Antiproliferative Effects Of Interferon Research Articles

Responsiveness to interferon treatment of human melanoma cells correlates to immunophenotype.

Clinical trials with interferon (INF) treatment in patients with advanced metastatic melanoma have met with limited success, suggesting that only a small subgroup of patients with melanoma is sensitive to INF therapy. For therapeutic strategies, it would be of great value to discriminate and define markers related to the anti-proliferative effects of INF. In the present study, we report on the in vitro growth inhibitory effects of INF in six human melanoma cell lines; this is associated with a modulation of cell surface markers. The responsiveness of human melanoma cell lines in vitro to INF-alpha shows a good correlation with a specific immunophenotype (TA99-/EGF-R+ or HLA-DR+). No marker correlated with sensitivity or resistance to INF-beta or INF-gamma. This is the first report identifying cell surface markers of human melanoma cells which might have a predictive value for the clinical response to INF-alpha.

Anti-proliferative effect of interferon in human melanoma cell lines

Anti-proliferative effect of interferon in human melanoma cell lines

Antiproliferative effects of interferons -alpha and -beta in combination with 5-fluorouracil, cisplatin, and cis- and trans-retinoic acid in three human lung carcinoma cell lines.

We assessed the antiproliferative effect of human recombinant interferon -alpha (IFN-alpha) or -beta in combination with 5-fluorouracil (5-FU), cisplatin, or cis- or trans-retinoic acid on two human nonsmall cell lung carcinoma cell lines (SK-LU-1 and SK-MES-1) and on one human small cell lung carcinoma cell line (NCI-H69). Results were obtained by direct cell count and/or by the clonigenic assay. The three cell lines differed in their sensitivities to the antiproliferative effects of the different agents. However, both NSCLC cell lines were more responsive to IFN-beta than to IFN-alpha. The SK-MES cell line was more resistant to both IFNs than the SK-LU-1. The NCI-H69 cells were resistant to all the drugs tested, except trans-retinoic acid. The dose and time of exposure were found to be important factors in the case of IFNs and cytotoxic agents, with lower surviving fractions obtained with the higher doses and longer exposures. This finding, however, did not hold true for the retinoic acids, which showed no antiproliferative effect. Within the sensitivity of our system, we did not identify any synergistic interaction in any of the cell lines with IFN-alpha or IFN-beta and 5-FU or cisplatin. A slight synergistic interaction was observed with IFN and cis- or trans-retinoic acid in the SK-LU-1 cell line which was not thought to be clinically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.

The double-stranded RNA-dependent protein kinase (dsRNA-PK) is thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs). Studies examining the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Autophosphorylation and consequent activation of dsRNA-PK in vitro and in vivo result in phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) and inhibition of protein synthesis. Expression of a functionally defective mutant of human dsRNA-PK in NIH 3T3 cells resulted in malignant transformation, suggesting that dsRNA-PK may function as a suppressor of cell proliferation and tumorigenesis.

Induction of growth suppression and modification of gene expression in multi-drug-resistant human glioblastoma multiforme cells by recombinant human fibroblast and immune interferon.

The combination of recombinant human fibroblast (IFN-beta) and immune (IFN-gamma) interferon induces enhanced growth suppression and modifies the antigenic phenotype in parental and multi-drug-resistant (MDR) human glioblastoma multiforme (GBM) cells. The present study was conducted to explore the mechanism underlying this cooperative interaction between interferons in inducing growth suppression in MDR-GBM cells. For this analysis we have utilized 2 MDR-GBM cell lines which display a differential sensitivity to growth suppression when exposed to IFN-beta or IFN-gamma. GBM-18-B3 (MDR) cells are more sensitive to growth inhibition by IFN-gamma than by IFN-beta, whereas GBM-18-A3 (MDR) cells are inhibited to a greater degree by IFN-beta than by IFN-gamma. In both cell types, however, growth is suppressed to a greater degree by the combination of interferons than by equivalent concentrations of either type of interferon used alone. Growth suppression induced by the interferons, alone or in combination, was not associated with comparable changes in the steady-state level of MDRI mRNA. In addition, the anti-proliferative effect of interferon was similar in GBM-18 (MDR) cells grown in the presence or absence of colchicine. GBM-18-A3 and GBM-18-B3 cells differed in their de novo and interferon-inducible expression levels of IFN-beta-responsive genes, isg-15 and isg-54. In contrast, both cell types responded in a similar manner with respect to expression of the IFN-gamma-responsive gene, HLA Class II (HLA-DR beta), and HLA Class I, fibronectin and ICAM-I. No further increase in expression of any of the genes was observed which was unique to the combination of interferons.

Proliferative and antiproliferative effects of interferon-'Y and tumor necrosis factor-α. on cell lines derived from cervical and ovarian malignancies

Four human cell lines derived from cervical carcinomas (ME-180, SiHa, HT-3, and MS751) and three human cell lines derived from ovarian carcinomas (SK-OV-3, Caov-3, and NIH:OVCAR-3) were analyzed in vitro to determine the effect of recombinant interferon-y and recombinant human tumor necrosis factor-oc on cell growth and survival. The effects of interferon-y, tumor necrosis factor, and both interferon--y and tumor necrosis factor-a on cell growth were measured after.24 and 72 hours of incubation by the incorporation of chromium 51. The results of this analysis showed that all seven cell lines were resistant to the antiproliferative action of tumor necrosis factor-a, that the growth of most cell lines was inhibited by interferon-y by 72 hours of incubation, and that after 72 hours of incubation all cell lines demonstrated a synergistic antiproliferative response to the combination of interferon--y and tumor necrosis factor-oa. However, the effects of these cytokines on cell growth were found to differ among cell lines and varied with the concentration and the duration of incubation. The growth of one cell line (Caov-3) was stimulated by both tumor necrosis factor-a and interferon-y. These results suggest that the clinical effects of these cytokines on the growth of gynecologic cancers may be more complex than previously supposed.

Biochemical characterization of the membrane-associated phosphorylating proteins involved in the anti-proliferative effect of human recombinant interferon-alpha 2a (rIFN-.ALPHA.2a) in daudi cells.

The anti-proliferative effect of interferons (IFNs) was investigated, focusing on the physiological activities of membrane-associated proteins involved in the progress of cell proliferation. In preliminary screening of the inhibitory effect of IFNs against the cell growth of various human hematopoietic tumor cells, Daudi cell was the most sensitive to rIFN-alpha 2a. SDS-PAGE followed by autoradiography detected that treatment of Daudi cells with rIFN-alpha 2a significantly reduced phosphorylation of the membrane-associated Mr 98,000 and 70,000 polypeptides. To determine the physiological significance of these phosphorylating polypeptides in mediation of the IFN effects, they were purified from Daudi cells. It was found that the molecular weight of the purified polypeptides was approximately 330,000 and it (designated 330-kDa protein) was consist of two distinct subunits [alpha-subunit (Mr 98,000) and beta-subunit (Mr 70,000)]. The biochemical properties, such as subunit structure, subunit phosphorylation, requirements for phosphorylating activity and protease sensitivity of the 330-kDa protein were similar to those reported for Na+, K(+)-ATPase. Evidence provided here suggests that the anti-proliferative action of IFNs may, at least in part, be implicated in the IFN-induced physiological impairment of the membrane-associated 330-kDa protein.

Human lung cancer nodules in organotypic culture: No evidence of correlation between the antiproliferative effects of interferons and the induction of 2′, 5′-oligoadenylate synthetase : Martyre M-C, Beaupain R, Falcoff E. U 196 INSERM, Institut Curie, F-75231 Paris Cedex 05. Tumor Biol 1988;9:263-9

Human lung cancer nodules in organotypic culture: No evidence of correlation between the antiproliferative effects of interferons and the induction of 2′, 5′-oligoadenylate synthetase : Martyre M-C, Beaupain R, Falcoff E. U 196 INSERM, Institut Curie, F-75231 Paris Cedex 05. Tumor Biol 1988;9:263-9

Antiproliferative effect of interferons on human prostate carcinoma cell lines.

The effect of purified human fibroblast beta-interferon (B-IFN) and recombinant alpha-2b-interferon (A-IFN) on cell proliferation was investigated in two human prostate carcinoma cell lines, named PC-3 and DU-145. Both cell lines respond to the antiproliferative action of interferon, B-IFN being more effective than A-IFN. PC-3 is more sensitive than DU-145 cell line, showing 95% inhibition of cell proliferation at the highest concentration of B-IFN. As interferons, besides reducing cell growth, are able to modify steroid receptor content in different hormone-sensitive human tumours, our results may be of some relevance as these drugs might be used to regulate both cell proliferation and hormone-sensitivity in prostate cancer.

Human lung cancer nodules in organotypic culture: No evidence of correlation between the antiproliferative effects of interferons and the induction of 2′,5′-oligoadenylate synthetase : Martyre M-C, Beaupain R, Falcoff E. U 196 INSERM, Institut Curie, F-75231 Paris Cedex 05. Tumor Biol 1988;9:263-9

Human lung cancer nodules in organotypic culture: No evidence of correlation between the antiproliferative effects of interferons and the induction of 2′,5′-oligoadenylate synthetase : Martyre M-C, Beaupain R, Falcoff E. U 196 INSERM, Institut Curie, F-75231 Paris Cedex 05. Tumor Biol 1988;9:263-9

Effect of Interferon on Secretion of Proteins by Various Murine Cell Lines

The interferons (IFNs) have been shown to be antagonistic to the growth stimulatory effects of mitogens on cultured cells. A report of the interactions of IFN-beta and platelet-derived growth factor on BALB/c-3T3 mouse cells established that IFN itself induced the secretion of a limited number of proteins from this cell line. The present work was undertaken to determine if other murine cell lines treated with homologous IFN-beta also secreted new or additional protein(s) in response to this agent and if this response correlated with other phenotypic properties of the cells. The cell lines examined included L929 cells and two derivatives of this line (GM347 and WDIFN), CAK-TK-, Swiss-3T3, and BALB/c-3T3. Each line was exposed to [35S]methionine in the absence and in the presence of IFN-beta, the supernatant fluids collected, and the radioactive, secreted proteins examined by fluorography after electrophoresis through SDS-containing polyacrylamide gels. Two cell lines (GM347 and Swiss-3T3) did not appear to secrete new or additional proteins after IFN treatment. However, four lines (L929, WDIFN, CAK-TK-, and BALB/c-3T3) did secrete new or additional proteins in response to IFN. Thus IFN-induced secretion of protein appeared to be a common but not universal phenomenon. In addition, although the number and apparent size(s) of the IFN-induced, secreted proteins were different in these various lines, one protein (Mr = 89-90,000) appeared to be secreted by each of them. In this respect it was unique. Moreover the IFN-induced secretion of protein did not appear to correlate with the antiviral or antiproliferative effects of IFN.

Antiproliferative Effects of Interferons against Human Bladder Carcinoma Cell Lines in vitro

In order to evaluate the antiproliferative effects of recombinant human interferon-gamma 2c (rHu IFN-gamma 2c), recombinant human interferon-gamma (rHu IFN-gamma), natural interferon-beta (IFN-beta), and their combination with cytotoxic agents, 17 different human bladder carcinoma cell lines were tested in vitro. The antiproliferative effects were compared in evaluating the tumor cell inhibiting potency of the different interferon (IFN) classes. It could be demonstrated that interferons have inhibiting effects on the bladder cancer cell multiplication rate, yet there are significant differences in the susceptibility of different IFN preparations on different cell lines. The cell lines BT1, RT4, EJ, 468P, 253J, SD, TCCSUP, and SW1738 can be defined as sensitive, T24, 647V, VM-CUB2, and J82 as semisensitive, HT1376, 5637, VM-CUB1, 639V and SW1710 as resistant upon treatment with IFN. The combination of rHu IFN-alpha 2c, IFN-beta, and rHu IFN-gamma seems to be more effective than treatment with rHu IFN-alpha 2c alone. The cytotoxic effect of doxorubicin on bladder cancer cells can be intensified by combining it with IFN.

Human Lung Cancer Nodules in Organotypic Culture: No Evidence of Correlation between the Antiproliferative Effects of Interferons and the Induction of 2′,5′-Oligoadenylate Synthetase

Alveolar II pulmonary tumor cells (A549), maintained in continuous tridimensional organotypic culture, were used in an attempt to investigate whether there could be a relationship of the 2',5'-oligoadenylate (2,5A) synthetase pathway to the antiproliferative activity of interferons (IFNs) in this particular tumor cell model. IFN-alpha 2, -beta and -gamma were used separately and in combinations. IFN-alpha 2 and -gamma demonstrated an inhibitory effect on the nodule growth, whereas IFN-beta did not. Moreover, combinations of IFN-alpha 2 and -gamma resulted in a significant synergistic antiproliferative activity; IFN-beta only potentiated slightly the effect of IFN-gamma. All three IFNs induced an increase in the 2,5A synthetase activity, indicating a discrepancy with the pattern of anticellular activity. Furthermore, whereas the combination of IFN-alpha 2 and -gamma resulted in a synergistic antiproliferative effect, no synergism was observed in the induction of the enzyme. These results show that there is a lack of correlation between the sensitivity or the resistance to IFNs of A549 tumor nodules and the induction of the 2,5A synthetase activity.

Induction of B-cell differentiation antigens in interferon- or phorbol ester-treated Daudi cells is impaired by inhibitors of ADP-ribosyltransferase.

Treatment of the Daudi Burkitt lymphoma-derived cell line with human interferon alpha, which inhibits cell proliferation in this system, induces differentiation of these B-lymphoid cells into cells with a plasmacytoid phenotype. This differentiation, quantified by the appearance of surface antigens characteristic of mature plasma cells, is impaired by addition to the culture medium of the ADP-ribosyltransferase (ADPRT; EC 2.4.2.30) inhibitors 3-methoxybenzamide or 3-aminobenzamide. These agents also protect the cells against the inhibition of proliferation induced by low doses of interferon alpha. In contrast, the large inhibition of thymidine incorporation into DNA caused by interferon treatment is not affected by the ADPRT inhibitors. The phorbol ester phorbol 12-tetradecanoate 13-acetate induces the same plasma cell surface antigens that are induced by interferon treatment, and this effect is also impaired by the ADPRT inhibitors. These results suggest that interferons and phorbol esters share a mechanism of action that requires ADPRT activity. Protection of the cells against the antiproliferative effect of interferons by the ADPRT inhibitors suggests that growth inhibition may be a consequence of cell differentiation. In contrast, the inhibition of thymidine incorporation alone is not sufficient for the cessation of cell proliferation and is not a true reflection of the rate of DNA synthesis.

Interferon inhibits preferentially the synthesis of proteins associated with growth stimulation of Swiss 3T3 mouse fibroblasts

The putative role of inhibition of protein synthesis within the antiproliferative effect of interferon was analyzed in serum-stimulated Swiss 3T3 mouse fibroblasts. We observed an apparent coupling between protein-synthesis inhibition during G 1 and a delayed entry into the S-phase. To reveal any specificity in the protein-synthesis inhibition, we measured the amounts of synthesis of 56 major individual proteins, by using isotope double-labelling and two-dimensional gel electrophoresis. Interferon inhibited preferentially the synthesis of proteins which were increased after serum stimulation, whereas proteins synthesized in constant or decreased amounts after serum stimulation were significantly more resistant. The effects of interferon were also compared to those of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), an inhibitor of transcription. All interferon-sensitive proteins studied were inhibited by DRB treatment, but in addition DRB also inhibited several proteins which were completely resistant to interferon. We conclude that interferon primarily inhibits protein synthesis originating from a subset of newly transcribed messenger RNAs. The mechanism(s) for inhibition of protein synthesis and the possible relationship to the antiproliferative and antiviral effects of interferon are discussed.

Synergistic antiproliferative effect of human interferons in combination with mismatched double‐stranded RNA on human tumor cells

Four human tumor cell lines were studied for their response to antiproliferative effects of various interferons (IFNs) alone and in combination with the novel mismatched dsRNA, r(I)n r(C12,U)n (Ampligen). RT4 cells (bladder carcinoma) were resistant to Ampligen alone, while A2182 (lung carcinoma), HT 1080 C14 (fibrosarcoma) and RT112 (bladder carcinoma) cells were inhibited in a dose-dependent manner. In contrast, RT4 cells were sensitive to the antitumor effects of IFNs as were HT1080 C14 and RT112 cells, while A2182 cells were resistant. In 3 of 4 cell lines, the recombinant IFNs were less effective than the corresponding natural IFNs when compared by analysis of variance on an IRU/ml basis over a range of concentrations. In all cell lines, a synergistic antiproliferative effect was seen with all IFN preparations studied in combination with Ampligen, as calculated by the isobole method according to Berenbaum (1981). The antiproliferative effect of IFN was potentiated greater than 3.3- to greater than 250-fold, depending on the cell lines, IFN, and concentrations used. Varying the concentration of beta ser-IFN while holding the Ampligen concentration constant gave synergy at all of the physiologically achievable concentrations tested in RT4 cells. These results indicate that: Ampligen worked synergistically with all IFNs in all cell lines studied; growth inhibition of cells resistant to IFNs can be potentiated by low doses of Ampligen; the antiproliferative effect of IFNs can be potentiated by Ampligen in Ampligen-resistant cells; and Ampligen may work by a mechanism other than, or in addition to, the induction of IFNs.

Antiviral and antiproliferative effects of interferons in quiescent fibroblasts are dissociable

Quiescent mouse Swiss 3T3 cells or human fibroblasts treated with interferon for only 2 hr before the addition of serum are effectively protected against virus infection for up to 24 hr after the addition of serum and the removal of interferon. In sharp contrast, exposure of quiescent fibroblasts to interferon only before addition of serum or the peptides epidermal growth factor and insulin, has no inhibitory effect on cell proliferation. Further, even when interferon is added after the addition of growth factors, an exposure of several hours is required to inhibit stimulation of DNA synthesis. These findings strongly suggest that the antiviral action of interferons is dissociable from their ability to inhibit cell proliferation. The implications of these findings for elucidating the molecular events leading to the biological responses elicited by interferon are discussed.

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events: Studies with vascular smooth muscle cells and endothelial cells

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [ 3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFNα, recombinant human IFNα2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.

Characteristics of the Induction of Two Double-Stranded RNA-Dependent Enzymes by Interferon in Parental and Variant L Cells

The interferon (IFN)-dependent induction of two double-stranded RNA-dependent enzymes was examined in L cells and an L-cell variant (WDIFN) which is highly resistant to the inhibitory effects of IFN on cellular multiplication. IFN, in a concentration-dependent manner, inhibited the multiplication of parental L cells and induced increased levels of the double-stranded RNA-dependent enzymes in parental L cells. Although WDIFN cells were resistant to the antiproliferative effects of IFN, the cells responded to IFN by increasing their levels of the double-stranded RNA-dependent enzymes. However, the level of activity of each enzyme was lower in the WDIFN line than the parental line when both lines were treated with similar concentrations of IFN. The reduced response of the WDIFN line was not the result of the line being a heterogeneous population of cells nor of IFN being more unstable in the presence of WDIFN cells. In addition there was no evidence that WDIFN cells produced a mitogenic factor that could overcome the antiproliferative effects of IFN, nor that sodium butyrate could increase the sensitivity of WDIFN cells to the antiproliferative effects of IFN.

Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells.

We have analyzed the effects of various human interferons produced in bacteria and the antileukemic compound mezerein (MEZ) on growth and melanogenesis in human melanoma cells. In four human melanoma cell lines, recombinant human fibroblast interferon (IFN-beta) was more active than recombinant human leukocyte interferons (IFN-alpha A, IFN-alpha D, or IFN-alpha A/D (Bgl] in inhibiting cellular proliferation. When monolayer cultures were exposed to 1000 IU/ml IFN-beta for four days the degree of growth inhibition in the different melanoma cell lines varied between 94 and 26%. Similarly, four days growth in medium containing 10 ng/ml MEZ resulted in either no inhibition of growth or as much as 53% inhibition of growth, depending on the specific melanoma cell line tested. MEZ induced dendrite-like processes, cytoplasmic projections morphologically similar to those normally found in neurons and melanocytes, in all four melanoma cell lines, whereas none of the interferons tested had this effect. The combination of interferon and MEZ resulted in a dramatic inhibition in cellular proliferation in all four melanoma cell lines. When cell extracts were assayed for melanin content, a marker of melanoma cell differentiation, the combination of IFN-beta and MEZ resulted in higher levels of melanin than with either agent alone. Dendrite-like formation was also prominent in the cultures treated with this combination. These results indicate that the antiproliferative effect of interferon toward human melanoma dells can be enhanced by treatment with MEZ and that this effect is associated with an enhancement of terminal differentiation.