Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.     

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.     

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.     

T-cell restricted and unrestricted expression of transfected human interleukin-2 gene: phorbol ester- and calcium-inducible versus constitutive expression

Interleukin-2 (IL-2) gene expression is tightly controlled and generally limited to antigenic stimulation of T cells. To study the cell-specific expression of the IL-2 gene, we transfected the intact human IL-2 gene, including 2.0 kb of 5' and 0.3 kb of 3' flanking sequences, into mouse NIH-3T3 fibroblasts and BFS lymphoma T cells and into human epithelial HeLa cells. Stable transformants (NIH-3T3,HeLa and BFS cells) carried an intact transfected IL-2 gene and constitutively expressed cytoplasmic human IL-2 mRNA which was not detected in vector-transfected cells. Constitutive expression of IL-2 mRNA in human IL-2 gene-transfected NIH-3T3 and HeLa cells was associated to the secretion of bioactive IL-2 protein, while no IL-2 production was observed in untransfected or vector-transfected cells. Cytoplasmic IL-2 mRNA observed in transfectants was larger (1.4 kb) than endogenous IL-2 mRNA of human T cells, although smaller than RNA containing unspliced intact introns. No alternative promoters or polyadenylation signals were used by these cells, but some intronic sequences were present in the 1.4 kb mRNA. Phorbol ester and calcium ionophore did not modulate the expression of the transfected IL-2 gene in NIH-3T3 and HeLa cells, while these agents increased its expression in transfected BFS lymphoma T cells. We conclude that when transfected into lymphoid and non-lymphoid cells the intact human IL-2 gene is constitutively expressed, while its phorbol ester/calcium-mediated inducible expression is restricted to T cells. This suggests that the constitutive and inducible expression of the IL-2 gene can be dissociated and are presumably subjected to separate regulatory pathways.     

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Insulin-like growth factor II overexpression does not affect sorting of lysosomal enzymes in NIH-3T3 cells.

The sorting of newly synthesized mannose 6-phosphate (M6P)-containing proteins and of the major excreted protein (MEP), a lysosomal thiol proteinase, was studied in NIH-3T3 cells transfected with the cDNA of human insulin-like growth factor II (IGF II) or with the vector alone. Extracts from media and cells labelled with [35S] methionine were used for chromatography on a M6P/IGF II receptor affinity matrix or for immunoprecipitation to assess the distribution of newly synthesized M6P-containing proteins and MEP, respectively. The results indicate that the overexpression of IGF II did not affect the synthesis and the sorting of M6P-containing proteins and of MEP. The binding and uptake of the lysosomal enzyme arylsulfatase A were not affected in IGF II overexpressing cells.     

Construction of tumor-specific cells expressing a membrane-anchored single-chain Fv of anti-ErbB-2 antibody

Cells expressing a membrane-anchored single-chain fragment variable (scFv) domain against a tumor-specific antibody were fabricated. These cells were able to bind to cells of a human colon cancer line (BM314) expressing the erbB-2 proto-oncogene. A plasmid, pMFverbB, was first constructed in which the anti-ErbB-2 scFv gene was cloned in-frame between a signal peptide sequence and the platelet-derived growth factor receptor (PDGFR) transmembrane domain gene to express scFv on the cell surface. An African green monkey cell line, COS-1, was stably transfected with pMFverbB. Immunofluorescence assay experiments and microscopic observation showed that the cells expressing scFv bound to the human tumor cells overexpressing the ErbB-2 protein as well as to cells of a mouse fibroblast line (NIH-3T3) transfected with the erbB-2 gene. The cells expressing scFv could take up magnetite cationic liposomes as a model of particle-type drug and retained the ability to target ErbB-2-expressing cells. The fabricated cells have the potential to serve as drug carriers in drug targeting applications.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin.

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3' non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

An acid protease secreted by transformed cells interferes with antigen processing

The major excreted protein of malignantly transformed mouse fibroblasts (MEP), which is the precursor to lysosomal cathepsin L, was used to study the effect of exogenous acid proteases on antigen processing. When MEP and native pigeon cytochrome c were added to Chinese hamster ovary (CHO) cells expressing transfected major histocompatability complex class II gene products, the antigen-specific T-cell hybridoma 2B4 did not respond to the antigen. MEP appears to destroy the antigen in an acid compartment of the presenting cell because: (a) MEP is only active as a protease under acid conditions; (b) mannose 6-phosphate inhibited the internalization of MEP and blocked its effect on antigen processing; (c) the destruction required the simultaneous entry of the antigen and MEP into the cells; and (d) cytochrome c fragment 66-104 which does not need to be processed stimulated 2B4 in the presence of MEP. These results support the hypothesis that antigen processing requires internalization of the antigen into an acidic compartment, and they provide a new model for the investigation of the contribution of acid proteases to the reduced immunocompetence of tumor-bearing animals.     

Alternative Splicing of the Cytoplasmic Domain of Neural Cell Adhesion Molecule Alters Its Ability to Act as a Substrate for Neurite Outgrowth

A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.     

Activation of Nonexpressed Endogenous Ecotropic Murine Leukemia Virus by Transfection of Genomic DNA into Embryo Cells

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c x B6)F(1) mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).     

Structure and Growth-Dependent Regulation of the Human Cyclin B1 Promoter

As a step toward defining the signals important for the regulation of cyclin B1 expression, we cloned, sequenced, and partially characterized a 1012-bp genomic fragment encompassing the human cyclin B1 promoter. By transient expression experiments, we found that promoter activity resides within a -150/+182-bp DNA fragment. The activity of this promoter fragment was high in asynchronous NIH-3T3 cells, but dramatically decreased in quiescent cells. Time-course experiments, using stable transfectants expressing the CAT gene under the control of this fragment, were performed after releasing the cells from serum starvation. The results showed that the promoter becomes active at the end of the S phase and its activity increases during the cell cycle. Similar experiments performed with a shorter promoter region (-58/+182) showed that this 5′ deletion mutant is active throughout the cell cycle. In good agreement with promoter activity, Northern analysis indicated that the endogenous gene is negatively regulated in quiescent murine NIH-3T3 cells. The data presented here demonstrate that in NIH-3T3 cells the cyclin B1 promoter is growth regulated, and important regulatory elements must exist in the region spanning - 150 to 58 bp.     

Cloning and expression of the human substance K receptor and analysis of its role in mitogenesis.

The primary structure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfected NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I-labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substance K and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfected cells led to a rapid increase in the production of total inositol phosphates and the release of Ca2+ from internal stores. Growth of the cells transfected with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth factor receptor when expressed in mouse 3T3 cells.     

Sodium butyrate suppresses the transforming activity of an activated N-ras oncogene in human colon carcinoma cells

The transforming activity of DNA from a newly established undifferentiated human colon carcinoma cell line (MIP-101) was tested in the NIH-3T3 transfection assay. Southern blot analysis of the transfectant DNA revealed the presence of a human N-ras oncogene. Treatment of MIP-101 cells with the maturational agent sodium butyrate induced a more normal phenotype, including diminished growth rate, elimination of anchorage independent growth, and decreased tumorigenicity (R. Niles, S. Wilhelm, P. Thomas, and N. Zamcheck (1988) J. Cancer Invest. 6, 39). Here we report that there is a significant reduction in the transforming efficiency of the DNA from butyrate-treated MIP-101 cells. A nonspecific reduction in total DNA uptake as an explanation for these findings was eliminated by showing that there was similar uptake and expression of the thymidine kinase gene from the DNA of butyrate-treated and control MIP cells. Butyrate treatment had no detectable effect on the overall structure, methylation, and level of expression of the human N-ras gene from MIP-101 cells. An NIH-3T3 transformant ability after treatment with sodium butyrate. Although butyrate suppressed several transformed properties similar to MIP-101 cells, DNA from control and treated cultures had an identical level of transforming activity. The results suggest that the environment of the MIP cells may contain additional elements not present in the NIH-3T3 transformants which are required to observe the effect of butyrate on reduction of transforming activity.