Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

Cytokine regulation of the interleukin-1 receptor antagonist protein in U937 cells.

A naturally occurring receptor-level antagonist of interleukin-1 (IRAP or IL-1 ra) has recently been cloned. To determine what stimuli might regulate this inhibitor, cytokines were tested for their effects on the steady-state level of IRAP mRNA in phorbol ester-differentiated U937 cells. The cytokines tested fell into one of three groups: (a) inducers: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, (b) weak inducers (< 2-fold stimulation): [IL-1 alpha, IL-1 beta, and transforming growth factor-beta (TGF-beta)] and (c) cytokines with no effect: (IL-2, platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, granulocyte colony-stimulating factor, IL-3, IL-5, IL-6, interferon-gamma, multi-colony stimulating factor, tumor necrosis factor-alpha and IRAP itself. One hundred U/ml of either GM-CSF or IL-4 was the dose inducing peak IRAP mRNA expression; that peak expression occurred 12 h after addition of cytokine. GM-CSF induced a 34 +/- 15-fold increase in IRAP mRNA, and IL-4 induced a 15 +/- 6-fold increase. In the same RNA samples, GM-CSF increased IL-1 beta mRNA 5.9 +/- 1.7-fold, but IL-4 decreased IL-1 beta mRNA to half that of control levels (0.45 +/- 0.17). Thus, a single stimulus (IL-4) decreased the expression of an agonist (IL-1) while it increased the expression of an antagonist (IRAP). When U937 cells were treated with both IL-4 and GM-CSF, the level of IRAP mRNA induced was additive, suggesting that the cytokines acted differently to increase IRAP mRNA levels. The level of IL-1 mRNA in cells treated with both IL-4 and GM-CSF was intermediate. Dexamethasone and cycloheximide inhibited all mRNA increases and did not reverse IL-4-induced decreases in IL-1 mRNA. These studies have identified two cytokines which induce IRAP in the monocytic cells studied, and have partially characterized the differential regulation of IL-1 and its antagonist, IRAP.

Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein.

A human myelomonocytic cell line, U937, produced an interleukin-1 (IL-1) receptor antagonist protein (IRAP) which was purified and partially sequenced. A complementary DNA coding for IRAP was cloned and sequenced. The mature translation product of the cDNA has been expressed in Escherichia coli and was an active competitive inhibitor of the binding of IL-1 to the T-cell/fibroblast form of the IL-1 receptor. Recombinant IRAP specifically inhibited IL-1 bioactivity on T cells and endothelial cells in vitro and was a potent inhibitor of IL-1 induced corticosterone production in vivo.

Elevated transferrin receptor content in human prostate cancer cell lines assessed in vitro and in vivo.

Transferrin receptors (TfR) were measured in benign and malignant prostatic cells by performing Scatchard analysis following the administration of 125I-transferrin. Established human prostate cancer cell lines (PC-3 and DU-145) as well as biologically aggressive variants (PC-3 ASC and PC-3 DES) were shown to possess significant levels of high affinity TfR when assessed in vitro. In contrast, TfR content was negligible in cultured stromal cell fractions derived from human benign prostatic hyperplasia (BPH) specimens. Scatchard analysis was also performed on in vivo derived prostatic tissues: tumors resulting from the subcutaneous xenografting of PC-3 ASC cells into athymic, nude mice and fresh BPH surgical specimens. These tissues were dissociated and their stromal and epithelial components separated. TfR were only detected in the epithelial component of both malignant and benign epithelial cells. PC-3 ASC tumor cells exhibited TfR levels comparable to their in vitro expression and these levels were 10-fold greater than in the BPH cells. These findings suggest that elevated TfRs may serve as another useful marker of the transformed phenotype within human prostate tumor systems.

Two-dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate.

Studies were undertaken to compare and contrast the two-dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two-dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]-methionine-labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cells proteins by the use of an anti-vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma-derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two-dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.

Gene manipulation by homologous recombination in Escherichia coli.

The use of homologous recombination in Escherichia coli is described as a tool for DNA manipulation. The utility of the method is illustrated by the addition of 3'-flanking sequences to a dhfr minigene by plasmid-phage recombination involving a supF-containing dhfr minigene plasmid and a lambda Charon4A phage containing the 3' end of the dhfr gene. In addition, other uses of both plasmid-phage and phage-phage recombination in gene manipulation are described.

A rapid in vitro assay for quantitating the invasive potential of tumor cells.

We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells.

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.

Control of differentiation of thymocyte precursors in the bone marrow by thymic hormones.

Terminal deoxynucleotidyl transferase (TdT) is uniquely associated with the major thymocyte population and a minor bone marrow population which is separable on bovine serum albumin gradients. The expression of TdT in the bone marrow subpopulation is under thymic regulation in that this population is significantly reduced in nude mice or is lost with time after thymectomy. The expression of TdT in athymic mice can be induced both in vivo and in vitro by thymosin fraction 5 or by beta3, a peptide purified from thymosin fraction 5. The optimal concentration of beta3 in the in vitro induction of TdT is approximately 10 ng/ml. These results demonstrate that thymic hormones control the early differentiation of prothymocytes in the bone marrow.

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.