Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.

Growth control in cultured 3T3 fibroblasts II. Molecular properties of a fraction enriched in growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium. This growth inhibitory activity was fractionated by ammonium sulfate precipitation and gel filtration, yielding one fraction that was 35-fold enriched in specific activity. Analysis of the chemical and biological properties of this highly active fraction indicated that: (a) it is an endogenous cell product, synthesized by the 3T3 cells and shed into the medium; (b) it is a protein and its activity is sensitive to treatment with pronase; (c) the constituent polypeptide chains have molecular weights of 10,000 and 13,000; and (d) it is not cytotoxic and its effect on target cells are reversible. These results suggest that we have partially purified from conditioned medium an endogenous growth regulatory factor that may play a role in density-dependent inhibition of growth in cultured fibroblasts. We propose the term Fibroblast Growth Regulator to describe this class of molecules.

Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.

Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli.

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Galectin-3: differential accumulation of distinct mRNAs in serum-stimulated mouse 3T3 fibroblasts.

The murine Galectin-3 gene spans approximately 12 kb of DNA and contains six exons, with the translation initiation codon located in exon II. On the basis of restriction mapping and sequence analysis of the DNA upstream of exon II, primer extension assays, rapid amplification of cDNA ends, and ribonuclease protection assays were designed and carried out to determine the initiation site of transcription and the sequence of exon I. The results revealed at least two transcription initiation sites (alpha and delta), each of which appears to be specifically associated with the use of alternative donor splice sites, resulting in distinct mRNA species. Type I message initiates at transcription start site delta, uses splice donor site No. 2, retaining a 27 bp sequence, whereas type II message initiates at transcription start site alpha, uses splice donor site No. 1, resulting in the loss of the 27 bp sequence. Primer extension assays carried out with mRNA isolated from 3T3 fibroblasts at various times after serum stimulation indicate that while the type II message varies in level only a little over the first 20 h, there is dramatic accumulation of the type I message, which peaks at 16 h post mitogen addition.

Carbohydrate-binding protein 35. Levels of transcription and mRNA accumulation in quiescent and proliferating cells.

In previous studies, we observed proliferation-dependent expression and nuclear localization of the lectin, designated carbohydrate-binding protein 35 (CBP35), in 3T3 fibroblasts at the polypeptide level by Western blot and immunofluorescence analysis. In the present study, we have compared the expression of the CBP35 gene in quiescent and proliferative 3T3 cells at the levels of (a) accumulated mRNA by Northern blotting and (b) nuclear transcription by run-off assays. When serum-starved, quiescent cultures of 3T3 cells were stimulated by the addition of serum, there was an increase in the nuclear transcription of the CBP35 gene and in the accumulation of its mRNA early (1-3 h) in the activation process, well before the first wave of DNA synthesis. These increases were not dependent on de novo protein synthesis inasmuch as they occurred even in the presence of cycloheximide. There was also an elevated transcription rate and mRNA level in transformed cells when compared to their normal counterparts. Finally, the expression of CBP35 was compared between sparse, proliferating cultures of 3T3 cells and density-inhibited confluent monolayers of the same cells. Although the rate of transcription of the CBP35 gene was approximately the same in the two cultures, there was a higher level of CBP35 mRNA in the dense cells. Thus, it appears that post-transcriptional mechanisms may be involved in the accumulation of mRNA.

Procedure for selecting monoclonal antibodies for use in a ligand displacement assay of serum antibody levels.

Monoclonal antibodies for use in a ligand displacement assay were selected for specificity and affinity/avidity properties that result in their release and displacement in the presence of specific sample antibody but not in the presence of antibodies against other antigens. A screening process is described which involves measurement of displacement of antibody by an ELISA procedure using an enzyme labeled anti-immunoglobulin, providing a means of demonstrating usefulness of a candidate antibody in a ligand displacement format without necessitating the production of enzyme conjugates of each candidate antibody to be screened. The procedure was used to screen a set of eleven monoclonal antibodies (initially selected for anti-Trichinella spiralis specificity by conventional screening methods), and successfully discriminated between antibodies which were useful in the ligand displacement format and those which were not.

Export of galectin-3 from nuclei of digitonin-permeabilized mouse 3T3 fibroblasts.

Galectin-3 is a galactose-/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. Immunofluorescence staining revealed that galectin-3 distributes differentially between the nucleus and the cytoplasm, depending on the proliferative state of the cells under analysis. Using digitonin-permeabilized mouse 3T3 fibroblasts, we provide evidence that galectin-3 is rapidly and selectively exported from the nucleus. Although both phosphorylated and nonphosphorylated isoforms of galectin-3 are found in the nuclear fraction, only phosphorylated galectin-3 is identified in the exported fraction, implying that phosphorylation is important for the nuclear export of the protein. The rate of galectin-3 export is temperature dependent and is decreased by the addition of wheat germ agglutinin. More strikingly, galectin-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1). Indeed, a putative leucine-rich nuclear export signal can be found in residues 241-249 of the murine galectin-3 sequence. Finally, gel filtration of the exported material showed that galectin-3 can be found in at least two high molecular weight complexes (approximately 650 and approximately 60 kDa), both of which can be disrupted by lactose.