Nuclear chromatin changes during erythroid differentiation of friend virus induced leukemic cells

Friend leukemia (FL) cells grown in the presence of dimethylsulfoxide (DMSO) undergo erythroid differentiation. Acridine orange (AO) binding to DNA and thermal denaturation of DNA in situ were studied in differentiated and non-differentiated FL cells using flow-through cytofluorometry. The differentiated cells bind less AO than do the non-differentiated ones. The difference in AO binding is higher in the spectrum of emission characteristic for AO intercalation (at 530 nm) than for AO stacking (>600 nm) and depends on AO concentration. The difference is abolished after extraction of acid-soluble macromolecules from cells. During stepwise extraction by lowering pH, there is a progressive increase of AO binding to DNA. Most of the increase in AO binding of the non-differentiated cells occurs at pH 2.5-2.0; of the differentiated cells at pH 1.75-1.50. There are differences in sensitivity of DNA in situ to heat-denaturation between differentiated and non-differentiated cells, as evidenced by the variation in height and in position of the melting bands on derivative melting profiles. The changes described suggest that a profound modulation of chromatin structure, perhaps involving altered DNA-histone interactions, occurs during the DMSO-induced erythroid differentiation of FL cells.     

Phenotypic variation in clonal Abelson virus lymphoma cells

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.     

Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.     

Abelson virus-infected cells can exhibit restricted in vitro growth and low oncogenic potential.

We have designed a method for growing bone marrow cells infected with Abelson murine leukemia virus which permits examination of target cell growth early after infection. This culture system increases the efficiency of target cell growth by favoring rapid growth of a mixed population of adherent cells in the primary culture. The nonadherent Abelson virus-infected cell populations expressed pre-B-cell differentiation markers characteristic of Abelson virus-transformed cells (mu-heavy chains of immunoglobulin M and terminal deoxynucleotidyltransferase). Early after infection, these cell populations exhibited restricted in vitro and in vivo growth properties which differed from those of an established Abelson virus-transformed cell line, 2M3. These included a marked dependency upon the adherent cell layer for growth and viability, a lower efficiency of agar colony formation, and a lower capacity for tumor production in syngeneic animals. Growth of the early populations could be maintained in the absence of the adherent cell layer by using conditioned medium from long-term adherent cell cultures established in the absence of viral infection. After passage of the populations for several weeks, the in vitro growth properties gradually shifted toward that of the 2M3 cell line. Twelve-week-old populations grew independently of the adherent cell layer and showed an increased efficiency of agar colony formation. These data indicate that many lymphoid target cells exhibit an intermediate transformed phenotype when infected with Abelson virus. Growth of these cells in culture is mediated via a synergistic interaction between intracellular expression of the viral transforming gene and an exogenous growth-promoting activity which can be provided by cultures of adherent bone marrow cells.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Transformation of T-lymphoid cells by Abelson murine leukemia virus.

Abelson leukemia virus (A-MuLV) is an oncogenic murine retrovirus whose genome contains sequences homologous to those of a normal cellular gene, c-abl. It has been demonstrated to cause rapid transformation of several cell types, including pre-B lymphocytes, macrophages, and fibroblasts. More recently, A-MuLV has been reported to induce thymic tumors in a mouse strain (C57BL/Ka) previously thought to be resistant to disease induction. We showed that the masses occurring after intrathymic injection of the virus were composed of lymphocytes of a previously described immature T-cell phenotype. This phenotype has been defined here by flow cytometry of 10 primary tumor samples stained with antibodies to several thymocyte differentiation antigens. Hybridization of DNAs from these tumors with v-abl, immunoglobulin mu, and T-cell antigen receptor beta-chain probes confirmed the T-lymphoid, polyclonal nature of the primary tumor cells. The primary tumors were malignant, as clearly shown by reinjection into Thy-congenic host animals. Further, four Thy- in vitro cell lines derived from three tumors differed from the majority of primary tumor cells and were similar to previously described A-MuLV-transformed pre-B cells. The consistent T-lymphoid phenotype exhibited by primary A-MuLV thymomas may represent one stage of normal thymocyte differentiation.     

Erythropoietin stimulates 45Ca2+ uptake in Friend virus-infected erythroid cells

It has been shown previously in this laboratory that in vitro infection of mouse bone marrow cells with the anemia strain of Friend leukemia virus leads to growth of large bursts of erythroid cells which are arrested in development prior to hemoglobin synthesis but can respond to erythropoietin (EP) to complete the late stage of erythroblast differentiation. In this study, the effect of EP on the metabolism of 45Ca2+ in these cells was examined. At 4 degrees C, an increased rate of 45Ca2+ uptake and efflux as well as an increase in the steady state level of 45Ca2+ in treated cells was observed. Exchange of 45Ca2+ from preloaded cells at 4 degrees C indicated that treatment with EP increased the size of a rapidly exchanging pool of 45Ca2+ from 5 to 12% of total 45Ca2+ in the cell. The effect of treatment with EP can be seen as increased exchange of extracellular 45Ca2+ with cellular Ca2+; however, an effect of EP on the net level of Ca2+ in these cells cannot be excluded. This investigation demonstrates one of the earliest effects of EP on erythroid cells and suggests that alterations in Ca2+ metabolism may contribute to the progression of erythroid cells to their final development.     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

Regulation of differentiation in normal and transformed erythroid cells

Summary Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products. Presented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. These studies were supported in part by grants and contracts from the National Institutes of Health (GM-14552, CA-13696, CA-18314, NO1-CB-4008 and NO1-CP-1008) and the National Science Foundation (NSF-PCM-75-08696). E.F. and R.C.R. are fellows of the Schultz Foundation; A.B. was supported in part as an American Cancer Society Scholar; J.E.S. was supported by a USPHS Medical Scientist Training Grant; and M.T. and G.M.M. are Hirschl Trust Scholars.     

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.     

Study of markers of human erythroid differentiation in hybrid cells

Summary Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. Some of this work was conducted during the tenure of a National Research Fellowship 1 F32 AM05080-01 held by A.D.     

Regulation of differentiation in normal and transformed erythroid cells.

Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro

Summary The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.     

Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus.

The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation.