Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.     

Transforming growth factor-beta 1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) stimulated DNA synthesis (3-fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF-beta 1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8-fold increases in ribonucleotide reductase activity following TGF-beta 1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5-fold) and R1 gene expression (2,2.5, and 2.6-fold) were observed following 6, 12, and 24 hours of TGF-beta 1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1-fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF-beta 1, whereas 2.6 and 3.3-fold elevations in R1 protein levels were observed at 12 and 24 hours post-TGF-beta 1 exposure. These TGF-beta 1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3-fold after 6, 12, and 24 hours of TGF-beta 1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF-beta 1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF-beta 1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation.     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

Internalization of transforming growth factor-beta and its receptor in BALB/c 3T3 fibroblasts

The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.     

Cathepsin D precursors in clathrin-coated organelles from human fibroblasts

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.     

Plate-Induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes,chondrocytes, and fibroblasts

The BALB/3T3 clone A31 mouse embryo cell line has been used by many investigators as a model “normal” “fibroblast” line for a variety of in vitro studies. It has been shown, however, that these cells are not “normal” because they will produce tumors within 2–4 months if 3 × 10⁴ cells are implanted subcutaneously in BALB/c mice attached to 0.2 × 5 × 10-mm plastic plates. Previous studies also suggested that these cells were not fibroblasts because they gave rise to tumors with the characteristics of vascular endothelium not fibroblasts. We now report that BALB/3T3 (clone A31), BALB/3T3-T, a proadipocyte subclone of clone A31 cells, and six recent subclones of BALB/3T3-T cells show additional differentiation patterns when tumors derived by implantation of these cells attached to plastic plates are examined. Differentiation into pericytes, chondrocytes, and fibroblasts was observed. We conclude that the BALB/3T3 clone A31 cell line and related lines are multipotent mesenchymal cells which are capable of differentiation into a variety of cell types.     

Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region

The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.     

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.     

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.     

Adsorptive endocytosis of lysosomal enzymes by human fibroblasts: presence of two different functional systems that deliver an acid hydrolase to lysosomes

Endocytosis of human spleen beta-glucuronidase by human fibroblasts can be completely impaired by the competitive inhibitor mannose 6-phosphate or by pretreatment with acid phosphatase or endoglycosidases H or F. However, endocytosis of bovine spleen and liver beta-glucuronidase is partially impaired by the same treatments, suggesting that the bovine enzyme contains two endocytosis recognition markers located in separate enzyme domains. The mannose 6-phosphate recognition marker seems to be responsible for approximately 23% of the bovine enzyme endocytosis. The existence of two lysosomal endocytosis systems in human fibroblasts is supported by the following facts: (a) the rate of endocytosis of mannose 6-phosphate-containing human beta-glucuronidase was not affected by the presence of high levels of the bovine enzyme (which has only the other marker). (b) Anti-215K mannose 6-phosphate receptor antibodies selectively impair the endocytosis of the beta-glucuronidase containing mannose 6-phosphate. (c) Weak bases exert a differential effect on human and bovine endocytosis. beta-Glucuronidase internalized by either system is targeted to secondary lysosomes of human beta-glucuronidase-deficient fibroblasts, where it is able to degrade accumulated glycosaminoglycans. These results suggest that human fibroblasts have two different and independent endocytic systems for targeting of acid hydrolases to lysosomes.     

Mannose 6-phosphate/insulin-like growth factor II receptor in I-cell disease fibroblasts: increased synthesis and defective regulation of cell surface expression.

The amount of mannose 6-phosphate/IGF II receptors in fibroblasts from five I-cell patients was about 2-fold higher than in control fibroblasts. The elevated receptor concentration, which led to a higher binding and uptake of mannose 6-phosphate containing ligands and to a higher binding of IGF II resulted from an increased rate of synthesis, while the stability of the receptor was comparable to that in control fibroblasts. Control fibroblasts respond to mannose 6-phosphate, IGF I, IGF II and tumor promoting phorbol esters with a rapid redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface. In I-cell fibroblasts only a moderate increase in cell surface receptors was seen after exposure to these effectors. In contrast to control fibroblasts the treatment of I-cell fibroblasts with lysosomotropic amines failed to affect the mannose 6-phosphate containing ligand binding to the receptor. These data provide evidence for multiple potential regulatory sites in intracellular mannose 6-phosphate/IGF II receptor pathway which differ in control and I-cell fibroblasts.     

Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts

We have tested whether growth factors added to serum-deprived BALB/c 3T3 fibroblasts alter the casein kinase II activity measured in cell extracts. A rapid phosphocellulose chromatography method was developed that provides a 40-fold partial purification of casein kinase II activity assayed with the specific substrate peptide Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. Using this technique, kinase activity is stimulated 1.6-2.5-fold when isolated from fibroblasts treated with insulin or insulin-like growth factor I (IGF-I). The activated kinase activity exhibits the specific properties of casein kinase II such as the ability to utilize [gamma-32P]GTP as phosphate donor and marked inhibition by low concentrations of heparin. Activation of casein kinase II appears specific for these hormones because epidermal growth factor and platelet-derived growth factor have no effect on the kinase activity when added to fibroblasts under conditions where they markedly stimulate [3H]thymidine incorporation into DNA. Increases of casein kinase II activity by insulin and IGF-I were detected within 1 min of their addition to cell cultures. IGF-I is more potent in stimulating casein kinase II than insulin in mouse fibroblasts. These results demonstrate that casein kinase II is a selective target for insulin and IGF-I action in BALB/c fibroblasts, consistent with the hypothesis that this kinase plays a role in cellular signaling by these hormones.     

Low temperature blocks transport and sorting of cathepsin D in fibroblasts

The transport of newly synthesized cathepsin D in fibroblasts at 16-28 degrees C was compared to that at 37 degrees C. At 37 degrees C newly synthesized cathepsin D passes the trans Golgi within 30-60 min, becomes segregated from the secretory route into prelysosomal organelles within 1-2 h and processed to mature forms in dense lysosomes within 1.5-3 h after synthesis. The small fraction of cathepsin D that escapes transport into lysosomes is secreted within less than 2 h. At 16-28 degrees C the transport of cathepsin D to lysosomes is inhibited in a temperature-dependent manner. At 16-28 degrees C cathepsin D precursors are slowly transported to the trans Golgi. The cathepsin D precursors accumulate at a site that is in continuity with the secretory pathway and located within or distal of the trans Golgi and proximal to the site where cathepsin D precursors leave the secretory pathway as complexes with mannose 6-phosphate receptors. The arrest at this site is not complete. The receptor-dependent segregation of the cathepsin D precursors released from the block is impaired at less than or equal to 26 degrees C. The inhibition of segregation results in an increased, albeit retarded secretion of cathepsin D. The fraction of cathepsin D precursors that is segregated from the secretory pathway encounters a further low temperature block in prelysosomal organelles. There cathepsin D precursors are proteolytically processed to an intermediate form, which accumulates transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.     

Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor. Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase

beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor. Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase. Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms. About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate. The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column. The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase. This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme. However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase. The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase. These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.     

T cells from Leishmania major-susceptible BALB/c mice have a defect in efficiently up-regulating CXCR3 upon activation

Genetic background influences the outcome of Leishmania major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-gamma-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes (LN) early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-gamma-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-gamma-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the LN and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared the ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3(+) T cells in regional LN and lesions, whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naive BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-gamma and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major susceptibility.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

Possible requirement of serum progression factors for transformation of BALB/c 3T3 fibroblasts by v-ras p21.

To form colonies in soft agar, ras-transformed 3T3 fibroblasts require serum. We examined what growth factors in serum were essential for ras-induced transformation. Temperature-sensitive (ts) v-Ki-ras-transfected BALB/c 3T3 cells were used to strictly control both the activity of the ras protein and the cell cycle. When G0-arrested ts cells were cultured with 10% serum at a permissive temperature, greater than 50% of cells formed colonies. A similar colony-forming activity was observed in the presence of 10% platelet-poor plasma, but not in the presence of 10% plasma isolated from hypophysectomized rats. Inhibitors of IGF signals attenuated colony formation in the presence of serum. These data suggest that progression factors, probably IGFs, are essential components in serum for ras-induced transformation of 3T3 fibroblasts.     

Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.     

Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression.

Induction of quiescent BALB/c 3T3 murine fibroblasts by platelet-derived growth factor (PDGF) or fibroblast growth factor (FGFs) is accompanied by induction of c-myc gene expression. To study the role of c-myc in cell growth, we transfected BALB/c 3T3 cells with a plasmid construct containing a glucocorticoid-inducible c-myc gene. When these transfected cells were growth arrested in PDGF-FGF-freedefined medium, glucocorticoid treatment induced S-phase DNA synthesis. This induction of DNA synthesis was inefficient, and cell proliferation was not evident, suggesting that growth factors act through stimulation of c-myc expression together with other intracellular events.