide (HMBA) is one of the most potent inducers (18).…”

“…They can be induced to differentiate and synthesize hemoglobin in vitro when grown in the presence of a variety of structurally unrelated agents (2, 3, 11,14,18,19,22). Among the agents particularly active, hexamethylene-bis-acetamide (HMBA) is one of the most potent inducers (18). The expression of globin genes during cell differentiation can be suppressed by other unrelated agents (16,20,23,29).…”