Friend cell

In the absence of inducer, Friend cells divide normally and frequently but in the presence of inducer, although a tenfold increase in cell number may occur cell viability is markedly reduced and a terminally differentiated, non-dividing cell population is produced. 

MEL cells grow very rapidly and require subculture, by dilution (1 to 10) at least every other day to maintain viability. They can be 

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Table I represents the sequence changes that have been identified in H2A variants. The ratio of the different variants changes from species to species. Furthermore, the ratio H2A1/H2AI changes from 3 1 to 1 1 in normal cells or Friend cells after proliferation induced by DMSO treatment (Blankstein and Levy, 1976).

Nonphysiological compounds have also been described as influencing the overall metabolism of sialic acid. Administration of ethanol (2 g/kg) to rats significantly decreases the sialic acid content of brain tissue.246 Convulsions induced by pentylenetetrazole (6,7,8,9-tetrahy-dro-5/f-tetrazoloazepine) are accompanied by a diminution in the rate of biosynthesis of polysialogangliosicles GT, and GQn, in rat brain.227 Such ManNAc analogs as 2-acetamido-l,3,4,6-tetra-0-acetyl-2-deoxy-D-mannopyranose or the 2-(trifluoroacetamido) derivative lead to a marked lowering of the incorporation of radioactivity from labelled ManNAc into glycoconjugate sialic acids of murine, erythroleukemia (Friend) cells.247... 

Several other cultured cell lines were affected by butyrate (5). These included rat glial C6 cells (12-fold increase) and Friend erythroleukemic cells (4-fold increase). The increase in choleragen receptors in Friend cells was also time dependent (Table III). In addition, butyrate appeared to be specific dimethyl-sulfoxide (DMSO) induces erythroid differentiation (23) as does butyrate (24) but it did not cause an increase in toxin receptors (Table 111)7... 

The above results indicate that the increased choleragen binding to butyrate-treated HeLa cells is associated with increased GMl content. This was confirmed with Friend erythroleu-kemic cells (Table VII). Untreated Friend cells have measurable... 

Friend cells are murine, virus-transformed, erythroleukaemic (MEL) cells which grow in culture in suspension and exhibit a limited degree of differentiation along the erythroid line (Friend et al., 1966 Patuleia and Friend, 1967). The target cell for the Friend... 

J.D. Laskey, P. Ponka, H.M. Schulman (1986). Control of heme synthesis during friend cell differentiation role of iron and transferrin. J. Cell Physiol., 129, 185-192. 

Brae T, Ebisuzaki K. Inhibitors of poly(ADP-ribose) polymerase prevent Friend cell differentiation. In Althaus FR, Hilz H, Shall S, eds. ADP-Ribosylation of Proteins. Berlin-Heidelberg-New York-Tokyo Springer, 1985 446-452. 

Inhibitors of Poly(ADP-Ribose) Polymerase Prevent Friend Cell Differentiation... 

Friend et al. [1] made the striking observation that dimethyl sulfoxide (DMSO)-treated Friend erythroleukemia cells (FEE) differentiate in vitro. Subsequently, a number of other chemicals, including butyric acid,hypoxanthine andhexamethylene bis-acetamide were shown to induce Friend cells [2], These inducers appear to remove a block in the differentiation process but the mechanisms involved are unknown. The finding that benzamide and nicotinamide induced Friend cells [3, 4] suggested that since both compounds were inhibitors of poly(ADP-ribose) polymerase, poly(ADP-ribosylation) may have a role in the differentiation process. Furthermore, since poly(ADP-ribose) polymerase requires DNA strand breaks for activity [5], these observations implicated DNA strand breaks in FEE differentiation. 

The possible involvement of DNA strand breaks and poly(ADP-ribosylation) reactions has been indicated in other differentiating cell types such as myoblasts and lymphocytes [6-8] as well as in DNA repair [9]. However, the existence of DNA strand breaks in differentiating Friend cells [10, 11] is controversial since recent studies have shown that induction does not result in detectable strand breaks [12-15]. 

Our studies have been concerned with the effect of inhibitors of poly(ADP-ribose) polymerase on the differentiation of Friend cells. We have also investigated the question of DNA strand breaks, particularly as it relates to poly(ADP-ribose) polymerase inhibitors and DNA repair. 

Logarithmically growing Friend cells were labeled with [ C]-thymidine (0.03 juCi ml ) for 16 h, then diluted into fresh medium containing test chemicals. Subsequently these cells were removed at various times following the treatment and analyzed for DNA strand breaks using the alkaline elution method [18]. 

Since these results appear to contradict those reported previously [3, 4], we reexamined the effects of benzamide on Friend cell differentiation. As shown previously [3] at higher concentrations, benzamide was a weak inducer (Table 2). However, at concentrations which did not inhibit cell growth, benzamide inhibited induction of FEL differentiation (Table 2). 

These results indicate that some inhibitors of poly(ADP-ribose) polymerase also function as inhibitors of differentiation of Friend cells. Since some models of differentiation have invoked DNA rearrangements [8] and since DNA strand breaks are required for poly(ADP-ribose) polymerase activity [5], we examined the relationship of inducers of differentiation and poly(ADP-ribose) polymerase inhibitors for DNA... 

Table 1. Inhibitors of poly(ADP-ribose) polymerase prevent Friend cell differentiation ...

Friend cell differentiation was examined by benzidine staining 5 days after culturing of the cells with inducers and inhibitors... 

If poly(ADP-ribose) polymerase was involved in differentiation, would activation of the polymerase by DNA damaging agents alter the course of differentiation The alkylating agent, dimethyl sulfate, caused DNA single-strand breaks in Friend cells which were repaired (Fig. 2). Addition of 10 mM 3AB potentiated the DNA damage at each time-point examined. To confirm the observations that dimethyl sulfate increased the number of benzidine-positive cells (about 3% compared to 1% for controls), hemoglobin was also measured spectrophotometrically from cell extracts. 

Fig. 1. Dimethyl sulfoxide does not induce DNA single-strand breaks in Friend cells. Friend cells (10 ), prelabeled with C]-thymidine, were treated with 1.5% DMSO 24 h plus ( ) and minus...

SAB and analyzed by alkaline elution. Control cells had no treatment (O), and 100 rad of gamma rays from Cesium ( ). The elution profiles of Friend cell DNA treated DMSO for 0, 2, 4, 6, 8 and 12 h were not different from the controls... 

Fig. 2. Dimethyl sulfate causes DNA single-strand breaks in Friend cells that are potentiated by SAB. [ Cl-thymidine labeled Friend cells, treated with 50 yM dimethyl sulfate for 30 min, were centrifuged and resuspended in fresh media with or without SAB. Samples were removed at 0, S and 7 h after treatment and analyzed by alkaline elution. Numbers refer to h and + to presence of SAB...

Friend cells were treated with 50 yM dimethyl sulfate (DMS) for 30 min 10 mM SAB. Parallel cultures were induced with 1.5% DMSO and 10 mM SAB. After 5 days the cells were counted, lysed and centrifuged. The supernatants were analyzed for hemoglobin content. Uninduced cells contained 3.8 yg hemoglobin 10" cells... 

For example, since benzamide and nicotinamide induce Friend cell differentiation, it has been suggested that induction resulted from the inhibition of poly(ADP-ribose) polymerase [3,4]. However, these investigators noted that the inhibition of poly(ADP-ribose) polymerase was not a prerequisite of inducers since N -methylnicotinamide, a strong inducer, was not an inhibitor of this enzyme in vitro [3]. We have found that 3 AB inhibited FEL differentiation while benzamide had dual effects, inducing at high concentrations and blocking differentiation at low concentrations (see Results). 

These observations could be understood if two different reactions were involved in prevention and induction of differentiation. Benzamide at high concentrations might function as an inducer of Friend cell differentiation based on its polar-planar structure [20]. Inducers are typically most active at high concentrations. At lower concentrations, the primary effect of benzamide might be through its inhibition of poly(ADP-ribose) polymerase [21]. The dual effect of induction and inhibition of differentiation is a property of other FEL inducers such as bromodeoxyuridine [22]. 

Inhibitors of poly(ADP-ribose) polymerase prevent differentiation in other cells [6, 7]. The previous reports showing that benzamide was an inducer of FEL differentiation suggested that poly(ADP-ribose) polymerase might have a different type of role in Friend cells. However, our results show that Friend cell differentiation is not exceptional and that common mechanisms may be involved in differentiation of diverse cell types. 

If poly(ADP-ribose) polymerase was involved in the differentiation of Friend cells, then the role of DNA strand breaks must be examined since strand breaks modulate enzyme activity [5]. In a provocative model for differentiation, it has been suggested that DNA transposition reactions accompanied by DNA strand breaks are involved in gene activation [8]. In turn, these DNA strand breaks might trigger ADP-ribosylation reactions. We have found that treatment of Friend cells with dimethyl sulfate, which causes strand breaks, weakly induces FEL differentiation. However, the addition of 3AB blocked the differentiation process despite the increase in strand breaks. Therefore, DNA strand breaks by themselves do not stimulate differentiation but they may contribute to the differentiation process by activating poly(ADP-ribose) polymerase. 

Several studies have shown that inducers of Friend cells increased DNA strand breaks when assayed by sucrose density gradient centrifugation [10, 11]. However, strand breaks were not detected when alternate analytical methods were used [12-15]. Our study shows that the treatment of Friend cells with DMSO, sodium butyrate or N -methylnicotinamide separately or with 3AB did not induce DNA strand breaks. If DNA strand breaks are involved in differentiation of Friend cells, these breaks are either very transient or few in number. 

In mouse L cells, addition of one or two minor species of mammalian leucyl-tRNA were shown to be sufficient to restore translation (27). A similar conclusion was reached with yeast tRNA (28), even with poly(U,C) as template. The type of tRNA to be added appears to depend on the mRNA used (25, 27) and also on the extract Friend cells were reported to require lysyl-tRNA (29). These tRNA species were, however, not absent from interferon-treated cell extracts (24, 27, 50), l> u.t had to be added in excess over what is normally needed. A decreased charging of leu-tRNA or an increased deacylation were reported (5I, 32). Prolonged pre-incubation for 1-2 h of the extracts can increase the interferon-induced elongation block reversed by tRNA (55)-have recently observed that ATP is required to activate the translational inhibition seen in interferon-treated cell extracts without dsRNA. This could be shown by comparing extracts freed of endogenous mRNA activity either by micrococcal nuclease (54) or by pre-incubation with ATP. Table 5 shows that tRNA reversible-inhibition developed when ATP was present. It is not clear yet whether the protein kinase, or E and F may play a role in this inhibition without dsRNA. We also observed an inhibitor of leu-tRNA charging in ribosomal extracts of interferon-treated cells (Schmidt, A. and Revel, M., impublished). 

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