Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

Inhibition of Friend erythroleukaemia-cell tumours in vivo by a synthetic analogue of prostaglandin E2.

The effect of 16,16-dimethyl-PGE2-methyl ester (di-M-PGE2), a long-acting synthetic analogue of prostaglandin E2, on the replication of Friend erythroleukaemia cells (FLC) in vivo has been studied. Pre-treatment in vitro of both undifferentiated and differentiated FLC with di-M-PGE2 (1 microgram/ml) did not alter rates of tumour appearance or growth, but increased the median survival of DBA/2J mice. Systemic administration of di-M-PGE2 (10 microgram/mouse/day) was not toxic to the mice, but significantly inhibited tumour growth and increased median survival in mice injected s.c. with undifferentiated FLC. These effects of di-M-PGE2 were much more pronounced in mice receiving differentiated (DMSO-treated) FLC. In this latter group, the appearance of tumour was also significantly delayed by di-M-PGE2. The different effects of di-M-PGE2 treatment on tumours derived from undifferentiated and differentiated cells suggest that the analogue is acting directly on tumour-cell replication rather than on factors related to the host response.     

Growth inhibition of Friend erythroleukaemia cell tumours in vivo by a synthetic analogue of prostaglandin A: an action independent of natural killer-activity

Prostaglandins of the A series (PGAs) have been previously shown to inhibit the growth and to stimulate the differentiation of Friend erythroleukaemic cells (FLC) in vitro. In the present report we analysed the effect of PGA treatment in vitro on FLC tumorigenicity, and in vivo on FLC proliferation and on natural killer (NK) activity. PGA1 pretreatment of FLC in vitro for 5 days before inoculation into syngeneic mice slightly delayed tumour appearance, but did not significantly alter the pattern of tumour growth or mice survival, indicating that PGA1, at least in the conditions studied, did not affect FLC tumorigenicity. Daily treatment of mice with a long-acting synthetic analogue of PGA2 (16, 16 dimethyl-PGA2-methyl ester, di-M-PGA2) delayed tumour appearance, inhibited tumour growth, as measured by tumour weight and diameter, and increased the median mice survival time by 15-35%, depending on the schedule of treatment. Daily treatment with di-M-PGA2 strongly suppressed NK activity in normal mice but had no significant effect in tumour-bearing immunodepressed mice. PGA treatment of effector or target cells in vitro, or PGA added during the NK assay, had no effect on NK activity. We suggest that the chemotherapeutic effect of PGA is due to a direct action on tumour cell replication rather than to a stimulation of the host NK activity.     

Effect of endogenous and exogenous prostaglandin E on Friend erythroleukaemia cell growth and differentiation.

The effect of exogenous and endogenous prostaglandins on the patterns of growth and differentiation of Friend erythroleukaemia cells (FLC) were studied. During the differentiation process, DMSO stimulated PGE synthesis by an average of 95%. The addition of a long-acting synthetic analogue of PGE2,16,16-dimethyl-PGE2-methyl ester (di-M-PGE2) to the culture medium only slightly and temporarily slowed cell growth, with no appreciable induction of differentiation. However, in the presence of DMSO, the same concentration of di-M-PGE2 produced 73% inhibition of cell growth and accelerated and potently stimulated haemoglobin production. The action of both di-M-PGE2 and DMSO on cell proliferation was dependent upon the state of cell growth at the time of the administration of these compounds. FLC cultures treated with DMSO + di-M-PGE2 produced considerable amounts of haemoglobin before even one duplication cycle was completed. Both DMSO and di-M-PGE2 stimulated endogenous PGE biosynthesis, and the biosynthetic effect of these compounds was synergistic. Inhibition of endogenous prostaglandin synthesis by indomethacin completely abolished the effects produced by DMSO + di-M-PGE2 on the growth, and substantially reduced the stimulated differentiation of FLC. These data suggest that an endogenously synthesized prostaglandin plays a significant role in both the inhibition of replication and in the stimulation of differentiation induced by DMSO and di-M-PGE2 in Friend erythroleukaemia cells.     

The essential role of endogenous IFN α/β in the anti-metastatic action of sensitized T lymphocytes in mice injected with friend erythroleukemia cells

Adoptive transfer of splenic T lymphocytes from DBA/2 mice immunized against Friend erythroleukemia cells (FLC) inhibited the development of visceral metastases and increased the survival time of DBA/2 mice challenged i.v. with parental FLC 24 hr to 2 months later. Immune spleen cells were ineffective in mice pre-treated with potent neutralizing antibody to mouse IFN α/β (but not to IFN γ), demonstrating the essential participation of endogenous IFN α/β in the inhibitory action of immune T lymphocytes against FLC metastases. These findings suggest that the reported inability of immune T lymphocytes to exert an anti-FLC effect in immunodeficient DBA/2 mutant beige (bg/bg) mice (unless these mice had also been treated with IFN α/β), may have been due to lower levels of endogenous IFN α/β in DBA/2 bg/bg mice than in normal DBA/2 +/bg mice. Experimental results in support of this hypothesis are presented. © 1995 Wiley-Liss, Inc.     

Hyposialylation of high-molecular-weight membrane glycoproteins parallels the loss of metastatic potential in wheat-germ agglutinin-resistant Friend leukemia cells

From the highly metastatic in vivo-passaged Friend leukemia cells (FLC), WGA-resistant (WR) tumor cell variants were selected. These WR FLC had lost their capacity to metastasize when injected i.v. or s.c. into DBA/2 mice. We have characterized the plasma membrane glycoproteins of the different FLC types by: (i) metabolic labelling with (3H)-galactose; (ii) surface labelling with galactose oxidase-borohydride; (iii) direct binding of (125I)-lectins on glycoproteins separated by SDS-PAGE. The ensemble of these approaches showed that the 100- to 200-kDa glycoproteins of in vivo-passaged FLC and WR FLC exhibited a very similar distribution of the terminal galactose in their oligosaccharide moieties. In contrast, the expression of terminal sialic acid was reduced in WR FLC with respect to in vivo-passaged counterparts as appreciated by: (i) binding experiments with (125I)-WGA; (ii) cathodic shift of the 100- to 200-kDa glycoproteins in 2-dimensional electrophoresis studies, and (iii) thiobarbituric acid assay after FLC treatment with neuraminidase. Moreover, binding experiments with (125I)-LPHA, (125I)-ConA and (125I)-WGA (after Smith degradation) indicated that, in the 100- to 200-kDa region, virtually identical asparagine-linked tri- or tetra-antennary complex-type oligosaccharides were expressed in both cell types. We conclude that the sialylation of high-molecular-weight surface glycoproteins (particularly in the 150-kDa region) is strongly associated with the metastatic potential of FLC, especially to the liver.     

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. V. Comparisons with the action of tumor necrosis factor

A number of similarities and dissimilarities in the anti-tumor effects of TNF and interferon alpha/beta have been observed in DBA/2 mice injected with Friend erythroleukemia cells (FLC). Mouse TNF exerted marked anti-tumor effects in mice injected either s.c. or i.p. with FLC lines 3C18 or 3 gamma R8 resistant in vitro to the cytotoxic effects of TNF. Likewise, mouse interferon alpha/beta had anti-tumor activity in mice injected with these FLC, resistant to the action of interferon alpha/beta or gamma in vitro. The results of histopathologic examination and 31P nuclear magnetic resonance analyses of 3C18 FLC s.c. tumors injected with TNF resembled the results previously obtained for 3C18 FLC tumors injected with interferon alpha/beta, although the effects of TNF occurred more rapidly. Injection of mice with antibody to mouse interferon alpha/beta or gamma did not abrogate the anti-tumor effects of TNF in mice injected i.p. with FLC. Our results suggest that in this experimental system the anti-tumor effects of TNF, like interferon alpha/beta, do not result from a direct effect on the tumor cells themselves but are host-mediated.     

Tumor necrosis factor alpha induces early morphologic and metabolic alterations in Friend leukemia cell tumors and fibrosarcomas in mice

Morphologic and metabolic studies have been carried out on Friend leukemia cell (FLC) tumors (grown in DBA/2 mice) or fibrosarcomas (grown in C3H/HeN or C3H/HeJ mice) shortly after peritumoral injection of recombinant mouse tumor necrosis factor (TNF) alpha. Marked vascular congestion and focal extravasation of erythrocytes were observed as soon as 1 hr after injection of either FLC tumors or fibrosarcomas with TNF. Focal areas of disaggregation of tumor cells were observed 1 hr after injection of TNF. Intraluminal thrombi (composed of degranulated platelets and fibrin) were detected 3 and 6 hr after TNF treatment, and were associated with areas of depletion of endothelial cell cytoplasm. To correlate these morphologic changes in the tumor with alterations in tumor metabolism, NMR spectroscopy and biochemical studies were undertaken on freshly dissected FLC tumors and fibrosarcomas shortly after injection of TNF. The earliest metabolic alterations observed after 1 hr in TNF-treated FLC tumors of fibrosarcomas were: (i) increase in the average intratumoral pH; (ii) decrease in the levels of ATP. These phenomena were not associated with a reduced glycolytic capacity of TNF-treated tumors as, at these early times after injection, the levels of lactic acid were virtually the same for TNF-treated or control treated tumors. Alterations in the levels of some products of phospholipid degradation (GroPCho, GroPEtn, GroP and Cho) also occurred in these tumors as early as 3 hr after TNF treatment. These metabolic changes were not observed in ascitic FLC tumors after TNF treatment. We suggest that TNF induces alterations in tumor blood vessels which subsequently lead to changes in tumor metabolism and tumor degeneration.     

An anti-cancer derivative of butyric acid (pivalyloxmethyl buterate) and daunorubicin cooperatively prolong survival of mice inoculated with monocytic leukaemia cells.

A derivative of butyric acid, pivalyloxymethyl butyrate (AN-9), inhibited the proliferation and induced apoptosis of mouse monocytic leukaemia Mm-A cells, although sodium butyrate, but not AN-9, induced differentiation of the cells. AN-9 and DNA-specific antineoplastic agents synergistically inhibited the growth of Mm-A cells, and the simultaneous treatment was required to evoke the maximum growth-inhibitory effect. On the other hand, there was no synergy between butyrate and the drugs, or AN-9 and anti-metabolic agents in inhibiting the growth of the cells, suggesting that the synergistic effect is specific to AN-9 and DNA-reacting agents. AN-9 as a single agent prolonged the survival of mice inoculated with Mm-A cells in a dose-dependent manner. Moreover, administration of AN-9 plus daunorubicin (DNR) markedly prolonged their survival. These results suggest that combination with AN-9 and DNR entails an obvious therapeutic potential.     

Interferon treatment markedly inhibits the development of tumor metastases in the liver and spleen and increases survival time of mice after intravenous inoculation of Friend erythroleukemia cells

To investigate the effect of interferon treatment on the development of tumor metastases, DBA/2 mice were injected i.v. with 2 X 10(6) Friend erythroleukemia cells (FLC) (equivalent to about 5 X 10(5) LD50). FLC multiplied rapidly in the liver and spleen and all untreated or control treated mice died between 7 and 12 days. Daily treatment of mice with potent preparations of mouse interferon alpha/beta was initiated 3 to 72 hr after i.v. inoculation of tumor cells, at times when FLC were already present in the liver and spleen. Interferon treatment resulted in a 100 to 1,000-fold inhibition of the multiplication of FLC in the liver and spleen and a marked increase in mean survival time. Small numbers of tumor cells persisted in the liver and spleen in some interferon-treated mice and could be recovered by bioassay several weeks after tumor inoculation. Most interferon-treated mice died with tumor in the ensuing months. Three of 34 interferon-treated mice were considered cured as they were alive at 386, 325 and 284 days after tumor inoculation. Daily treatment of tumor-inoculated mice with human recombinant interferons alpha D and alpha BDDD, which had antiviral activity on mouse cells in culture, also increased the survival time of mice injected i.v. with FLC. The use of the interferon-resistant 3C18 line of FLC suggests that the marked inhibition of development of established liver and spleen metastases was not due to a direct effect of interferon on the tumor cells, but was host-mediated.     

Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors

We have studied the anti-tumor effects of human recombinant IL-2, alone or in association with LAK cells, in mice transplanted subcutaneously (s.c.) with the following syngeneic tumors: highly metastatic Friend leukemia cells (FLC), nonmetastatic FLC, lymphoma RBL-5 cells and HeJ16 fibrosarcoma cells. In these tumor models, peri-tumoral injections of IL-2 were more effective in inhibiting tumor growth than a systemic treatment. Although s.c. IL-2 treatment resulted in marked inhibition of tumor growth in mice injected s.c. with highly metastatic FLC, it was not effective in inhibiting growth of FLC in the liver and spleen. IL-2 therapy was more effective at increasing survival time in mice transplanted with non-metastatic FLC or with RBL-5 cells. In mice transplanted with HeJ16 fibrosarcomas, s.c. IL-2 treatment resulted in highly significant anti-tumor effect and survival of 70% of tumor-injected mice. No general correlation was found between in vitro sensitivity or resistance to the cytolytic activity of LAK cells and the anti-tumor effects observed in vivo. Subcutaneous injection of IL-1 beta in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Combined treatment of IL-1 beta and IL-2 produced a synergistic anti-tumor effect: 60% of mice injected with highly metastatic FLC survived. Combined IL-1/IL-2 treatments exerted no anti-tumor activity either in DBA/2 mice injected with antibody to Thy 1.2 antigen or in nude mice, indicating that T cells play important roles during IL-1/IL-2 therapy. In vitro treatment of FLC with IL-1 beta resulted in a slight inhibition of cell multiplication, whereas even high doses of IL-2 did not affect FLC multiplication. Our results indicate that local combined treatments with IL-1 and IL-2 can induce potent, host-dependent (T cell-mediated) anti-tumor effects against highly malignant tumors.     

Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. I

Interferon-sensitive (745) and interferon-resistant (3C1-8) Friend leukemia cells (FLC) are highly tumorigenic for DBA/2 mice. The phenotype of interferon sensitivity or resistance does not change with in vivo passage. Daily administration of mouse interferon markedly enhanced the survival time of mice injected with either 745 or 3C1-8 cells. Use of quantitative methods for determining the number of FLC (colony formation in agarose and immunofluorescence) permitted us to show that potent, partially purified or highly purified mouse interferon (s.a. 0.5 to 1 X 10(9) u/mg protein) induced a 100- to 1,000-fold decrease in the number of tumor cells in the peritoneal cavity in the days following inoculation of 745 or 3C1-8 cells. Interferon decreased the number of FLC even when treatment was initiated at a time when tumor cells were multiplying exponentially in the peritoneal cavity. There was no evidence that interferon acted as an inducer of FLC differentiation in vivo. The finding that interferon was equally effective in mice inoculated with interferon-resistant cells as in mice inoculated with interferon-sensitive cells suggests that in this experimental system interferon does not act directly on the tumor cells, but that the interferon-induced antitumor activity is mediated by the host.     

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.     

Sensitized T lymphocytes render DBA/2 beige mice responsive to IFN α/β therapy of friend erythroleukemia visceral metastases

Interferon α/β (IFN α/β) is highly effective in inhibiting the development of Friend erythroleukemia cell (FLC) visceral metastases in DBA/2 mice injected intravenously (i.v.) with FLC, but does not protect FLC-injected DBA/2 beige (bg/bg) mice. Use of IFN α/β-resistant FLC indicated that IFN was acting through host mechanisms in DBA/2 mice and thus pointed to a defect in some host mechanism in bg/bg mice essential for IFN's anti-metastatic action. We undertook experiments to restore in bg/bg mice the marked anti-FLC meta-static effect of IFN a/p observed in DBA/2 and +/bg mice. Adoptive transfer of spleen cells from normal syngeneic mice to IFN-treated bg/bg mice was ineffective, but the transfer of splenic T lymphocytes from FLC-immunized DBA/2 or +/bg mice markedly increased the survival time of FLC-injected bg/bg mice provided that these mice were also treated with IFN a/p. Neither treatment alone resulted in an increase in survival time. As few as 1 × 10⁷ immune spleen cells were effective in IFN-treated FLC-injected bg/bg mice. The T-cell immune response to FLC of bg/bg mice was diminished compared with that of +/bg mice. Likewise, only combination therapy of immune spleen cells and IFN α/β resulted in an increased survival time of ESb-lymphoma-injected bg/bg mice. Our results indicate the essential participation both of T-cell-mediated immune mechanisms and of IFN α/βin the inhibition of FLC visceral metastases.     

Employment of a [3H]thymidine-incorporation assay to distinguish the effects of different Friend erythroleukemia-inducing retroviruses on erythroid cell proliferation

Spleen cells taken from mice infected as adults with two different variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, as well as spleen cells taken from mice infected as newborns with Friend murine leukemia virus (F-MuLV) were assayed in a proliferation assay in the presence or absence of the erythroid hormone erythropoietin (Epo). Infection of NIH Swiss mice with SFFV resulted in excessive proliferation of erythroid cells that could still differentiate, and spleen cells taken from these mice were able to incorporate high levels of tritiated thymidine ([3H]dThd) in the absence of Epo, even in the presence of antibodies to Epo. In contrast, the level of proliferation of spleen cells from SFFVA-infected mice, but not those from SFFVP-infected mice, could be greatly enhanced by the addition of Epo to the cultures. Infection of newborn mice with F-MuLV resulted in the generation of Friend mink cell focus-inducing virus, which caused excessive proliferation of erythroid cells that appeared to be blocked in differentiation, resulting in severe anemia. Spleen cells from these mice were unable to proliferate in the absence of Epo. However, when increasing doses of Epo were added to the cultures, the cells proliferated at levels equivalent to the levels seen with SFFV. These results indicate that a proliferation assay based on the incorporation of [3H]dThd into spleen cells in response to Epo can be used as a quantitative means of assessing and comparing the effects of erythroleukemia-inducing retroviruses on the proliferation of their target cells.     

Interaction of IFN α/β with host cells essential to the early inhibition of friend erythroleukemia visceral metastases in mice

We have previously shown that an intact immune system was essential to the increase in survival time of IFN-α/β-treated mice injected i.v. with an IFN-α/β-resistant line of Friend erythroleukemia cells (FLC) highly metastatic to the liver and spleen. Here, we have investigated the early interactions of IFN α/β with host cells prior to the development of the immune response. IFN α/β treatment resulted in 50- to 100-fold inhibition of FLC multiplication in the liver and spleen of normal DBA/2 mice shortly after tumor inoculation, as evaluated by colony formation in agarose. IFN treatment was far less effective in inhibiting the multiplication of FLC in the livers of NK-cell-deficient DBA/2 beige mice, or in immunocompetent DBA/2 mice treated with antibody to asialo GMI, or silica, or in mice subjected to sub-lethal irradiation. Injection of antibody to CD4 or CD5 did not affect the early inhibitory action of IFN α/β on FLC multiplication but did decrease survival time. Light- and electron-microscope examination of the livers of IFN-treated, FLC-injected mice confirmed the early inhibition of FLC multiplication in the liver and spleen. Our results indicate that IFN α/β inhibits the development of FLC visceral metastases by acting first on host cells, such as NK cells and macrophages, and then continues to act in consort with the developing immune response. © 1994 Wiley-Liss, Inc.     

Changes in biophysical parameters and in phospholipid composition associated with resistance to doxorubicin

Friend leukemia cells (FLC) resistant to different concentrations of doxorubicin were used to investigate the biochemical and biophysical changes associated with resistance. We have found that fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene analyzed on single cell level increased in resistant as compared to sensitive FLC. Furthermore, phospholipid analysis of sensitive and doxorubicin-resistant cells revealed changes in ratios of phosphatidyl-choline to phosphatidyl-ethanolamine and phosphatidyl-choline to sphingomyelin. These results correlate with decreased electrophoretic mobility in resistant cells. Our results indicate that changes in cell structure occur with the level of resistance to doxorubicin. These changes are probably the consequence rather than the cause of resistance.     

Importance of interferon alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend erythroleukemia cells in the liver

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. Our results indicate that interferon alpha (IFN-alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver. (a) After i.v. inoculation of FLC there was an inverse correlation between the presence of IFN-alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC-injected adult C57B1/6 mice had circulating IFN-alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC-injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice. (b) FLC did multiply in the livers of newborn C57B1/6 mice and in the livers of irradiated adult C57B1/6 mice, and IFN-alpha was not detected in their sera. In contrast, after i.v. inoculation of FLC, IFN-alpha was detected in the sera of 3-week-old and athymic nu/nu adult C57B1/6 mice while FLC failed to multiply in the liver. (c) FLC also induced IFN-alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver. We suggest that, depending on the site of tumor implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumor cells, and that IFN-alpha is particularly important when FLC are injected i.v.     

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.     

31P-nuclear magnetic resonance analysis of interferon-induced alterations of phospholipid metabolites in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice

Adult DBA/2 mice were given injections s.c. with either interferon-sensitive (745) or -resistant (3Cl-8) Friend erythroleukemia cells (FLC). After tumor nodules had developed, mouse interferon-alpha/beta was injected daily into the tumor. 31P-Nuclear magnetic resonance (NMR) spectroscopy examinations were undertaken on freshly dissected tumors at different days of treatment with either interferon or control preparations. Analysis of 745 FLC tumors in untreated mice at different days of tumor growth (day 8 to 13 after tumor implantation) showed marked increases in the levels of phosphorylcholine (PCho), glycerophosphorylethanolamine (GroPEtn) and glycerophosphorylcholine (GroPCho). In contrast high levels of PCho, GroPEtn and GroPCho were already detectable in the 3Cl-8 FLC tumors on day 8, and no significant changes were observed during subsequent tumor growth. The intracellular pH value remained practically constant in both FLC tumors. Daily intratumoral administration of either partially purified (10(7) IU/mg of protein) or highly purified (10(9) IU/mg of protein) mouse interferon-alpha/beta to both cell tumors resulted in decreases in the levels of PCho, GroPEtn and GroPCho and in increases in the intracellular pH with respect to tumors treated with control preparations or left untreated. Two days of daily treatment of mice with interferon sufficed to induce these metabolic changes which preceded the appearance of necrosis in the tumors. Treatment of FLC tumors with X-rays on day 12 of tumor growth did not result in any comparable metabolic changes 2 days after irradiation. Changes in the levels of phospholipid metabolites were not observed when 745 or 3Cl-8 cells were cultivated in the presence of interferon. As interferon induced these changes in both interferon-sensitive and -resistant tumors we conclude that interferon treatment results in host-mediated effects on the biosynthesis and/or catabolism of tumor cell phospholipids.