Hepatic binding proteins translocating azo dye carcinogen metabolites from cytoplasm into nucleus in rats.

When liver cytosol prepared from rats administered [(14)C]-3'-Methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks of radioactivity containing proteins (Peak-I-IV) and one peak devoid of protein (Peak-V) were obtained. Translocation of azo dye metabolites from these various cytosolic fractions into nucleus was studied in an in vitro system and a maximum of about 10% of the radioactivity associated with a particular cytosolic fraction (Peak-II) could translocate into the nuclei. Radioactivity (%) translocated did not increase upon addition of excess nuclei. Passage of this protein fraction through an immobilized protease column reduced the azo dye metabolite translocation by 65%, concomitant with the degradation of proteins. Translocation was not observed with protein-free metabolites extracted from this cytosolic fraction; addition of proteins corresponding to peak-II from normal rat liver cytosol significantly restored the metabolite translocation. This observation suggests that specific cytosolic proteins are involved in the translocation of azo dye carcinogen metabolites from liver cytoplasm into the nucleus. When the liver cytosolic proteins corresponding to this fraction (Peak-II) were iodinated with (125)I-iodine and incubated with purified nuclei, translocation of three specific proteins into nucleus was observed as seen by SDS-PAGE and fluorography of nuclear proteins. Covalent binding of azo dye metabolites to DNA was not observed when cytosolic peak-II fraction containing azo dye metabolites was incubated with isolated liver DNA instead of liver nuclei. This suggests that the interaction of azo dye metabolites with nuclear macromolecules necessitate further prior processing which actually may occur in the nucleus.

The effect of cytokines on the migration of fibroblasts derived from different regions of the canine shoulder capsule.

This study examined the effect of several cytokines on the chemotactic migration of fibroblasts derived from 3 different parts of the canine shoulder: the upper part of the medial glenohumeral ligament (equivalent to the anterior part of the inferior glenohumeral ligament of the human shoulder); the inferior part of the medial glenohumeral ligament (equivalent to the axillary pouch of the human shoulder); and the posterior capsule (equivalent to the thin posterior capsule in the human shoulder). Platelet-derived growth factor-AB stimulated the migration of all 3 cell types in a dose-dependent manner, with increases from 150% to 300% at 1 ng/mL to 500% to 700% at 10 ng/mL. Hepatocyte growth factor also stimulated the migration of all 3 cell types in a dose-dependent manner (130% to 310%). Insulinlike growth factor-1 increased the migration of all 3 types of fibroblasts by 160% to 250%. Bone morphogenic protein-2, interleukin-1, and transforming growth factor-b had no significant effect on migration of shoulder capsular fibroblasts. These data demonstrate that capsular fibroblasts are responsive to specific growth factors and suggest the potential for use of growth factors to augment healing and/or remodeling of the shoulder capsule.

Insulin-like growth factor-I is expressed by avian flexor tendon cells.

Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48+/-0.05 ng/g tissue for the epitenon and 3.83+/-0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive form, most likely bound to insulin-like growth factor-binding proteins.

Differential expression of integrin subunits in canine knee ligament fibroblasts.

A method for measuring the expression of integrin subunits on the cell surface of knee ligament fibroblasts was developed with use of flow cytometry and immunofluorescence. The ligament cells exhibited uniform size and density, as shown by forward and side-scatter properties, and showed minimal nonspecific binding of isotype control antibodies compared with unstained cells. All cells expressed the alpha5 integrin subunit; lateral collateral ligament cells stained with antibody to alpha5 showed a mean fluorescence intensity 2-fold higher than that of medial collateral ligament cells, 1.5-fold higher than that of posterior cruciate ligament cells, and 3-fold higher than that of anterior cruciate ligament cells, indicating a greater expression of the alpha5 subunit by lateral collateral ligament cells than by medial collateral, posterior cruciate, and anterior cruciate ligament cells. All cells expressed the beta1 integrin subunit; the expression by posterior cruciate ligament cells was 3-fold higher than that by medial collateral ligament or lateral collateral ligament cells and 5-fold higher than that by anterior cruciate ligament cells. All cells expressed the beta3 integrin subunit; the expression by posterior cruciate ligament cells was 1.5, 3, and 4.5-fold greater than that by lateral collateral, anterior cruciate, and medial collateral ligament cells, respectively. Our data suggest there is a differential expression of integrin subunits in knee ligament fibroblasts, and this in part may explain differences in their attachment and adherence to extracellular matrix molecules.

The effect of cytokines on the proliferation and migration of bovine meniscal cells.

We determined the effect of cytokines on the proliferation and migration of cells isolated from the inner-third (white-white), middle-third (red-white), and outer-third (red-red) regions of bovine meniscus. Cells from the outer, or peripheral, region of the meniscus exhibited higher DNA synthesis in the presence of 10% serum compared with cells from the inner or central regions. Recombinant human platelet-derived growth factor-AB, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated DNA synthesis of all meniscal cells in a dose-dependent manner, with a two- to threefold maximal stimulation at 10 ng/ml. Cell migration was also stimulated by addition of cytokines. Platelet-derived growth factor and hepatocyte growth factor caused an increase in the migration of cells derived from all three zones, while interleukin-1 selectively stimulated the migration of outer-zone meniscal cells. Epidermal growth factor was much less effective and stimulated the migration of cells in the inner and outer zones by 40% to 50%, while bone morphogenic protein-2 and insulin-like growth factor-1 stimulated the migration of meniscal cells from the middle zone by 40% to 50%. The identification of cytokines that stimulate both the growth and migration of meniscal cells may provide new tools for modulation of meniscal healing.

Characterization of chemotactic migration and growth kinetics of canine knee ligament fibroblasts.

Migration and proliferation of ligament fibroblasts are essential for the healing of ligament injuries. This study was designed to evaluate the migration of intraarticular (anterior and posterior cruciate) and extraarticular (medial and lateral collateral) ligament fibroblasts in response to cytokines and to determine the effect of cell passage on cell proliferation. Recombinant human platelet-derived growth factor, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated the migration of all ligament cells in a dose-dependent manner, with optimal migration at 10 ng/ml. Recombinant human epithelial growth factor preferentially stimulated the migration of intraarticular ligament fibroblasts, whereas recombinant human interleukin-1 was more effective with extraarticular ligament fibroblasts. Recombinant human insulin-like growth factor-1, insulin-like growth factor-2, transforming growth factor-beta, and fibroblast growth factor had no significant effect on the migration of ligament-derived fibroblasts. These data suggest that specific cytokines stimulate the migration of knee ligament fibroblasts and provide a rationale for possible therapeutic approaches to optimize ligament healing. Fibroblasts derived from the anterior cruciate ligament have been shown to proliferate at a slower rate than those derived from the medial collateral ligament. We have extended these observations and have demonstrated that fibroblasts from both the posterior and anterior cruciate ligaments proliferate at a slower rate than lateral and medial collateral ligament-derived fibroblasts. The differences between the growth rates of intraarticular and extraarticular fibroblasts become insignificant with serial passaging of the cells.

Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5-15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals.

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells.

Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C2, and H5-6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of "fast" and "slow" moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C2 and H5-6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C2 and H5-6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.

Rat placental hepatocyte growth factor/scatter factor: purification, characterization, and developmental regulation.

In view of significant species-specific sequence differences between human and rat placental hepatocyte growth factor (HGF)/scatter factor (SF), the rat placental HGF/SF (rpSF) was purified, and its properties compared with human placental HGF/SF (hpSF). Like hpSF, rpSF scattered Madin-Darby canine kidney cells at 1-2 ng/ml and is composed of two subunits of 60 kDa and 30 kDa. Higher amounts (> 50%) of uncleaved 90-kDa form was present in the HGF/SF preparations from both human and rat placentas. Rat placental SF reacts with antibodies raised against hpSF in rabbits and chickens. The SF activity when expressed per gram rat placental tissue rises rapidly up to 9 days and then levels off. When expressed per milligram tissue protein it also increases rapidly up to 9 days and then declines. The expression of HGF/SF mRNA during development parallels that of HGF/SF activity. The specific activity of HGF/SF receptor (c-met) mRNA also appears to peak at 6 days. These findings suggest that (i) in spite of significant (> 10%) sequence differences between rpSF and hpSF, they exhibit similar structural, biologic, and immunologic characteristics and (ii) HGF/SF and its receptor are expressed in high amounts on Day 6 and then decline in developing placenta.

Scatter factor induces blood vessel formation in vivo.

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.

Scatter factor (hepatocyte growth factor) is a potent angiogenesis factor in vivo.

Scatter factor (SF), a fibroblast-derived cytokine characterized by its ability to convert non-motile epithelial cells to a motile fibroblast-like phenotype, is identical to hepatocyte growth factor (HGF), a broad-spectrum mitogen. SF is a heterodimeric glycoprotein that is homologous to plasminogen and other blood coagulation proteases but lacks proteolytic activity. Its receptor is the c-met proto-oncogene product, a growth factor receptor-like transmembrane tyrosine kinase. This unique cytokine is also synthesized and secreted by vascular smooth muscle cells and acts on endothelial cells to stimulate migration, protease production, invasion, proliferation, and differentiation into capillary-like tubes in vitro. SF-containing implants in mouse subcutaneous tissue and rat cornea induce directed ingrowth of new blood vessels from surrounding tissue, with maximal angiogenic responses at doses of 100-200 ng of SF. Immunoreactive SF is expressed at sites of neovascularization within human psoriatic plaques. These findings suggest that SF may play a significant role in the formation and repair of blood vessels under physiologic and pathologic conditions.

HGF-SF: effects on motility and morphology of normal and tumor cells.

HGF-SF is a recently discovered cytokine which has both mitogenic and motogenic effects on a wide variety of cells. We used a computerized digital imaging system to measure motility and morphology of isolated cells. In this chapter we will describe the effect of HGF-SF and of other growth factors on the velocity, area, circularity, and flatness of normal and tumor cells. We will then discuss the possible mechanism of HGF-SF induced motility and the possible role of this factor in biological and pathological processes.

The interaction of HGF-SF with other cytokines in tumor invasion and angiogenesis.

Scatter factor (SF) is a glycoprotein which is secreted by mesenchymal cells and which causes cohesive epithelial cell colonies to spread out, separate into individual cells, and assume a fibroblastic morphology (i.e., to "scatter"). SF is now known to be identical or nearly identical to hepatocyte growth factor, a serum-derived mitogen for various normal cell types. SF, tumor necrosis factor-alpha (TNFa), and interleukin-1 (IL1) share the ability to stimulate scattering, motility, and protease production in a variety of human tumor cell types. SF and TNFa stimulate vascular endothelial cell motility in vitro and induce angiogenesis, the formation of new blood vessels, in vivo. These factors may participate in a cytokine network which regulates tumor invasion and metastasis directly by enhancing the malignant epithelial phenotype and indirectly by inducing tumor neovascularization.

Effect of scatter factor and hepatocyte growth factor on motility and morphology of MDCK cells.

We investigated the effects of human placental scatter factor (hSF), mouse scatter factor (mSF) and recombinant human hepatocyte growth factor (HGF) on motility and morphology of individual Madin-Darby canine kidney cells using a computerized cell tracking system. All three factors increased the velocity of individual cells and the ratio of moving to stationary cells. Similarly, all three factors caused changes in morphologic features of cells, leading to increased area, flatness, and polarity. Increases in area and flatness but not polarity were slightly greater with HGF than with hSF or mSF. These results suggest that SFs and HGF have similar effects on motility and morphology of isolated epithelial cells.

Scatter factor is a glycoprotein but glycosylation is not required for its activity.

Scatter factor (SF) is a protein produced by fibroblasts, smooth muscle cells, and human placenta which scatter cohesive epithelial cell colonies and increases cellular motility. SF bound to concanavalin A and other lectins with high affinity. SF could also be stained with a glycoprotein specific stain. Incubation of producer cells (N-ras-transformed 3T3), with tunicamycin homolog A1 did not have any significant effect on the secretory activity of SF. The treatment of SF with N- and O-glycanases as well as endoglycosidase H had no effect on its activity. However, treatment of target (Madin Darby canine kidney) cells with tunicamycin A1, abolished the scattering response. These studies suggest that scatter factor is a glycoprotein, but glycosylation is not required for its secretion or activity by the producer cells; however, glycosylation of proteins in the target cells is required for SF action.

Protein binding, nuclear translocation and biliary secretion of metabolites of 3'-methyl-N,N-dimethyl-4-aminoazobenzene during hepatocarcinogenesis in rats.

1. The hepatic content, biliary excretion, cytosolic protein binding and nuclear translocation of metabolites of i.v. administered 14C-3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-methyl-DAB) were investigated in rats at various stages of 2-acetamidofluorene (AAF)-induced hepatocarcinogenesis. 2. At nodular and post-nodular stages biliary excretion of radioactive metabolites was decreased, although hepatic content of radioactivity was similar to controls not dosed with AAF. The secretion in bile of a major azo dye binding protein was also decreased at these stages. 3. Binding of dye metabolites to cytosolic proteins was decreased by 40% at nodular and post-nodular stages compared to controls. 4. Translocation in vitro of dye metabolites from cytosol to nucleus at nodular and post-nodular stages was 40% less than that of controls. Since specific soluble proteins control translocation from cytosol into the nucleus (and bile), this decreased binding of metabolites may explain the diminished translocation of carcinogen metabolites into the nucleus.

Purification, characterization and mechanism of action of scatter factor from human placenta.

Scatter factor (SF) causes contiguous sheets of epithelium to spread and cells to separate from each other. SF also increases the velocity, area, and reduces the circularity of individual cells. These changes are mediated in part by alterations in protein synthesis, protein phosphorylation, cytoskeletal reorganization, and cell surface components. SF has been purified from the conditioned medium of ras transformed 3T3 cells and human placenta. Sequence information suggests that SF from 3T3 cells is closely related to hepatocyte growth factor. SF is a glycoprotein, but glycosylation is not necessary for its activity. Glycosylation of target cell proteins, however, is required for SF action.

Site-directed inactivation of human lung acidic glutathione S-transferase by 1-chloro-2,4-dinitrobenzene in the absence of glutathione.

Human lung acidic glutathione S-transferase is irreversibly inhibited by 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first-order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. The Ki was 0.14 mM; and kobs was 0.32 min-1 at 0.6 mM CDNB. The enzyme was protected against CDNB inactivation by GSH. The other two classes of glutathione S-transferase, the basic and near-neutral, are not significantly inactivated by CDNB. Incubation with [14C]CDNB indicated covalent binding to all three classes of transferase. One peptide fraction was found to be radiolabelled in both the basic and acidic transferases when these were incubated with [14C]CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC. Incubation in the absence of GSH yielded one and two additional labelled peptide fractions for the basic and acidic transferases, respectively. Our results suggest that while CDNB arylates all three classes of human transferases, only the acidic transferase possesses a specific GSH-sensitive CDNB binding site, binding to which leads to time-dependent inactivation.

Binding of sulfobromophthalein to rat and human ligandins: characterization of a binding-site peptide.

Photoaffinity techniques were employed to affect the covalent binding of [35S]sulfobromophthalein to proteins of rat and human liver cytosol. In rat liver cytosol at low concentrations, sulfobromophthalein bound to the 22 kDa subunit of ligandin. In human liver cytosol, binding to a 23.5 kDa subunit was observed. At higher concentrations, sulfobromophthalein also bound to 12, 23.5, 37, and 42 kDa peptides. When the peptides resulting from CNBr cleavage of [35S]sulfobromophthalein-ligandin complex were resolved by high-performance liquid chromatography, radioactivity was associated with two peptides. The peptide containing 80% of the radioactivity was isolated and characterized. Its molecular weight is 3.4 kDa, it contains the single tryptophan residue of ligandin and has a glutamate (glutamine) as the N-terminal amino acid.

Noncovalent binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene and its metabolites to liver cytosolic proteins and its role in their nuclear translocation.

When liver cytosol prepared from rats administered [14C]3'-methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak (V) of radioactivity devoid of protein were obtained. Forty to fifty-five per cent of the radioactivity in the protein peaks was butanol-extractable. When the protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 90% of the radioactivity was separated from the proteins, indicating lack of covalent binding. Several differences in the metabolite patterns were seen when the butanol-extractable metabolites from the five chromatographic peaks were analyzed by TLC. When pooled fractions of the peaks were incubated with isolated rat liver nuclei, only radioactivity associated with peak II was translocated into the nucleus. Translocation was time- and temperature-dependent and was maximal at 40 min at 37 degrees C. Only 10 to 12% of the radioactivity associated with peak II could be translocated even in the presence of an excess of nuclei, indicating that specific protein metabolite adduct(s) present in this fraction is/are translocated. Five per cent of translocated radioactivity was irreversibly bound to DNA, 3% to RNA, 67% to non-histone proteins, and 7.5% to histones; the remaining was not associated with any of these macromolecules.