An intracellular factor that induces erythroid differentiation in mouse erythroleukemia (Friend) cells

We have previously shown that in vitro erythroid differentiation of mouse Friend cells is a result of a synergistic action of two distinctive intracellular reactions. We now have evidence that a factor in the cell free extract is involved in one of the reactions. This factor triggers erythroid differentiation when introduced into undifferentiated mouse Friend cells, provided the cells have been briefly exposed to dimethyl sulfoxide. The factor is induced in nonerythroid cells as well following treatment of the cells by agents that affect DNA replication. Cycloheximide inhibited the induction of the factor. The factor, which is in the cytoplasm, was partially purified and proteinaceous. When introduced into the cells the partially purified factor converts 60% to 70% of undifferentiated Friend cells to erythroid cells, at an efficiency almost equivalent to the efficiencies achieved by typical inducing agents. The factor's biochemical characteristics and possible role in erythroid differentiation are also discussed.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

INDUCTION OF ERYTHROID MATURATION BY DIMETHYL SULFOXIDE IN FRIEND LEUKEMIC CELLS

Enhancement of the erythroid maturation in Friend virus-induced leukemic cells has been examined in vitro by the treatment with dimethyl sulfoxide (DMSO). Although the cell growth was inhibited in the medium containing 2% DMSO, many cells remained viable for a week. By the 3rd day of the culture, the cells treated with DMSO became more strongly agglutinated by phytohemagglutinin than the cells incubated without DMSO. Mouse erythrocyte membrane-specific antigens were also detectable at the 4th day. At the 8th day of the culture hemoglobin synthesis was apparently demonstrated in the cells treated with DMSO, which could not be seen in the untreated cells. Maturation or differentiation along the erythroid pathway in Friend leukemic cells by DMSO is discussed on these markers.     

Autocrine differentiation-inhibiting factor (ADIF) from chicken erythroleukemia cells acts on human and mouse early BFU-E erythroid progenitors

tsAEV-LSCC HD3 chicken erythroid cells transformed by the avian erythroblastosis virus (AEV) secrete an autocrine differentiation-inhibiting factor, ADIF, which blocks differentiation without affecting proliferation of the chicken erythroid cells that synthesize and secrete it into the culture medium. The chicken erythroleukemia cell ADIF activity is not restricted to avians. It prevents dimethylsulfoxide (DMSO) from stimulating murine Friend erythroleukemia cells to synthesize hemoglobin. ADIF also blocks erythroid differentiation in normal human and murine bone marrow where it selectively targets the early BFU-E (burst-forming) erythroid precursor cells without affecting the more advanced CFU-E erythroid precursor cells or cells of the different granulocyte-macrophage lineage.     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

Inhibition by 1 alpha,25-dihydroxyvitamin D3 of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Regulation of erythroid differentiation by vitamin D3 derivatives was examined in Friend erythroleukemia cells. After Friend cells were cultured for 5 days with 1.5% dimethyl sulfoxide (DMSO), as much as 70% of the cells became benzidine-positive and the hemoglobin content increased in parallel with the increase of benzidine-positive cells. The DMSO-induced erythroid differentiation was markedly inhibited by concurrent addition of the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent in inhibiting DMSO-induced erythroid differentiation. 1 alpha,25(OH)2D3 alone was totally ineffective in both cell growth and erythroid differentiation. These results together with our previous reports indicate that 1 alpha,25(OH)2D3 is somehow involved not only in myeloid differentiation, but also in erythroid differentiation.     

Subcellular distribution of 2'',5''-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2'-5')(A)n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2'-5')(A)n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2'-5')(A)n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2'-5')(A)n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.     

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.     

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.     

Friend erythroleukemia cell membrane transferrin receptors

We have compared the uptake of transferrin by murine Friend erythroleukemia cells with the uptake of transferrin by murine reticulocytes. Friend cells which had been induced to erythroid differentiation by dimethyl sulfoxide took up transferrin in a manner qualitatively and quantitatively similar to the uptake of transferrin by reticulocytes, while uninduced Friend cells took up only negligible amounts of transferrin. Specific transferrin-binding activity could be demonstrated in detergent extracts of membranes from induced cells and this activity was isolated from membrane extracts by the use of antibody to transferrin. The isolated membrane component(s) with transferrin-binding activity migrated electrophoretically as a single protein on sodium dodecyl sulfate gels and had similar properties to a transferrin-binding protein isolated previously from reticulocytes.     

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.     

Dexamethasone inhibition of DMSO-induced transglutaminase activity and differentiation of leukemic cells

Treatment of the Friend erythroleukemic (FL) cell line GM979 with dimethyl sulfoxide (DMSO) or n-butyric acid induced erythroid differentiation. Transglutaminase (TGase) activity also increased in these treated cells. Glucocortical steroids, i.e., dexamethasone (DEX) and triamcinolone acetonide, when added to the cultured medium, inhibited the DMSO-induced hemoglobin synthesis but not n-butyric acid-induced hemoglobin synthesis. Similarly, these steroids inhibited DMSO-increased TGase activity but not n-butyric acid-increased TGase activity in intact FL cells. Neither the differentiation-inducing agents nor the steroids had any effect on TGase activity when they were directly added to cell lysates. These results support the view that the increase of TGase activity may be related to erythroid differentiation of FL cells and of its possible role of this enzyme in FL cell-induced differentiation.     

Further studies on the biological properties of Friend virus-induced leukemic cells differentiating along the erythrocytic pathway

When Friend virus-induced leukemic cell lines were injected into irradiated hosts after the second radiation dose, the colony-forming unit (CFU) in the recipient spleens per 10⁴ cells was found to be 7-fold higher than the CFU obtained when the second radiation dose had been given shortly after the inoculation of the cells. Serial passage of the cells from the spleen colonies to irradiated hosts resulted in a marked increase of the CFU value, indicating that this cell population was capable of both self-replication and erythroid differentiation. The “f” fraction, which indicates the percentage of the inoculated cells that reach the spleen in the irradiated recipients, was found to be approximately 15%. If the highest CFU value obtained from serial colony-to-colony passages is corrected by this factor, a final cloning efficiency of about 18% is demonstrated. Neither induced plethora nor the administration of erythropoietin (1 u/mouse/for 2 days) appeared to affect the spleen colony-forming ability of the leukemic cells. Erythroid differentiation is not detectable in the transplantable subcutaneous tumors which were used to initiate the tissue culture lines and which also are capable of inducing erythroid spleen clones in irradiated recipients. This lends support to the theory of the influence of “microenvironmental factors” on the fate of stem cells with potential for differentiation.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.     

Proliferative activity and the rate of differentiation of rat bone marrow cells in normal conditions and in early periods of acute radiation sickness

The autoradiographic method was used to study proliferative activity of cells of a myeloid and erythroid compartments of the bone marrow of intact and irradiated (a single exposure of 10 Gy) rats. The proliferative activity and differentiation rate of cells decreased at early times after irradiation. It was shown that giant neutrophils had heavy tracer nuclei, were formed from myeloblasts, being at the most active phase of the S stage at the time of irradiation, and had a very low differentiation rate.     

Co-expression of spectrin and fodrin in friend erythroleukemic cells treated with DMSO

Friend erythroleukemic cells can be used as a model of erythroid cell differentiation with the synthesis of the erythrocyte-specific products hemoglobin and spectrin stimulated by agents such as DMSO. In the present study we investigated the expression of both erythroid spectrin and non-erythroid fodrin in uninduced and DMSO-treated Friend cells. We report that both spectrin and fodrin co-exist at low levels in uninduced Friend cells and both are induced by treatment with DMSO. After longer times both spectrin and fodrin appear to undergo rearrangements into submembranous ‘patches’ and ‘caps’. Although both molecules co-localize in most of these cells, they can be independently immunoprecipitated, suggesting that significant amounts of hybrid molecules are not formed.     

Selective gene mutation in MEL cells.

MEL cells, undergoing erythroid differentiation and parasynchronized by dimethyl sulfoxide (DMSO) induction, were irradiated with a 3-s pulse of UV light at sublethal dose. A large number of clones deficient in different gene functions are found in the progeny of the treated cells, if the pulse irradiation is performed 18-24 h from the start of DMSO induction. Kinetics of thymidine incorporation into DNA show that the period of sensitivity corresponds to the S phase. The results show that the activities of the tested genes are differently affected depending on the exact time of cell irradiation. Maximum percent inhibition of cells not expressing glucose-6-phosphate dehydrogenase (G-6-PD) (70%) is produced by irradiating at 20 h from the start of DMSO induction; 6-phosphogluconate dehydrogenase (6-PGD) (55%), and hypoxanthine (guanine) phosphoribosyltransferase (HPRT) (33%), at 21 h; hemoglobin (50%), at 22 h. The time difference in the sensitivity to UV light is highly reproducible and has been exploited to isolate, with high efficiency, cellular clones deficient in any one of the tested functions. Determinations of enzymatic activities on cell lysates show that the expression of tested genes is actually altered in cells that, on the basis of cytochemical tests, appear unaffected by UV irradiation. While the production of mutant clones is observed only during the S phase of the cell cycle, immediate statistical damage of the cellular DNA is produced at all times of irradiation. This finding excludes that the two types of phenotypic alterations, blocked or altered gene expression, both propagated in the progeny of the cells as clonal properties, may derive from a preferential alteration of those functions during the S phase.