Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Early plasma membrane depolarization by alpha interferon: biologic correlation with antiproliferative signal

Daudi lymphoma cells, of a line sensitive to growth inhibition by alpha interferon, showed dose-dependent plasma membrane depolarization within 10 min after exposure to natural or recombinant alpha interferons (10 to 1000 IU/ml). This biophysical change was detected flow cytometrically by measuring the intensity of fluorescent emission from cells stained with dye indicators of membrane potential. Subclones of Daudi lymphoma cells, resistant to growth inhibition by alpha interferon, showed no membrane depolarization. Parallel results were obtained in initial tests of an isologous pair of T cell and B cell lines which differ in sensitivity to growth inhibition. Thus, decreased membrane potential may herald an interferon signal for antiproliferative action.     

Comparative antiproliferative and receptor binding activities of interferons alpha and beta on lymphoblastoid and melanoma cells

The relative antiproliferative and receptor binding characteristics of the hitherto little-characterized interferon alpha 4a on cells of lymphoid and epithelial origin are compared with two other type I interferons, alpha 2 a and beta. Using the lymphoblastoid cell line, Daudi, interferons alpha 4 a and alpha 2 b had similar antiproliferative activity, and were about 10-fold more active than IFN beta. By contrast, using the melanoma cell line Sk-Mel-28, IFN beta was the most active, whereas IFN alpha 2b and IFN alpha 4a were respectively 60-fold and greater than 1000-fold less active than on Daudi cells. Receptor binding did not correlate with antiproliferative sensitivities, but confirmed a shared receptor component for these three interferons. These results indicate that the antiproliferative activities of three type I IFNs differs markedly on different cell types and that this is unlikely to be due to receptor binding, but more likely a post receptor binding event.     

Effects of alpha, beta and gamma recombinant interferons on CEA production from HT-29 human colon adenocarcinoma cell line

The human colon adenocarcinoma derived cell line HT-29 is a good in vitro model for the study of CEA production and release under various experimental conditions. Many studies indicate that CEA secretion is correlated with cell proliferation and seems to depend on the growth conditions and differentiation characteristics induced by the culture medium. The present study demonstrates that recombinant interferons alpha, beta and gamma (rIFN alpha, rIFN beta, rIFN gamma) can modify CEA production and release by HT-29 cell-line. rIFN gamma in particular causes an enhancement of CEA production and release in the culture medium. This dose-depending effect is in some way correlated to cell growth inhibition since the enhancement of CEA expression in the interferon treated cells is evident in the presence of a reduction in cell proliferation. The activity of rIFN alpha and rIFN beta on CEA release is much less remarkable than that demonstrated by rIFN gamma, and is probably only due to the fact that HT-29 colon adenocarcinoma cells respond poorly to the effects of rIFN alpha and rIFN beta at the doses we used. These findings suggest that CEA production, expression and release can be modulated in a variety of ways under the influence of different rIFN treatment and this situation must be taken into account in immunodiagnostic and immunotherapeutic applications of anti-CEA monoclonal antibodies in the cancer patient.     

Prostaglandin E2 acts at two distinct pathways of T lymphocyte activation: inhibition of interleukin 2 production and down-regulation of transferrin receptor expression

The mechanism by which prostaglandin E2 (PGE2) inhibits human T lymphocyte activation and proliferation was studied. We analyzed the effect of physiologic concentrations of PGE2 on interleukin 2 (IL 2) production, expression of IL 2 receptor (Tac antigen), and expression of the transferrin receptor after in vitro activation with phytohemagglutinin. PGE2 inhibited T lymphocyte proliferation by 80 to 90% of control values. This was associated with a similar degree of inhibition of IL 2 production while the expression of IL 2 receptor was not affected. This was in marked contrast to the expression of the transferrin receptor, which was inhibited 65% after 72 hr of in vitro activation. The addition of exogenous, purified IL 2 reconstituted lymphocyte proliferation to 50% of control values, but had no effect on transferrin receptor expression. Because PGE2 is known to increase the intracellular concentration of 3',5' cyclic adenosine monophosphate (cAMP), we investigated the effect of another adenylate cyclase activator, i.e., isoproterenol, as well as the effect of extracellular administration of the cAMP derivative dibutyryl cAMP (dBcAMP) on IL 2 production, Tac antigen expression, and transferrin receptor expression. It was demonstrated that isoproterenol, as well as dBcAMP, inhibited transferrin receptor expression on PHA-activated T lymphocytes to the same extent as PGE2, and exogenous IL 2 could not counteract the down-regulation of the receptor expression. In contrast, neither isoproterenol nor dBcAMP had any significant effect on IL 2 receptor expression. Prostaglandin F2 alpha (PGF2 alpha), which has been reported to elevate intracellular cyclic GMP levels, had no effect on lymphocyte activation and proliferation, and did not counteract the PGE2-induced depression in IL 2 production. In contrast to its effect on peripheral blood lymphocytes, PGE2 had no effect on transferrin receptor expression or cell proliferation by IL 2-dependent T cell clones and IL 2-independent T cell lines. These studies demonstrate that PGE2 exerts its inhibitory effects on T cell activation and proliferation via two distinct pathways: inhibition of IL 2 production and inhibition of transferrin receptor expression. The transferrin receptor inhibition is mediated via the cAMP pathway and is IL 2-independent.     

The biology of interferon actions

The interferons comprise a group of proteins which were first identified by their ability to protect cells against virus infections. They are synthesized and secreted by a variety of cell types in response to various inducers and exert their effects in vivo by interaction with specific cellular receptors. In this sense the interferons are analogous to polypeptide hormones. In recent years it has become clear that the interferons are capable of influencing cellular physiology and behavior in a number of ways. Their effects include antiviral actions, inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation and activation of various cell types in the immune system. This review aims to summarize the current state of biology of interferon actions with special emphasis on the hemopoetic system.     

Modulation of cell-surface transferrin receptor by the imino sugar N-butyldeoxynojirimycin.

The imino sugar, N-butyldeoxynojirimycin, is an inhibitor of the glycoprotein-processing enzyme glucosidase I and exhibits anti-(human immunodeficiency virus) activity in vitro. We have investigated the effect(s) of this compound on cell-surface glycoproteins by flow cytometry. We observed selective modulation of the transferrin receptor in response to treatment with 0.5 mM N-butyldeoxynojirimycin resulting in reduced cell-surface transferrin-receptor expression. The receptor modulation was dose dependent, resulted in reduced 59Fe uptake by treated cells and was fully reversible within 24 h of culture in the absence of the compound. Pulse/chase analysis in conjunction with endoglycosidase-H digestion demonstrated that transferrin-receptor glycosylation was altered following N-butyldeoxynojirimycin treatment, which is compatible with glucosidase inhibition. In addition, modulation of transferrin receptor in response to N-butyldeoxynojirimycin was not confined to a single cell line, but was also observed with certain human lymphoid and myeloid cell lines. Mechanism(s) of action of the imino sugar resulting in reduced cell-surface transferrin-receptor expression are discussed.     

Interferons and bone. A comparison of the effects of interferon-alpha and interferon-gamma in cultures of human bone-derived cells and an osteosarcoma cell line

Recombinant human interferon-alpha 2C and recombinant human interferon-gamma (5-1000 U/ml) inhibit the proliferation of normal human bone-derived cells and a human osteosarcoma cell line. In the bone-derived cells the inhibitory effect of interferon-gamma was significantly greater than that of interferon-alpha, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon-alpha did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon-gamma affected the activity of the enzyme in both cell types: in the bone-derived cells the effect of interferon-gamma was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon-gamma selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone-derived cells, however, interferon-alpha decreased proline incorporation into collagen and non-collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two-dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14-87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon-alpha or interferon-gamma and differential induction in normal and transformed bone-derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone-derived cells and demonstrate significant differences between the responses of normal cells and transformed bone-derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone-derived cells in general.     

Limitin: An interferon-like cytokine that preferentially influences B-lymphocyte precursors

We have identified an interferon-like cytokine, limitin, on the basis of its ability to arrest the growth of or kill lympho-hematopoietic cells. Limitin strongly inhibited B lymphopoiesis in vitro and in vivo but had little influence on either myelopoiesis or erythropoiesis. Because limitin uses the interferon alpha/beta receptors and induces interferon regulatory factor-1, it may represent a previously unknown type I interferon prototype. However, preferential B-lineage growth inhibition and activation of Janus kinase 2 in a myelomonocytic leukemia line have not been described for previously known interferons.     

Interferon-gamma induces altered oncogene expression and terminal differentiation in A431 cells

In tumor cell lines in which oncogene expression is abnormal, modulation of the expression of the oncogene (myc, src, or ras) by interferons (IFNs) has been observed concurrently with cell growth inhibition or phenotypic reversion. Oncogene expression has also been reported to vary during the differentiation of several neoplastic cell lines. Treatment of monolayer cultures of A431, a human epidermoid carcinoma cell line, with IFN-gamma resulted in rapid morphological alterations and cell death not seen with either IFN-alpha or IFN-beta. These changes were accompanied by elevated expression of mRNA's for p21 (the c-ras gene product) and the epidermal growth factor receptor as well as increases in the biosynthetic rate of their respective proteins. These effects likewise appeared to be specific for IFN-gamma. Growth inhibition by IFN-gamma was also observed when A431 cells were grown in a three dimensional in vitro culture system. Immunohistochemical staining of these "tumoroids" with a differentiation specific, anti-keratin antibody indicated that IFN-gamma enhanced expression of this keratin. This observation suggests that the killing by IFN-gamma of A431 cells may result from an acceleration of terminal differentiation.     

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.     

Interferon impedes an early step of hepatitis delta virus infection

Hepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection is poor, with a high risk of cirrhosis and hepatocellular carcinoma. The best antiviral therapy is weekly administration for at least one year of high doses of interferon alpha. This efficacy of interferon therapy has been puzzling in that HDV replication in transfected cell lines is reported as insensitive to administration of interferon alpha or gamma. Similarly, this study shows that even when an interferon response was induced by transfection of poly(IC) into a cell line, HDV RNA accumulation was only modestly inhibited. However, when the HDV replication was initiated by infection of primary human hepatocytes, simultaneous addition of interferons alpha or gamma at 600 units/ml, a concentration comparable to that achieved in treated patients, the subsequent HDV RNA accumulation was inhibited by at least 80%. These interferon treatments were shown to produce significant time-dependent increases of host response proteins such as for Stat-1, phosphoStat-1, Mx1/2/3 and PKR, and yet interferon pretreatment of hepatocytes did not confer an increased inhibition of HDV replication over interferon treatment at the time of (or after) infection. These and other data support the interpretation that interferon action against HDV replication can occur and is largely mediated at the level of entry into primary human hepatocytes. Thus in vivo, the success of long-term interferon therapy for chronic HDV, may likewise involve blocking HDV spread by interfering with the initiation of productive infection of naïve hepatocytes.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

Regulation of murine plasmacytoma transferrin receptor expression and G1 traversal by plasmacytoma cell growth factor

Many plasmacytomas arising in BALB/c mice require a specific, macrophage-derived growth factor in order to proliferate in vitro. Since transferrin receptor expression is normally regulated by tissue-specific growth factors and because expression of these receptors is required for cell proliferation, we examined the interaction of plasmacytoma growth factor (PCT-GF) on transferrin receptor expression and cell cycle progression in several PCT-GF-dependent and independent plasmacytoma cell lines maintained in vitro. We found that removal of PCT-GF results in a rapid and specific loss of transferrin receptor expression with concomitant G1 arrest in early G1. The time required for G1 arrest to become maximal correlates closely to the initial level of surface transferrin receptor expression and the rate of decay following removal of PCT-GF. The calcium channel blocker diltiazem interferes with the ability of PCT-GF to maintain transferrin receptor expression in PCT-GF-dependent cell lines and causes a G1 arrest of the cell population. When added to a PCT-GF-independent cell line, diltiazem also inhibited transferrin receptor expression and caused G1 arrest. Thus, both PCT-GF-dependent and -independent plasmacytoma cell lines require transferrin receptor expression for growth. In factor dependent cell lines, transferrin receptor expression requires exogenous PCT-GF, while in factor-independent cells, transferrin receptor expression is constitutive. In both cell types, intracellular calcium levels may play a role in receptor expression.     

Interferon inhibitor in the blood of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) patients at advanced stages of the disease have an interferon inhibitor in the blood circulation. This inhibitor can block antiviral activity of all three types of human interferons and can significantly reduce the synthesis of interferon alpha by the treated lymphocytes obtained from normal healthy individuals. Available evidence suggests that inhibitor activity is neither because of the antibody to interferon nor due to high level of protease-like activity in the plasma. The inhibitor has also been shown to be effective in eliminating the interferon-mediated enhancement of natural killer cell activity. Interferon inhibitory activity was not detected in any of the sera taken from normal healthy individuals. Identification and characterization of interferon inhibitor has direct bearing upon effective utilization of interferons in the clinic.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.