Fibroblast Interferon Research Articles

Recent insights into apoptosis and toxic autophagy: The roles of MDA-7/IL-24, a multidimensional anti-cancer therapeutic.

Apoptosis and autophagy play seminal roles in maintaining organ homeostasis. Apoptosis represents canonical type I programmed cell death. Autophagy is viewed as pro-survival, however, excessive autophagy can promote type II cell death. Defective regulation of these two obligatory cellular pathways is linked to various diseases, including cancer. Biologic or chemotherapeutic agents, which can reprogram cancer cells to undergo apoptosis- or toxic autophagy-mediated cell death, are considered effective tools for treating cancer. Melanoma differentiation associated gene-7 (mda-7) selectively promotes these effects in cancer cells. mda-7 was identified more than two decades ago by subtraction hybridization showing elevated expression during induction of terminal differentiation of metastatic melanoma cells following treatment with recombinant fibroblast interferon and mezerein (a PKC activating agent). MDA-7 was classified as a member of the IL-10 gene family based on its chromosomal location, and the presence of an IL-10 signature motif and a secretory sequence, and re-named interleukin-24 (MDA-7/IL-24). Multiple studies have established MDA-7/IL-24 as a potent anti-cancer agent, which when administered at supra-physiological levels induces growth arrest and cell death through apoptosis and toxic autophagy in a wide variety of tumor cell types, but not in corresponding normal/non-transformed cells. Furthermore, in a phase I/II clinical trial, MDA-7/IL-24 administered by means of a non-replicating adenovirus was well tolerated and displayed significant clinical activity in patients with multiple advanced cancers. This review examines our current comprehension of the role of MDA-7/IL-24 in mediating cancer-specific cell death via apoptosis and toxic autophagy.

VITAMIN D NEPHROPROTECTIVE STRATEGY OF CHILDREN WITH CHRONIC RENAL DISEASE

According to current conception, the process of forming of nephrosclerosis despite the origins is detected by the force of the same cellular and molecular mechanisms. Hence primary triggers cause elevated production of range of cellular response mediators, cytokines and growth factors such as transforming plateled-derived growth factor, fibroblast growth factor, interferon gamma, nuclear factor and others, by means of sequential processes, is followed by the replacement of kidney tissue. One of the most studied aspects of the process of nephrosclerosis is the influence of anginotensin II. Therefore, despite the triggering factors, development and progression of nephrosclerosis all children with chronic renal disease are in a risk group with predisposition for renal insufficiency and it is required to prescribe nephroprotective therapy, i.e. the drugs that will influence one of the links of the nephrosclerosis, in order to slow down its progression. Currently, with nephroprotective goal in early age are used the following elements - angiotensin converting enzyme inhibitors and angiotensin receptors, antogonists (blockers). Lately, the active search for effective and safe drugs with nephroprotective effect on one side and great interest towards undiscovered before qualities of Vitamin D caused a huge amount of works that support the effect of Vitamin D. Prescription of Vitamin D to animals with uremia was followed by the drop of apoptosis of podocides and loss of the protein that is contained in the split diaphragm that caused decrease in proteinuria. Vitamin D suppresses protophibrotic TGF-B1 in tubular epithelial cells. The regression of tubulointerstitial phibrosis under the influence of Vitamin D was confirmed based on the cases of animals with obstruction of urethra. Along with the use of the mentioned above groups of drugs, which effectiveness was proved experimentally and clinically, we strongly believe the more thorough study of the Vitamin D drug use is needed, especially to study the prevention and slowing down of the progression of nephrosclerosis in the cases of children with chronic renal disease. It is crucial to detect the exact dosage of the drug, the prescription period and the methods for the control of the effective concentration of the drug.

Role of MDA-7/IL-24 a Multifunction Protein in Human Diseases.

Subtraction hybridization identified genes displaying differential expression as metastatic human melanoma cells terminally differentiated and lost tumorigenic properties by treatment with recombinant fibroblast interferon and mezerein. This approach permitted cloning of multiple genes displaying enhanced expression when melanoma cells terminally differentiated, called melanoma differentiation associated (mda) genes. One mda gene, mda-7, has risen to the top of the list based on its relevance to cancer and now inflammation and other pathological states, which based on presence of a secretory sequence, chromosomal location, and an IL-10 signature motif has been named interleukin-24 (MDA-7/IL-24). Discovered in the early 1990s, MDA-7/IL-24 has proven to be a potent, near ubiquitous cancer suppressor gene capable of inducing cancer cell death through apoptosis and toxic autophagy in cancer cells in vitro and in preclinical animal models in vivo. In addition, MDA-7/IL-24 embodied profound anticancer activity in a Phase I/II clinical trial following direct injection with an adenovirus (Ad.mda-7; INGN-241) in tumors in patients with advanced cancers. In multiple independent studies, MDA-7/IL-24 has been implicated in many pathological states involving inflammation and may play a role in inflammatory bowel disease, psoriasis, cardiovascular disease, rheumatoid arthritis, tuberculosis, and viral infection. This review provides an up-to-date review on the multifunctional gene mda-7/IL-24, which may hold potential for the therapy of not only cancer, but also other pathological states.

A functional in vitro model of heterotypic interactions reveals a role for interferon-positive carcinoma associated fibroblasts in breast cancer.

BackgroundCancer-associated fibroblasts (CAFs) play an important role in breast cancer pathogenesis by paracrine regulation of breast cancer cell biology. Several in vitro and mouse models have characterized the role of cell contact and cytokine molecules mediating this relationship, although few reports have used human CAFs from breast tumors.MethodsPrimary breast CAF cultures were established and gene expression profiles analysed in order to guide subsequent co-culture models. We used a combination of colorimetric proliferation assays and gene expression profiling to determine the effect of CAFs on the MCF-7 breast cancer cell in an indirect co-culture system.ResultsUsing gene expression profiling, we found that a subgroup of breast CAFs are positive for a type one interferon response, confirming previous reports of an activated type one interferon response in whole tumor datasets. Interferon positive breast cancer patients show a poor prognostic outcome in an independent microarray dataset. In addition, CAFs positive for the type one interferon response promoted the growth of the MCF-7 breast cancer cell line in an indirect co-culture model. The addition of a neutralizing antibody against the ligand mediating the type one response in fibroblasts, interferon-β, reverted this co-culture phenotype. CAFs not expressing the interferon response genes also promoted the growth of the MCF-7 breast cancer cell line but this phenotype was independent of the type one fibroblast interferon ligand.ConclusionsPrimary breast CAFs show inter-patient molecular heterogeneity as evidenced by interferon response gene elements activated in a subgroup of CAFs, which result in paracrine pro-proliferative effects in a breast cancer cell line co-culture model.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1117-0) contains supplementary material, which is available to authorized users.

Low concentration of PDGF-AB shows synergism with IFN-α in induction of IFN-β and -γ in MRC5 fibroblasts

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. Also it has been reported as a strong suppressor of interferon (IFN) expression. IFN-α has opposite effect on fibroblast function and IFN induction. Here is our early report on the effect of low concentration of PDGF-AB alone or in combination with IFN-α on IFN mRNA production in MRC5 fibroblasts. MRC5 cells incubated with IFN-α or PDGF-AB, alone or in combination, produced significant induction of IFN-α, -β and -γ mRNA in comparison with untreated cells. The induction was dose-dependent, with higher effect in cells treated with lower concentrations of PDGF-AB. Also, low concentration of PDGF-AB showed synergism with IFN-α in IFN-β and -γ induction. Results presented here open new possibilities in multi-cytokine therapy and expand previous results on PDGF activity.

Inhibition of Endogenous Interferon Beta by Neutralizing Antibodies Against Recombinant Interferon Beta

To determine if neutralizing antibodies (NAbs) against interferon beta from patients with multiple sclerosis (MS) cross-react with other type 1 interferons, especially endogenous interferon beta, and thus might impede the immune systems of affected patients. Masked serum samples from MS patients were challenged in vitro against recombinant interferon beta-1a and interferon beta-1b, as well as human leukocyte interferon and fibroblast interferon, the latter representing endogenous interferon. The neutralizing capacity of serum samples on these type 1 interferons was assessed using a luciferase reporter gene assay. Randomly selected samples were titrated to further delineate the cross-reactivity of antibodies. University medical center in Düsseldorf, Germany. We randomly selected 150 samples from interferon beta-treated MS patients who had previously been tested for the presence of binding antibodies and NAbs. Neutralization of interferon beta bioactivity and cross-reactivity of anti-interferon beta antibodies. Antibody-mediated neutralization of interferon beta bioactivity in vitro against recombinant interferon beta was observed in all serum samples that had previously tested positive for binding antibodies and NAbs. A neutralizing pattern comparable to that of recombinant interferon beta was observed when endogenous interferon was assessed, reflecting cross-reactivity of NAbs. No differences in neutralization between recombinant and endogenous interferon were observed with respect to the interferon beta preparation used for treatment. Furthermore, no neutralization of other type 1interferons by NAbs could be detected. A proportion of MS patients who are treated with recombinant interferon beta develop NAbs that also neutralize endogenous interferon. Because NAbs at high titers can persist for years, these antibodies may impede the immune system in affected MS patients regardless of their current treatment regimen.

Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-alpha 2b and human IFN-gamma in healthy blood donors

Recently, naturally occurring antibodies to IFN-alpha were discovered in a few systemic lupus erythematosus (SLE) and cancer patients; however, in most patients monitored for anti-IFN antibodies before treatment, no antibodies were found. In an attempt to explain the 'IFN-blocking effect' that we observed in all serum samples we investigated 200 sera from healthy blood donors. We isolated the globulin fraction, and used rabbit anti-human IgG and IgM columns, protein A columns and T-gel affinity chromatography to isolate human IgG and IgM. All sample fractions were tested in a biological IFN neutralization assay by means of a sensitive MTT-assay. We found that normal human serum contained autoantibodies to crude human leucocyte IFN, native human fibroblast IFN, recombinant human leucocyte IFN-alpha 2b and recombinant human IFN-gamma, and that these naturally occurring antibodies were biologically active immunoglobulins of IgG and IgM type. These anti-IFN antibodies were also present in purified human normal immunoglobulin pools. We conclude that all humans have naturally occurring anti-interferon antibodies in their serum, and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by immunoglobulins.

Intramuscular gene transfer of fibroblast growth factor-1 using improved pCOR plasmid design stimulates collateral formation in a rabbit ischemic hindlimb model

Fibroblast growth factor 1 (FGF1) is an angiogenic factor known to play a role in the growth of arteries. The purpose of this study was to evaluate the usefulness of direct intramuscular injection of an optimized expression plasmid encoding FGF1 to augment collateral formation and tissue perfusion in a rabbit ischemic hindlimb model. Truncated FGF1 fused to the human fibroblast interferon (FIN) signal peptide was expressed from a newly designed plasmid backbone with an improved safety profile for gene therapy applications. In vitro, optimization of plasmid design yielded in a dramatic increase in expression efficiency for FGF1, independent of the presence of a signal peptide, as analyzed by Western Blotting. In vivo, successful transgene expression could be demonstrated by FGF1 immunostaining after gene application. FGF1 plasmid containing FIN signal peptide (100, 500, and 1,000 mug), when injected into ischemic muscle areas of rabbits 10 days after ligation of the external iliac artery, exhibited a pronounced therapeutic effect on collateral formation to the ischemic hindlimb in a dose-depending manner, as assessed by physiological (blood pressure ratio, maximal intra-arterial Doppler flow) and anatomical (angiographic score, histologic evaluation of capillary density) measurements 30 days after therapy, compared to saline or lacZ control plasmid. FGF1 plasmid without a signal peptide sequence resulted in a comparable therapeutic effect on collateral formation at comparable doses (500 and 1,000 mug). Our results indicate that intramuscular FGF1 gene application could be useful to stimulate collateral formation in a situation of chronic peripheral ischemia. The presence of a signal peptide does not seem to be obligatory to achieve bioactivity of intramuscular transfected FGF1. An optimized vector design improved both biosafety of gene transfer and expression efficiency of the transgene, rendering this vector highly suitable for human gene therapy. Therefore, this new generation vector encoding FGF1 might be useful as an alternative treatment for patients with chronic ischemic disorders not amenable to conventional therapy.

Treatment of low-grade diffuse astrocytomas by surgery and human fibroblast interferon without radiation therapy.

Low-grade diffuse astrocytomas are slowly growing tumors with a relatively long overall survival. However, a substantial proportion undergoes dedifferentiation to a more malignant phenotype. Considerable controversy exists as to the best therapeutic management for patients with such tumors. Over the past decade, we have applied human fibroblast interferon (HFIF) therapy without radiation therapy to low-grade astrocytomas. We investigated 28 patients with WHO grade II astrocytomas of the cerebral hemispheres treated by surgery plus HFIF therapy. The overall response rate to the HFIF therapy was 36%. All side-effects of HFIF were transient, tolerable and manageable. The 5-year progression free survival and overall survival probabilities were 65% and 96%, respectively. Although our data from small cohort of patients may have modest value, our results suggest that HFIF may be useful in treating low-grade diffuse astrocytomas.

The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells.

Human melanoma cells growth arrest irreversibly, lose tumorigenic potential and terminally differentiate after treatment with a combination of fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ). Applying subtraction hybridization to this model differentiation system permitted cloning of melanoma differentiation associated gene-7, mda-7. Expression of mda-7 inversely correlates with melanoma development and progression, with elevated expression in normal melanocytes and nevi and increasingly reduced expression in radial growth phase, vertical growth phase and metastatic melanoma. When expressed by means of a replication incompetent adenovirus (Ad.mda-7) growth of melanoma, but not normal early passage or immortal human melanocytes, is dramatically suppressed and cells undergo programmed cell death (apoptosis). Infection of metastatic melanoma cells with Ad.mda-7 results in an increase in cells in the G(2)/M phase of the cell cycle and changes in the ratio of pro-apoptotic (BAX, BAK) to anti-apoptotic (BCL-2, BCL-XL) proteins. Ad.mda-7 infection results in a temporal increase in mda-7 mRNA and intracellular MDA-7 protein in most of the melanocyte/melanoma cell lines and secretion of MDA-7 protein is readily detected following Ad.mda-7 infection of both melanocytes and melanoma cells. The present studies document a differential response of melanocytes versus melanoma cells to ectopic expression of mda-7 and support future applications of mda-7 for the gene-based therapy of metastatic melanoma.

Cloning and characterization of human ubiquitin-processing protease-43 from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol RaSH

Defects in growth control and differentiation occur frequently in human cancers. In the case of human melanoma cells, treatment with a combination of fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss of proliferative potential and tumorigenic properties with a concomitant induction of terminal differentiation. These changes in cellular properties are associated with an induction and suppression in specific subsets of genes that occur in a temporal manner. To identify the complete repertoire of gene changes occurring during melanoma reversion to a more differentiated state a number of molecular approaches are being used. These include, subtraction hybridization using temporally spaced cDNA libraries, random cDNA isolation and evaluation by reverse Northern blotting and high throughput microarray analysis of subtracted cDNA clones. In the present study we have used a novel approach, rapid subtraction hybridization (RaSH), to identify and clone an additional gene of potential relevance to cancer growth control and terminal cell differentiation. RaSH has identified a human ubiquitin-processing protease gene, HuUBP43, that is differentially expressed in melanoma cells as a function of treatment with IFN-β or IFN-β + MEZ. HuUBP43 is a type I interferon inducible gene that is upregulated in a diverse panel of normal and tumor cells when treated with IFN-β via the JAK/STAT kinase pathway. This gene may contribute to the phenotypic changes induced by IFN-β during growth arrest and differentiation in human melanoma cells and other cell types as well as the antiviral and growth inhibitory effects of interferon.

Palliation by Intratumoral Infiltration with Natural Interferon-beta

Background: Curative approaches to tumor therapy have achieved greater importance through new developments such as cytostatic agents and their combination with other therapy concepts, but for the majority of tumor patients only palliative therapy is possible. Size or location of tumor manifestations can result in severe discomfort for patients, in some cases even in a reduction of functionality. Patients and Methods: For the purpose of this study, a total of 55 patients with a variety of advanced malignant diseases nonresponding or progressive under radio- and/or chemotherapy were treated by intratumoral injection of natural human fibroblast interferon (nIFN-β). nIFN-β was administered intralesionally 3 times per week for at least 4 weeks at doses of 2–8 MIU, depending on tumor size. Local tumor response was observed over a median follow-up period of 18 weeks. Results: In 37 patients (67%) a complete or partial remission of the local tumor manifestation was achieved. Survival times of these patients were improved compared with those of patients without local tumor remission. 16 patients without significant change of tumor volume benefited from the palliative (extensive analgesic) effect of the nIFN-β therapy. During treatment, none of the patients showed a progression of the locally treated tumor, even when the basic malignant disease progressed. The side effects of the nIFN-β therapy were tolerable, and no patient discontinued therapy. Conclusion: From these observations, intralesional nIFN-β therapy of malignant tumors appears to be a useful palliative addition to radio- and/or chemotherapy with the aim of local control of tumor growth.

Regulation of mda-7 gene expression during human melanoma differentiation

Induction of irreversible growth arrest and terminal differentiation in human melanoma cells following treatment with recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expression in differentiating human melanoma cells. The mda-7 gene is actively transcribed in uninduced HO-1 human melanoma cells and the rate of transcription of mda-7 is not significantly enhanced by treatment with IFN-beta, MEZ or IFN-beta+MEZ. The high basal activity of the mda-7 promoter in uninduced melanoma cells and the absence of enhancing effect upon treatment with differentiation inducers is corroborated by transfection studies using the promoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT - PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-beta+MEZ and to a lesser degree in MEZ treated cells. We show that induction of terminal differentiation of HO-1 cells with IFN-beta+MEZ dramatically increases the half-life of mda-7 mRNA while treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitutive activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda-7 expression during terminal differentiation. The 3' UTR region of mda-7 contains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled by a labile protein factor(s). Substitution of the SV40 polyadenylation signal sequence in the luciferase reporter plasmid with the mda-7-ARE-3'-UTR renders the Luciferase message unstable when expressed in proliferating and reversibly differentiated melanoma cells. In contrast, the luciferase message is stabilized when the mda-7-ARE-3'-UTR construct is expressed in terminally differentiated HO-1 cells. These results provide compelling evidence that mda-7 expression during terminal differentiation in human melanoma cells is regulated predominantly at a posttranscriptional level.

AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation.

Treatment of human melanoma cells with a combination of recombinant fibroblast interferon (IFN-beta) and the protein kinase C (PKC) activator mezerein (MEZ) causes a rapid and irreversible suppression in growth and terminal cell differentiation. Temporal subtraction hybridization combined with random clone selection, reverse Northern hybridization, high throughput microchip cDNA array screening, and serial cDNA library arrays permit the identification and cloning of genes that are differentially expressed during proliferative arrest and terminal differentiation in human melanoma cells. A specific melanoma differentiation associated (mda) gene, mda-7, exhibits reduced expression as a function of melanoma progression from melanocyte to metastatic melanoma. In contrast, treatment of metastatic melanoma cells with IFN-beta + MEZ results in expression of mda-7 mRNA and protein. To evaluate the mechanism underlying the differential expression of mda-7 as a function of melanoma progression and induction of growth arrest and differentiation in human melanoma cells the promoter region of this gene has been isolated from a human placental genomic library and characterized. Sequence analysis by GCG identifies multiple recognition sites for the AP-1 and C/EBP transcription factors. Employing a heterologous mda-7 luciferase gene reporter system, we demonstrate that ectopic expression of either AP-1/cJun or C/EBP can significantly enhance expression of the mda-7 promoter in melanoma cells. In contrast, a dominant negative mutant of cJun, TAM67, is devoid of promoter-enhancing ability. Western blot analyses reveals that cJun and the C/EBP family member C/EBP-beta are physiologically relevant transcription factors whose expression corresponds with mda-7 mRNA expression. Electrophoretic mobility shift assays (EMSA) performed using nuclear protein extracts from terminally differentiated human melanoma cells document binding to regions of the mda-7 promoter that correspond to consensus binding sites for AP-1 and C/EBP. These results provide further mechanistic insights into the regulation of the mda-7 gene during induction of terminal cell differentiation in human melanoma cells.

Differentiation induction subtraction hybridization (DISH): a strategy for cloning genes displaying differential expression during growth arrest and terminal differentiation.

Human cancers often display aberrant patterns of differentiation. By appropriate chemical manipulation, specific human cancers, such as human melanoma, leukemia and neuroblastoma, can be induced to lose growth potential irreversibly and terminally differentiate. Treatment of HO-1 human melanoma cells with a combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) results in irreversible growth arrest, a suppression in tumorigenic properties and terminal cell differentiation. A potential mechanism underlying these profound changes in cancer cell physiology is the activation of genes that can suppress the cancer phenotype and/or the inactivation of genes that promote the cancer state. To define the repertoire of genes modulated as a consequence of induction of growth arrest and terminal differentiation in human melanoma cells, we are using a differentiation induction subtraction hybridization (DISH) approach. A subtracted cDNA library, differentiation inducer treated cDNAs minus uninduced cDNAs, was constructed that uses temporally spaced mRNAs isolated from HO-1 cells treated with IFN-beta+MEZ. Approximately 400 random clones were isolated from the subtracted DISH library and analyzed by reverse Northern and Northern blotting approaches. These strategies resulted in the identification and cloning of both 30 known and 26 novel cDNAs displaying elevated expression in human melanoma cells induced to growth arrest and terminally differentiate by treatment with IFN-beta+MEZ. The DISH scheme and the genes presently identified using this approach should provide a framework for delineating the molecular basis of growth regulation, expression of the transformed phenotype and differentiation in melanoma and other cancers.

Identification and temporal expression pattern of genes modulated during irreversible growth arrest and terminal differentiation in human melanoma cells.

Abnormalities in differentiation are common occurrences in human cancers. Treatment of human melanoma cells with the combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) results in a loss of tumorigenic potential that correlates with an irreversible suppression in proliferative ability and induction of terminal differentiation. It is hypothesized that this is associated with the differential expression of genes that may directly regulate cancer cell growth and differentiation. To define the relevant gene expression changes that correlate with and potentially control these important cellular processes a differentiation induction subtraction hybridization (DISH) scheme is being used. A temporally spaced subtracted differentiation inducer treated (TSS) cDNA library was constructed and differentially expressed DISH clones were isolated and evaluated using a high throughput microchip cDNA (Synteni) array screening approach. Verification of differential gene expression for specific cDNAs was confirmed by Northern blotting. The temporal kinetics of regulation and the expression pattern of DISH genes were also evaluated by microchip cDNA array screening. Using this approach with 1000 DISH cDNA clones (approximately 10% of the DISH library) has resulted in the identification and cloning of both 26 known and 11 novel cDNAs of potential relevance to growth control and terminal differentiation in human melanoma cells.

Regulation of human fibroblast interferon gene expression by human leukocytic interferon and cytopathic α-virus

Natural human α-interferon and Sindbis α-virus induce production of ²1-interferon mRNA in human fibroblasts. Accumulation and regulation of ²1-interferon mRNA splicing after induction with α-interferon and virus were different. Expression of the ²1-interferon gene was assessed by conjugated reverse transcription and polymerase chain reaction (semiquantitative variant).

Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene

Subtraction hybridization using a cDNA library prepared from temporally spaced mRNAs from human melanoma cells treated with recombinant human fibroblast interferon (IFN- β) plus mezerein (MEZ) that induces terminal differentiation ( tester cDNA library) and a temporally spaced cDNA library prepared from actively proliferating melanoma cells ( driver cDNA library) produced a T emporally S paced S ubtracted ( TSS) cDNA library. This approach resulted in the identification of melanoma differentiation associated ( mda) genes displaying both enhanced and suppressed expression during growth inhibition and differentiation. In the present report, we describe a novel cDNA mda-9 that consists of 2084 nucleotides, and encodes a protein of 298 amino acids with a predicted M r of approx. 33 kDa. Treatment of human SV40-immortalized normal melanoma cells with immune interferon, INF- γ, induces growth suppression and enhances mda-9 expression without inducing terminal differentiation. These results establish that induction of terminal differentiation in human melanoma cells, using the combination of a type I interferon (IFN- β)+MEZ, can elicit signaling pathways and gene expression changes also regulated by type II immune interferon.

Chicken-embryo fibroblasts produce two types of interferon upon stimulation with Newcastle disease virus

Controversy has long surrounded the question of whether chickens, like mammals, can produce two types of interferon (IFN). Recently, type-I and type-II chicken IFNs have been cloned. Our study focuses on the further characterization of native fibroblast and spleen IFNs and shows that chicken embryo fibroblasts (CEFs) produce a mixture of type-I and type-II IFNs. IFN was purified by three different methods, controlled pore-glass chromatography, ion-exchange chromatography and preparative SDS-PAGE. Three protein bands showing IFN-like anti-viral activity, from CEFs which had been virus-stimulated for IFN production, were detected at 25, 27 and 29 kDa. Polyclonal antibodies produced against these bands showed partial cross-reaction with purified media from mitogen-stimulated spleen cells in ELISA, western blot analysis and anti-viral activity neutralization assay. Differences between purified conditioned media from CEF and spleen were found with respect to the stimulation of macrophages for nitric oxide production, pH stability and signal transduction pathways; only CEF IFN activated the IFN-stimulated gene factor-3 complex, whereas both CEF and spleen IFNs activated the IFN regulatory factor-1 gene. These findings concur with the differences that are known to exist between mammalian type-I and type-II IFNs. Attempts at sequencing the 25 and 27 kDa proteins by Edman degradation yielded evidence of N-terminal blockage.

Suppression of human ribosomal protein L23A expression during cell growth inhibition by interferon-beta.

Interferons inhibit cell growth in normal and tumor-derived cells. The molecular basis of interferons antiproliferative activity remains to be defined. Using subtraction hybridization, a human melanoma differentiation associated gene, mda-20, has been identified that is down-regulated by treatment with interferon. Sequence analysis indicates that mda-20 is human ribosomal protein L23a (rp L23a). The mRNA levels of rp L23a and growth are diminished in a variety of human tumor cell lines following treatment with human fibroblast interferon, interferon-beta (IFN-beta). Expression of rp L23a is also reduced in human melanoma cells treated with human leukocyte (IFN-alpha) and immune (IFN-gamma) interferons, but not by growth inhibition resulting from serum starvation. These findings suggest that growth suppression alone is not sufficient to reduce rp L23a expression. Instead, reduced rp L23a mRNA results from biochemical changes mediated by interferons. Ectopic expression of an antisense rp L23a sequence in human HeLa cervical carcinoma cells results in a reduction in colony formation indicating a direct antiproliferative effect by inhibiting rp L23a expression. The mechanism underlying inhibition in rp L23a expression in IFN-beta-treated cells may involve antisense rp L23a RNA. These results suggest that rp L23a may be one of the target molecules involved in mediating growth inhibition by interferon.