Human Interferon-beta Research Articles

A striking similarity in the organization of the E-selectin and beta interferon gene promoters.

Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.

An HMG-like protein that can switch a transcriptional activator to a repressor.

One protein can activate some genes and repress others in the same cell. The Drosophila protein Dorsal (which, like the human protein NF-kappa B3, is a member of the Rel family of transcriptional activators) activates the twist gene and represses the zen gene in the ventral region of early embryos. Here we describe a Drosophila HMG1 protein, called DSP1 (dorsal switch protein), that converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed a negative regulatory element (NRE), found adjacent to Dorsal-binding sites in the zen promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta (IFN-beta) enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription.

Viral induction of the human beta interferon promoter: modulation of transcription by NF-kappa B/rel proteins and interferon regulatory factors

Multiple regulatory domains within the -100 region of the beta interferon (IFN-beta) promoter control the inducible response of the IFN gene to virus infection. In this study, we demonstrate that the formation of NF-kappa B-specific complexes on the positive regulatory domain II (PRDII) precedes the onset of detectable IFN-beta transcription in Sendai virus-infected cells. By using NF-kappa B subunit-specific antibodies, a temporal shift in the composition of NF-kappa B subunits in association with the PRDII domain is detected as a function of time after virus infection. Furthermore, a virus-induced degradation of I kappa B alpha (MAD3) protein is observed between 2 and 8 h after infection; at later times, de novo synthesis of I kappa B alpha restores I kappa B alpha to levels found in uninduced cells and correlates with the down regulation of IFN-beta transcription. In cotransfection experiments using various NF-kappa B subunit expression plasmids and two copies of PRDII/NF-kappa B linked to a chloramphenicol acetyltransferase reporter gene, we demonstrate that expression of p65, c-Rel, or p50 or combinations of p50-p65 and p65-c-Rel differentially stimulated PRDII-dependent transcription. Coexpression of I kappa B alpha completely abrogated p65-, c-Rel-, or p65-p50-induced gene activity. When the entire IFN-beta promoter (-281 to +19) was used in coexpression studies, synergistic stimulation of IFN-beta promoter activity was obtained when NF-kappa B subunits were coexpressed together with the IFN regulatory factor 1 (IRF-1) transcription factor. Overexpression of either I kappa B or the IRF-2 repressor was able to abrogate inducibility of the IFN-beta promoter. Thus, multiple regulatory events--including differential activation of DNA-binding NF-kappa B heterodimers, degradation of I kappa B alpha, synergistic interaction between IRF-1 and NF-kappa B, and decreased repression by I kappa B and IRF-2--are all required for the transcriptional activation of the IFN-beta promoter.

Effect of cytokines and prostaglandins on the growth of chick retinal pigment epithelial cells.

The effect of cytokines and prostaglandins on the growth of chick retinal pigment epithelial (RPE) cells was studied in vitro. The growth of RPE cells was evaluated by [3H]-thymidine uptake. Human recombinant interleukin-1 beta, interferon-beta and tumor necrosis factor-alpha stimulated the growth of RPE cells at 50-200 units/ml. Prostaglandins such as PGE1, PGE2 and PGF2 alpha suppressed RPE cell growth at 10(-5) approximately (-6)M. Indomethacin, an inhibitor of cyclooxygenase, did not affect the spontaneous RPE cell growth at 10(-7) approximately (-9) M. However, 10(-7) M indomethacin enhanced RPE cell growth stimulated by cytokines synergistically. The addition of 10(-6) M PGF2 alpha suppressed the enhanced growth of RPE cells which was induced with cytokines and indomethacin. Furthermore, RPE cells stimulated with cytokines produced PGE2. These results suggest that RPE cells produce prostaglandins which have a negative regulatory role in RPE cell growth.

Pharmacokinetics and toxicity of inhaled human natural interferon-beta in patients with lung cancer.

Eight patients with a thoracic malignancy inhaled single doses of 3-100 x 10(6) IU of human natural interferon (IFN)-beta via a dosimeter-equipped jet nebulizer. After inhalation IFN-beta was not detectable in any of the serum samples as measured using an antiviral bioassay. Except for a slight body temperature rise and an 18% decrease in peak expiratory flow rate observed in 1 patient the patients did not experience any major side effects. Inhaled natural IFN-beta in this study was tolerated better than inhaled IFN-alpha or IFN-gamma in our previous studies. IFN-beta might thus be a promising local treatment for pulmonary diseases. Further work is needed to investigate the concentrations of biologically active IFN in alveolar epithelial lining fluid after inhalation and the therapeutic effect of inhaled IFN-beta.

Efficient transfection of human interferon-beta gene to human glioma cells by means of cationic multilamellar liposomes coupled with a monoclonal antibody [corrected

We have been devoting our efforts to develop the useful liposomes that have high potentials of gene transfer and we aim human gene therapy for malignant glioma with cytokine genes. In our previous study, we prepared our original reverse-phase evaporation vesicles (REV) by an improved procedure of reverse-phase evaporation method. However, this procedure was very complicate. In this paper, a simple procedure for the preparation of cationic multilamellar vesicles (MLV) was introduced, and efficient expression and growth-inhibitory effect of MLV with entrapped human interferon-beta gene to glioma cells was comparable to that obtained with REV. Considering the present experiments, MLV seem to be more preferable for clinical application.

Positive interactions between human interferon and cepharanthin against human cancer cells in vitro and in vivo.

A human tumor microcytotoxicity-viable cell-staining assay was used to test the antiproliferative effect of recombinant human interferon-beta or -gamma alone and in combination with bisbenzylisoquinoline alkaloid cepharanthin against four human tumor cell lines in vitro and in nude mice. Results obtained in the in vitro study indicate that combinations of interferon-beta/-gamma with cepharanthin show synergistic and, occasionally, additive antiproliferative effects in a dose-dependent manner on tumor viable cell-staining assay. Interferon-gamma combined with cepharanthin suppressed the growth of all four human tumor cell lines (RPMI 4788, PC 10, HeLa, ZR-75-1), and this enhanced antiproliferative effect was not dependent on the interferon species involved, including interferon-beta and -gamma. In an experimental model of pulmonary metastasis, in which human colon tumor cells were inoculated i.v. into nude mice, interferon-gamma alone exerted significant inhibitory activity against pulmonary metastasis in a dose-dependent manner, and cepharanthin alone also significantly inhibited metastasis. Furthermore, a combination of interferon-gamma with cepharanthin resulted in a considerable suppression of pulmonary metastasis. These studies indicate that due to their therapeutic potential, combinations of recombinant human interferon-beta or -gamma with cepharanthin might be a promising therapy for pulmonary metastasis of human cancers.

Combined betaseron R (recombinant human interferon beta) and radiation for inoperable non-small cell lung cancer

Purpose : Based on in vitro evidence of radiosensitization by Betaseron® (β-IFN), a Phase I/II study was undertaken to determine toxicity and response using combined radiation (RT) and B-IFN in patients with unresectable Stage III and nonsmall cell lung cancer. Methods and Materials : Varying doses of β-IFN(10 to 90 × 10 6 IU) were administered IV immediately preceding RT on the first three days of weeks 1, 3, and 5. The RT dose was 1.8 Gy/day, 5 days/week for a total of 54 or 59.4 Gy. Results : Thirty-nine patients were entered, 32 of whom were evaluable. The median follow-up time at time of analysis was 60 months. Responses were based on CT scan. The response rate for the total group was 81% with 44% achieving complete response. Seventy-eight percent of patients with complete response survived a minimum of 21 months. Twenty-six patients had Stage III A/B disease with a median tumor size of 6.5 cm. and median survival was 19.7 months. The 5-year actuarial survival for this group was 31%, with a plateau persisting after 3 years. There were no treatment related deaths nor any event of life threatening toxicity. Of eight patients surviving 3–5 years, no long-term toxicity has been observed. Karnofsky indices were 90–100 and respiratory symptoms were minimal. Conclusion : β-IFN is well-tolerated. Response and survival rates are sufficiently encouraging to warrant further investigation in a randomized trial which has been accepted as an RTOG study awaiting drug availability.

Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications.

Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced N-glycosylation or O-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3-5 amino acids were also absent in a small proportion of the purified proteins. The introduction of potential artificial O-glycosylation peptide sequences (..GGKAPTPPPK..), to the C-terminus or between positions 80 and 81 of the IL-2 polypeptide chain, resulted in the secretion of unglycosylated and O-glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(beta 1-3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL-2 mutant protein. The Gal(beta 1-3)GalNAc protein forms could be partially altered in vitro to mammalian-type glycoforms by porcine liver beta-galactoside alpha-2,3-sialyltransferase in the presence of CMP-N-acetylneuraminic acid. An IL-2 mutant form, with an 11-amino-acid peptide of human interferon-beta at position 4, which includes its only N-glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(alpha 1-6)]GlcNAc or Man2GlcNAc[Fuc(alpha 1-6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell-culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma.

Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. The in vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-alpha/beta (MulFN-alpha/beta) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [3H]-thymidine incorporation, respectively. Incubation (24h) of G-26 cells with As-S, As-P or MulFN-alpha/beta, resulted in a dose dependent decrease in cell viability (IC50 = 125 microM As-S; 175 microM As-P and 3.6 x 10(4) U/ml MulFN-alpha/beta) and proliferation (IC50 = 157 microM As-S; 185 microM As-P and 3.6 x 10(4) U/ml MulFN-alpha/beta). A combined exposure to 175 microM As-S and 800 U/ml of MulFN-alpha/beta resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-alpha/beta, but not with human interferon-alpha lymphoblastoid or human interferon-beta. Ascorbyl esters inhibited cytosolic GST activity (1-50 = 15.0 microM As-S and 28.5 microM As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 microM and 2.0 microM for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 microM As-S or 800 U/ml MulFN-alpha/beta.

Enhancement of human hepatocyte growth factor production by interleukin-1 alpha and -1 beta and tumor necrosis factor-alpha by fibroblasts in culture.

Human hepatocyte growth factor (hHGF) was first purified from plasma of patients with fulminant hepatic failure (Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S., and Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419) and is now identified to be the same protein as the scatter factor (Weidner, K. M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y., and Birchmeier, W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7001-7005) and tumor cytotoxic factor (Shima, N., Nakao, M., Ogaki, F., Tsuda, E., Murakami, A., and Higashio, K. (1991) Biochem. Biophys. Res. Commun. 180, 1151-1158), and it is known to be produced by fibroblasts in culture. Here we report that inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) stimulate production of hHGF from human embryonic lung fibroblasts, MRC-5, and human gingival fibroblasts, GF-5. Recombinant human IL-1 alpha (rhIL-1 alpha) and recombinant human TNF-alpha (rhTNF-alpha) increased hHGF levels in culture supernatants of MRC-5 and GF-5 cells dose-dependently as determined by an enzyme-linked immunosorbent assay for hHGF. The half-maximal stimulatory concentrations of rhIL-1 alpha and rhTNF-alpha were about 1ng/ml and 10 units/ml, respectively. rhIL-1 beta showed almost the same effect as IL-1 alpha on stimulation of production of immunoreactive hHGF from the two cell lines. However, rhIL-6 failed to show the stimulatory effect on hHGF production by the cells in the range of 2-200 units/ml. Human interferon-beta and -gamma also did not show the stimulatory activity. Stimulation of hHGF production was observed 6-12 h after addition of rhIL-1 alpha or rhTNF-alpha and lasted at least 48 h, and the observed stimulation of hHGF production by cytokines was suppressed by addition of corresponding antiserum. hHGF mRNA levels of MRC-5 cells increased by addition of rhIL-1 alpha and rhTNF-alpha in a dose-dependent manner as determined by Northern blot analysis using cDNA for hHGF as a probe. In addition, results from nuclear run-off transcription experiments showed that the two cytokines regulated increasing hHGF gene expression at transcriptional levels rather than a change in mRNA stability. These observations indicate that the inflammatory cytokines modulate the production and secretion of hHGF by fibroblasts and may play an important role for tissue repair and regeneration.

Protective effect of interferon beta on human T cell leukaemia virus type I infection of CD4+ T cells isolated from human cord blood.

The present study shows the effect of human interferon beta (IFN beta) on the susceptibility of highly purified cord blood CD4+ T cells to infection with the human T cell leukaemia virus type I (HTLV-I). Unfractionated cord blood mononuclear cells (CBMC), or a separated CD4+ T cell subpopulation (CBCD4) were exposed to HTLV-I by cocultivation with a chronically infected virus-donor cell line. The results show that presence of proviral DNA as well as virus transcription was markedly reduced by IFN beta in both populations, indicating that this cytokine protects not only unfractionated CBMC but also purified CBCD4 cells from virus infection. Moreover IFN beta treatment caused 60%-80% inhibition of virus expression in CBCD4, assayed as the presence of virus core protein p19. This study demonstrates that IFN beta is able to inhibit HTLV-I infection of CBMC through a mechanism that does not necessarily involve cell-mediated natural or antigen-dependent immunity afforded by CBMC subpopulations distinct from targets of HTLV-I infection. Therefore it is reasonable to conclude that IFN beta has a direct protective effect on CBCD4, through induction of antiviral resistance/activity in target cells.

Interferon-beta promoters contain a DNA element that acts as a position-independent silencer on the NF-kappa B site.

The human interferon-beta (IFN-beta) promoter contains several functional domains that contribute to its virus-inducible regulation. One of them, PRDII, and NF-kappa B-binding sequence, can function as a constitutively activating element. Due to the presence of a negative regulatory domain that mediates a constitutive repression the natural IFN-beta promoter is silent in the non-induced state. Within this domain we have delimited an 11 bp element that acts as a negative regulatory element (NRE) of PRDII. Although the NRE is physically overlapping with PRDII in the IFN-beta promoter, it acts as a position-independent silencer of PRDII. Virus infection, which leads to the transcriptional activation of the IFN-beta promoter, does not alter the negative activity of the NRE on an isolated PRDII. It is the cooperative effect of PRDI and PRDII that is able to overcome the NRE function after virus infection. By UV cross-linking analysis using uninduced and virus-induced nuclear extracts, we show that two factors with molecular masses of approximately 95 and 100 kDa bind to the NRE.

Human recombinant interferon-?SER and tamoxifen: growth suppressive effects for the human breast carcinoma MCF-7 grown in the athymic mouse

Tamoxifen is the endocrine treatment of choice for breast cancer. However, resistance to therapy and patient relapse inevitably occurs. In future treatment schedules, interferons could be administered with tamoxifen, in an attempt to prevent disease recurrence. Human recombinant interferon-beta SER (rIFN-beta SER) inhibited the growth in vitro of the estrogen receptor (ER) positive breast cancer cell line MCF-7 and the ER negative breast cancer cell line MDA-MB-231. This inhibitory effect was achieved at doses of 50 U/ml and above. The growth of MCF-7 tumors in estradiol-stimulated athymic mice was greatly inhibited by high dose rIFN-beta SER treatment (10(6)U/day). In spite of the impressive antitumor effects upon MCF-7 tumors, rIFN-beta SER had no effect upon ER levels within the tumors at either the RNA or protein level, as measured by Northern blotting and ER-EIA respectively. High dose rIFN-beta SER (10(6)U/day) did result in some inhibition in the growth in vivo of the tamoxifen-stimulated MCF-7 variant MCF-7 TAM, although not to the same extent as was observed with the estradiol-stimulated MCF-7 tumors. rIFN-beta SER was also administered to animals bearing MCF-7 tumors and treated with estradiol and tamoxifen. In the animals undergoing high dose therapy (10(6)U/day), tumor growth was completely suppressed. Furthermore, tumor growth continued to be suppressed in those animals in which the rIFN-beta SER therapy was halted and the tamoxifen capsule removed. No tumors were observed in spite of the environment of estradiol stimulation. Thus, the combination of interferon and tamoxifen was totally growth suppressive for MCF-7 xenografts in nude mice.

Interrelationship between O6-alkylguanine-DNA alkyltransferase activity and susceptibility to chloroethylnitrosoureas in several glioma cell lines.

In order to study the dynamic relationship in glioma cells between O6-alkylguanine-DNA alkyltransferase (AGT) activity and resistance to the cytotoxic effect of chloroethylnitrosoureas (CENUs), we investigated the changes in sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after modulation of AGT activity. In ACNU-resistant rat glioma cell lines (9LR1, 9LR3, and 9LR12) and a human glioma cell (HNG-1), O6-methylguanine enhanced cytotoxicity to ACNU following a depletion of AGT activity. But no enhancement of cytotoxicity was seen in an ACNU-sensitive rat glioma cell line (9L). In the 9L and 9LR12 cells, equivalently sublethal doses of ACNU similarly depleted AGT activity but the regeneration rates of this repair protein were different. In the case of a 7-day pretreatment with human recombinant interferon-beta (HuIFN-beta), although it could modulate AGT activity in HNG-1 cells, no definite influence on cellular sensitivity to CENUs was observed. However, a 50-day pretreatment with HuIFN-beta conferred resistance to CENUs on them despite its effect to reduce AGT activity. Thus, diversity was seen in the relation between AGT activity and resistance to CENUs when AGT activity was modulated by HuIFN-beta. The results of this study suggest that AGT activity is one of factors affecting cellular sensitivity to CENUs but that alternative mechanisms of tolerance may be induced depending upon some environmental effects.

Expression of active human interferon-beta in transgenic plants.

Tobacco plants were transformed with the human gene for interferon-beta (IFN-beta). Transformation was determined by the polymerase chain reaction (PCR), and expression was determined by Western blot analysis, by purifying the IFN from the transgenic plants, and by bioassays indicating its activity in human cells. Plants expressing IFN-beta were self-pollinated. IFN-beta-expressing progeny plants were selected and produced active IFN-beta, indicating stable transformation.

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

Abstract We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.

Evaluation of blood-brain barrier permeability and the effect of interferon in mouse glioma model.

The aim of this study was to evaluate whether interferon [IFN] can affect intracerebrally grown glioma and how alteration of the blood-brain barrier [BBB] may influence this effect. An intracerebrally implanted glioma G-26 (G-26) mouse brain-tumor model was developed and used in these studies. Histological characterization of this intracerebrally grown tumor revealed its anaplastic character. The astrocytic origin of G-26 was evidenced by glial fibrillary acidic protein staining and electron microscopic visualization of glial filaments. A study of tumor progression and animal survival showed development of a well defined tumor nodule within approximately seven days after the implantation. The median animal survival time was 27 +/- 3.8 days. The integrity of the blood-brain barrier [BBB] within the tumor was evaluated by the intravenous injection of horseradish peroxidase at days 3, 7, 10 and 20 after brain tumor implant and compared to 'sham' controls. The tumor-induced BBB alteration was progressive from day 3 to day 20. Glioma-26 subcutaneously passed in C57BL/6 mice was also continuously cultured in vitro. Its proliferation was inhibited by homologous mouse interferon alpha/beta [MuIFN alpha/beta] but not by human interferon alpha lymphoblastoid or human interferon beta. The in vivo studies of G-26 glioma treatment with MuIFN alpha/beta were performed using single bolus of IFN in osmotically altered animals or slow IFN infusion through osmotic micro-pumps. The slow infusion of IFN had no effect on animal survival. However, a statistically significant increase in animal survival was observed after single bolus IFN treatment following osmotic BBB alteration.