Friend cell differentiation

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... 

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). 

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... 

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. 

Included among other differentiating cell lines which have been established in culture, is the human promyelocytic cell line HL-60, which differentiates into more mature myeloid cells upon treatment with retinoic acid and prostaglandin E] (PGEi). Friend erythroleukemia cells differentiate into hemoglobin-producing cells when treated with either dimethyl sulfoxide, or hexamethylene bis-acetamide. 

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... 

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... 

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. 

The antifungal natural product TSA, originally isolated from a Streptomyces, was found to have reversible biological activity at low nanomolar concentrations. Yoshida and coworkers [4] demonstrated that TSA causes the induction of Friend leukemia cell differentiation as well as inhibition of the cell cycle of normal rat fibroblasts in the Gi and G2 phases. This initial work revealed that at low nanomolar concentrations, TSA induces the accumulation of acetylated histones because of inhibiting HD AC activity within the cell. 

HMG protein-induce erythroleukemia cell differentiation. Friend virus-induced mouse erythroleukemia cells differentiate under the effects of hexamethylene bisacetamide (HMBA) and HMGl protein. Ca" " ionophore induced excess HMGl protein and promoted leukemia cell differentiation. Further inducers of leukemia cell differentiation are HDAC inhibitors h)q)eracetylating N-terminal tails of core histones H3/4 (trichostatin), DMSO, phorbol myristate acetate (PMA), the activator of PKC. ... 

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. 

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... 

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. 

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. 

Bilello JA, Gauri KK, Kuhne J, Warnecke G, Koch G (1982) Induction and inhibition of Friend erythroleukemic cell differentiation by pyrimidine analogs analysis of the requirement for intracellular accumulation and incorporation into DNA. Mol Cell Biol 2 1020-1024... 

A particularly useful system for studies of cell differentiation was found to be murine erythroleukemia cells (Marks and Rifkind, 1978). These proerythroblasts, transformed by the Friend virus complex, can be permanently grown in a cell culture. Exposure to dimethylsulphoxide or a variety of other inducers stimulates MEL cells to make a transition to the more advanced stages of erythroid maturation. An analysis of the differentiated state of individual cells, based on cloning cells in a semi-solid medium, has revealed (Housman et al., 1978) that an irreversible commitment to differentiation occurs as a discrete event at a given time after the application of the inducer. Removal of the inducer prior to this event returns a cell to its initial state, whereas its removal after this event leaves it in the differentiated state. 

Trichostatin (Fig. 17) was the first HDAC inhibitor discovered and one of the first two compounds (SAHA being the other) to enter clinical trials. Trichostatin (TSA) was first isolated in 1976, by Tsuji and Kobayashi from Streptomyces hygroscopicus as an antifungal antibiotic against Trichophyton spp. In the 1980s, it demonstrated the ability to induce differentiation in Friend murine erythroleukemia (MEL) cells and inhibited proliferation. ... 

Yoshida M, Iwamoto Y, Uozumi T, Beeppu T. (1985) Trichostatin C, a new inducer of differentiation of friend leukemic cells. Agric Biol Chem 49 563-565. 

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