The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.     

Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells

The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-Cl2 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10^?4 M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-Cl2 cells to accumulate 8–9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30–40 hr after DMSO addition. Biliverdin induces 20–40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.     

5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells.

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediated precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 x 10(-4) M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.     

Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.     

Isolation and characterization of murine erythroleukemia cell variants nonresponsive to hemin for the expression of globin genes

Thirty-four cloned lines of mouse erythroleukemia (MEL) cells that showed impairment in hemoglobin sy nthesis induced by hemin were isolated from line 745. Among these lines, 75% showed a reduced or nil response to induction by dimethyl sulfoxide (DMSO) and/or hypoxanthine. Four of these clones (three lines nonresponsive to hemin and one not responsive to either hemin or DMSO) were further characterized for the amount of globin mRNA accumulated in the cytoplasm and for the rate of globin synthesis upon exposure to hemin or DMSO. None of the four were induced by hemin to accumulate globin mRNA and all had a reduced rate of globin synthesis by comparison with control line 745. Two of the four lines had a high uninduced level of cytoplasmic globin mRNA not matched by a corresponding rate of globin synthesis, suggesting that they may accumulate defective globin mRNAs.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

In many types of cells the synthesis of delta-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe-Tf (20 microM). Furthermore, in induced Friend cells 100 microM Fe-SIH stimulated 2-14C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. In contrast, Fe-SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe-SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.     

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.     

Can glutathione-S-transferases function as intracellular heme carriers?

The possibility that glutathione-S-transferases can serve as heme carriers in cells was studied via the following two characteristics: the ability to bind hemin reversibly and the coordination between heme and glutathione-S-transferases level in the cell. two erythroleukemic cell lines that can be induced to synthesize hemoglobin were studied, K-562 and Friend murine erythroleukemia cells. It was found that hemin-associated glutathione-S-transferase tends to lose its native structure as expressed by partial irreversible inhibition of glutathione conjugation activity. In K-562 cells, a small increase in heme synthesis was induced, but under no condition could glutathione-S-transferase be elevated. In addition, introduction of high hemin from without caused large hemoglobin production but did not induce changes in the glutathione-S-transferase content. Dimethyl sulfoxide-induced Friend murine erythroleukemia cells synthesized a large amount of endogenous hemin that had to be transported from the mitochondria for hemoglobin synthesis. Although a concomitant increase in glutathione-S-transferase level (20-40%) was observed, it was only short-lived, unlike hemin, which continued to increase. These data indicate a lack of correlation between glutathione-S-transferase and hemin or hemoglobin levels. Finally, dimethyl sulfoxide-induced cells were treated with succinyl acetone to inhibit heme synthesis. These cells showed the same increased levels and time-dependent pattern of glutathione-S-transferase as untreated cells. A similar phenomenon was observed when different substrates were used to measure the activities of glutathione-S-transferases. These results raise doubts about the possibility of glutathione-S-transferases functioning as heme carriers in cells.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

Stimulation of protein synthesis by hemin in extracts of Friend erythroleukemia cells.

Extracts prepared from Friend erythroleukemia cells were highly active in translating endogenous mRNA and a consistent 2-fold stimulation by hemin was observed. When extracts were treated with micrococcal nuclease and incorporation was dependent on exogenous globin mRNA, there was more significant stimulation by 37.5 micron hemin and greater than 10-fold stimulation by 75 or 150 micron hemin. The effects of hemin were not strikingly different in extracts of dimethyl-sulfoxide-induced or uninduced cells. The results could reflect an effect on initiation of protein synthesis analogous to that in rabbit reticulocytes.     

Erythroid differentiation in a friend erythroleukemic cell x lymphoma hybrid cell line is limited, possibly due to reduced hem levels

Induction of erythroid differentiation has been investigated in a cell hybrid formed between an inducible Friend cell and a lymphoma line (L5178Y) derived from the same strain of mouse (DBA/2). Although globin messenger RNA (mRNA) is induced by DMSO to a level similar to that in the inducible Friend cell parent (about 9 000 molecules/cell) haemoglobin does not accumulate in detectable amounts, nor do morphological changes characteristic of terminal differentiation occur. This failure to accumulate haemoglobin in response to DMSO is due to a reduced rate of globin chain synthesis (6% of total protein synthesis, compared to 25% for the parental Friend cell), and partly to inability of the globin chains synthesized to form tetrameric haemoglobin molecules. Globin chain instability is not the reason why haemoglobin does not accumulate. In comparison, treatment of the hybrid cells with haemin induces about 14% globin synthesis and about 13 000 globin mRNA molecules. These values are somewhat higher than with DMSO. Treatment of hybrid cells with haemin plus DMSO is even more effective; it induces 25% globin synthesis and about 30 000 globin mRNA molecules and terminal differentiation also occurs normally. Whether treated with DMSO or haemin or both, virtually all the globin mRNA molecules seem to be present in polysomes and are therefore presumably in the process of being translated. These results suggest that failure of differentiation in these hybrid cells is due to haem limitation which also prevents the expression of other co-ordinated erythroid functions.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.     

Effects of metalloporphyrins on hemoglobin formation in mouse Friend virus-transformed erythroleukemia cells. Stimulation of heme biosynthesis by cobalt protoporphyrin

Mouse Friend virus-transformed erythroleukemia cells in culture undergo erythroid differentiation when treated with a variety of compounds including iron protoporphyrin IX, i.e. hemin. Exogenous hemin is not only incorporated into hemoglobin in these cells but also stimulates heme biosynthesis (Granick, J. L., and Sassa, S. (1978) J. Biol. Chem. 253, 5402-5406). In this study, we examined whether metalloporphyrins other than hemin can also induce differentiation, and if so, whether they can also be incorporated into hemoglobin. Among eight metalloporphyrins examined in culture of these cells, i.e. Co, Mn, Cu, Mg, Ni, Zn, Sn, and Cd protoporphyrin IX, only Co protoporphyrin (10(-4) M) was found to significantly increase the biosynthesis of heme and hemoglobin. In contrast to hemin-mediated induction of erythroid differentiation, Co protoporphyrin was not incorporated into hemoglobin in Friend cells. These data indicate that Co protoporphyrin induces the formation of heme and hemoglobin in Friend cells and that these increases are due to the enhancement of heme biosynthetic activity.     

Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.     

Transferrin receptors and iron utilization in DMSO-inducible and -uninducible friend erythroleukemia cells

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.     

Effects of succinylacetone on dimethylsulfoxide-mediated induction of heme pathway enzymes in mouse Friend virus-transformed erythroleukemia cells

Heme has been reported to exert a control over its own biosynthesis and to affect the erythroid differentiation process at different sites. In this study, succinylacetone, a powerful inhibitor of δ-aminolevulinic acid dehydrase was used to block heme synthesis and to study the effects of heme depletion on the dimethylsulfoxide (DMSO)-mediated induction of the heme pathway enzymes in Friend virus-transformed erythroleukemia cells. The presence of succinylacetone in the medium during the DMSO treatment (1) potentiates the induction of δ-aminolevulinic acid synthetase (the first enzyme of the pathway) and this effect is reversed by the addition of exogenous hemin; (2) does not affect the induction of δ-aminolevulinic acid dehydrase (the second enzyme); (3) prevents the induction of porphobilinogen deaminase (the third enzyme), since no increase could be detected in either the enzyme activity or the immunoreactive protein and this effect could not be reversed by the addition of exogenous hemin; (4) does not affect the induction of ferrochelatase. The possible role of heme or of intermediate metabolites of the pathway on the induction of these enzymes during the erythroid differentiation process is discussed.