Biologic and biochemical differences between in vitro and in vivo passaged friend erythroleukemia cells. II. Changes in cell surface glycoproteins associated with a highly malignant phenotype

Friend erythroleukemia cells (FLC), serially passaged in vkroor by intraperitoneal injection in DBA/2 mice, exhibit markedly different tumorigenicity and capacity to metastasize. We have attempted to determine whether the differences in tumorigenicity between these two lines of FLC were correlated with any biochemical changes in their cell membranes. Although consistent modifications of FLC membrane gangliosides were detected after FLC multiplied in the peritoneum, the pattern of FLC gangliosides was not a stable characteristic and did not correlate with tumorigenicity. In contrast, analysis of FLC membrane glycoproteins by cell surface labelling techniques (i.e., galactose-oxidase-borohydride techniques and polyacrylamide gel electrophoresis-fluorography) or by metabolic labelling of glycoproteins with ³H-galactose, revealed consistent differences in the high MW region of the gels between parental in vitro passaged FLC (either 745 or 3C1-8 cells) and clones derived from in vivo passaged cells. No significant differences in the membrane proteins were detected between in vitro and in vivo passaged FLC when lactoperoxidase-catalyzed iodination and polyacrylamide gel electrophoresis-autoradiography were used. It is seen that repeated in vivo passages of FLC resulted in the appearance of different patterns of membrane glycoproteins and that these changes appeared to be associated consistently with the capacity of these cells to grow as tumor ascites and to metastasize to the liver and spleen.     

Lysis of FLD-3 Friend erythroleukemia cells in vitro and in vivo: effect of 89Sr treatment and Friend virus infection

The effector cells from non-immunized mice capable of lysing 51Cr-labelled FLD-3 BALB/c Friend virus-induced erythroleukemia cells in vitro and cells capable of clearing FLD-3 cells labelled with 5-iodo-2'-deoxyuridine-125I (125IdUrd) from the lungs in vivo were characterized and compared with natural killer (NK) cells reactive against YAC-I lymphoma cells. Unlike NK cells, the cells capable of lysing FLD-3 cells in vitro were insensitive to antibodies directed against NK-2.1 or Thy-1.2 antigens (plus complement) and to pretreatment of mice in vivo with silica particles, 89Sr or estradiol. Heat-killed C. parvum organism stimulated anti-FLD-3 effector cells without changing the slow rate (24 h) of lysis in vitro. The ability to clear FLD-3 and YAC-1 cells from the lung was normal and defective, respectively, in C57BL/6-bg/bg(beige) mice and in mice pretreated with 89Sr or estradiol. We conclude that natural cytotoxic (NC) cells lyse FLD-3 cells, Fv-2, which regulates resistance to leukemia induction by Friend virus, does not regulate NC(FLD-3) activity, and the virus does not affect NC(FLD-3) activity during the first several days of infection of normal genetically susceptible mice. However, infection of 89Sr-treated mice inhibits NC(FLD-3) function owing to the activation of suppressor cells. These data suggest (but do not prove) that effector cells similar or identical to NC(FLD-3) cells may function in vivo to resist the proliferation/survival of certain leukemia cells.     

Natural resistance in mice against Friend cells injected intravenously. III. Comparison between in vivo and in vitro passaged interferon-sensitive (745) and interferon-resistant (3Cl8) cell clones

In vitro (FLC-Vt) or in vivo (FLC-V) passaged Friend erythroleukaemia cells of DBA/2 origin were tested for susceptibility to natural resistance (NR) in vivo or to NK cell activity in vitro. Scarcely oncogenic FLC-Vt cells were highly susceptible to in vivo NR (measured as rapid organ clearance or growth inhibition in lethally irradiated mice) or to in vitro NK attack. Conversely, highly oncogenic FLC-V cells were weakly susceptible to NR and to NK as well. These data seem to point out that natural immunity, which is up-regulated by endogenous or exogenous interferons, can play a significant role in surveillance against mouse leukaemic cells of retrovirus origin.     

Structure-activity relationship between anthracycline-induced differentiation and inhibition of glycoprotein synthesis in Friend erythroleukemia cells

The effect of adriamycin, daunomycin, N,N-dimethyladriamycin, N,N-dimethyldaunomycin, pyrromycin, marcellomycin, and aclacinomycin A on erythroid differentiation and glycoprotein synthesis in Friend erythroleukemia cells, clone F4-6 was investigated. Whereas N-dimethylated natural anthracyclines, pyrromycin, marcellomycin, and aclacinomycin A stimulated erythroid differentiation (induction of hemoglobin synthesis), this was not seen with adriamycin, daunomycin and their N-dimethylated derivatives. The incorporation of 3H-mannose in glycoprotein was inhibited by the N-alkylated natural anthracyclines at a concentration at which they induced erythroid differentiation. N,N-Dimethyladriamycin and N,N-dimethyldaunomycin only inhibited 3H-mannose incorporation into glycoprotein at cytotoxic concentrations. However, adriamycin and daunomycin did not inhibit glycoprotein synthesis, even at high cytotoxic concentrations. Aclacinomycin A decreased the incorporation of 3H-mannose into proteins earlier than the incorporation into dolichol-linked oligosaccharide intermediates. Tunicamycin, a specific inhibitor of glycoprotein synthesis, failed to stimulate differentiation in Friend erythroleukemia cells. These results indicate a structure-specific induction of the differentiation and inhibition of glycoprotein synthesis in Friend cells by N-alkylated anthracyclines. The inhibition of glycoprotein synthesis may be involved in the induction of differentiation by N-alkylated anthracyclines, but it cannot be the only target for the differentiation-inducing effect of these substances.     

Growth and differentiation of Friend erythroleukemia cells in serum-free culture

A synthetic medium allowing indefinite optimal growth of Friend erythroleukemia cells (FLC) is described. It consists of Iscove's modified Dulbecco's medium supplemented with bovine serum albumin, transferrin, and a lipid mixture. Transferrin and lipids are essential for Friend cells growth. Under these conditions, FLC erythroid differentiation, promoted by a number of inducers, is less efficient than in cultures with serum-rich medium, suggesting that unknown serum factors may play an additional role in this phenomenon. Conversely, the enhancement of erythroid differentiation induced by low doses of Interferon is superimposable in both types of cultures.     

Plasminogen activator and its enhancement in differentiating mouse Friend erythroleukemia cells

Urokinase-type plasminogen activator (u-PA) activity was found in the medium as well as in the lysates of cultured uninduced Friend leukemia (FL) cells. PA activity progressively increased during the cell differentiation induced by dimethyl sulphoxide (DMSO), 4-hydroxy-3-methoxybenzaldehyde or hypoxanthine. Both the differentiation and the enhancement of PA activity in cultures of DMSO-induced cells were blocked by treating the cells with 1 microM dexamethasone. A highly significant correlation (rs = 0.93) was found between the number of hemoglobinized cells and the rate of PA secretion, indicating that the increase in PA activity coincides with late events of the differentiation process. FL cells specifically adhere to fibronectin-coated surfaces but tend to lose this property during the differentiation process. Anti-u-PA IgG antibodies promoted the attachment of differentiating cells to fibronectin-coated surfaces, suggesting that u-PA plays a role in the detachment of FL cells from fibronectin immobilized on the growth substratum.     

Dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells in the absence of cytokinesis.

Friend erythroleukemia cells grown in culture and induced to differentiate along the erythroid developmental pathway by dimethyl sulfoxide (DMSO) were used as a model system to investigate the requirement for cellular replication to express a differentiated erythroid phenotype. That cytokinesis is not essential for DMSO-induced erythroid differentiation as measured by the synthesis and accumulation of hemoglobin was shown by experiments using cytochalasin B. In these studies, hemoglobin was found to accumulate in Friend cells treated simultaneously with DMSO and cytochalasin B; such treatment caused cells to become enlarged and multinucleated due to inhibition of cytokinesis by cytochalasin B. In contrast, exposure of cells to cytochalasin B for at least 48 hr prior to DMSO caused significant inhibition of erythroid differentiation. The findings support the concept that cellular division and, thereby the production of new cellular types are not required for gene activation and the expression of an erythroid phenotype. These effects of cytochalasin B on DMSO-induced differentiation of Friend leukemia cells also suggest plasma membrane-cytoskeleton involvement in the initiation of the erythroid maturation process in this system.     

The effect of passage in vitro and in vivo on the properties of murine fibrosarcomas I. Tumorigenicity and immunogenicity

Cloned cell lines of chemically-induced murine fibrosarcomas maintained in tissue culture usually fail to grow when transplanted to normal syngeneic mice. They grow, however, in various categories of T cell deficient mice and after such passage grow readily in normal mice. Both cultured and mouse-passaged lines possess strong TATA. Three alternative explanations are suggested which might account for these findings. Emergence during the initial passage of a population of tumour cells resistant to NC cells. Acquisition during the initial passage of a protective surface molecule that interferes with the efferent side of the immune response when the tumour cells are subsequently transplanted to a normal host. Loss during the initial passage of a Class I MHC molecule which prevents dual recognition of the tumour cells by T cells when they are transplanted to a normal host. New experiments are proposed to distinguish between these possibilities.     

Cellular-oncogene expression in Friend erythroleukemia cells: relationship to differentiation, commitment and TPA effects

Induced differentiation of Friend erythroleukemia cells canbe continuously and reversibly inhibited by phorbol ester tumorpromoters, e.g. 12-O-tetradecanoylphorbol-13-acetate (TPA),for many years, allowing us to study the mechanisms of differentiationand its inhibition by TPA. We previously identified two stepsin the differentiation process, which can be inhibited by TPA,before and after commitment to differentiation. Using permanentlycommitted cells and TPA-resistant variants we examined the roleof cellular oncogenes in Friend cell differentiation control,and their possible modulation by tumor-promoting phorbol esters.We report here characteristic changes in myc, myb and fos mRNAlevels upon induction of differentiation by hexamethylene bisacetamidetreatment, and present evidence that c-myb mRNA decline is onefeature of Friend cell commitment to differentiation. In addition,using our TPA-sensitive and resistant cell lines, we observedthat the hexamethylene bis-acetamide induced pattern of oncogeneexpression is unperturbed by TPA, regardless of whether thecells are differentiating or not.     

Polyamine biosynthesis enzymes in the induction and inhibition of differentiation in Friend erythroleukemia cells

O-Ornithine decarboxylase (ODC) activity in cultures of Friend erythroleukemia cells induced to differentiate with various compounds was examined. Based on ability to stimulate ODC activity, the inducers tested could be divided into two categories. Inducers of the first class, among which were dimethyl sulfoxide and hexamethylene bisacetamide, stimulated ODC activity and were inhibited by dexamethasone and the phorbol diester, 12-O-tetradecanoylphorbol-13-acetate. Specific inhibitors of polyamine biosynthesis, such as methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and the ornithine analogs, alpha-methylornithine and alpha-hydrazinoornithine, also inhibited the induction of Friend erythroleukemia cell differentiation. The inhibition of induced differentiation by this class of compounds could be abrogated by spermine, spermidine, or putrescine. Inducers of the second class, among which were sodium butyrate, actinomycin D, and aminonucleoside of puromycin, had little or no stimulatory effect on ODC and were inhibited only by bromodeoxyuridine. The effect of bromodeoxyuridine, which inhibits inducers of both classes, was not abrogated by polyamines.     

In vitro and in vivo studies of poly-L-lysine as inducer of Friend leukemic cells differentiation

Poly-L-lysine, a synthetic cationic polypeptide known for its ability to bind to cell membranes, was found to induce differentiation of Friend leukemia cells "in vitro". Studies were extended to the same "in vitro" model, in order to examine the therapeutic potential of this new differentiating agent. The i.p. administration of the polymer (Mw 2700) at the maximal tolerated dose resulted in major alterations of disease-related parameters. In particular, a multiple treatment schedule on the advanced disease resulted in a successful reduction of target organ weight and peripheral white blood cell count and appreciable differentiation of spleen and bone marrow cells. Apparently, the effects of poly-L-lysine were superior to those produced by N-methyl-acetamide, a potent inducer of differentiation "in vitro".     

A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state

Friend murine erythroleukemia (MEL) cells are transformed erythroid precursors that are held in an immature and proliferating state but can be induced to differentiate in vivo by treatment with a variety of chemical agents such as N, N-hexamethylene bisacetamide (HMBA). To investigate the role of Myb proteins in maintaining MEL cells in an immature and proliferating state we have produced stable transfectants in the C19 MEL cell line that contain a dominant interfering Myb allele (MEnT) under the control of an inducible mouse metallothionein I promoter. When expression of MEnT protein was induced with ZnCl2, the stable transfectants differentiated with kinetics that were similar to wild type C19 MEL cells treated with HMBA, including induction of alpha-globin mRNA expression, assembly of hemoglobin and growth arrest. Expression of endogenous c-myb and c-myc was also decreased in response to MEnT. Expression of mad-1 mRNA was rapidly increased in response to expression of MEnT resulting in a shift from predominantly c-Myc/Max complexes to predominantly Mad/Max containing complexes. These results strongly suggest that C19 MEL cells are held in an immature and proliferating state by a pathway that is dependent on Myb activity.     

In vivo and in vitro interactions between human colon carcinoma cells and hepatic stellate cells.

Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM-H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed alpha-smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM-H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture-induced activation contained platelet-derived growth factor (PDGF)-AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF)-beta, and could augment LM-H3-cell proliferation and migration. Neutralizing antibodies against PDGF-AA and PDGF-BB and those against PDGF-BB and HGF inhibited proliferation and migration, respectively, of LM-H3 cells, whereas antibody against TGF-beta had no effect. LM-H3 cells expressed PDGF receptors-alpha and -beta and c-met. Conditioned medium of LM-H3 cells contained PDGF-AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti-PDGF-AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis.