Matrix metalloproteinase-9 associated with heparan sulphate chains of GPI-anchored cell surface proteoglycans mediates motility of murine colon adenocarcinoma cells

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells.     

Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells.

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of extracellular matrix-degrading activities between 64-kDa and 90-kDa gelatinases purified in inhibitor-free forms from human schwannoma cells.

Two kinds of gelatinases (or type IV collagenases), 90-kDa and 64-kDa gelatinases, were purified in a tissue inhibitor of metalloproteinases (TIMP)- or TIMP-2-free form from the serum-free conditioned medium of human schwannoma YST-3 cells, and their activities on extracellular matrix proteins were compared. Sequential chromatographies on a gelatin-Sepharose column, an LCA-agarose column, and a gel filtration column in the presence of 5 M urea yielded 600 micrograms of the 64-kDa enzyme and 45 micrograms of the 90-kDa enzyme from 2.8 liters of the conditioned medium. The purified enzymes showed high gelatinolytic activities without activation by p-aminophenyl mercuric acetate (APMA), indicating that 5 M urea used in the final chromatography not only dissociated the inhibitors from the progelatinases but also activated the proenzymes. The inhibitor-free gelatinases showed a much higher activity than the APMA-activated inhibitor-bound enzymes. The specific activity of the 90-kDa enzyme was nearly 25 times higher than that of the 64-kDa enzyme. The 90-kDa gelatinase hydrolyzed type I collagen as well as native and pepsin-treated type IV collagens at 30 degrees C, while at 37 degrees C it potently hydrolyzed types I, III, and IV collagens but not fibronectin or laminin. The 64-kDa gelatinase showed a similar substrate specificity to that of the 90-kDa enzyme, except that it did not hydrolyze type I collagen and native type IV collagen at 30 degrees C.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Vitamin C and thiol reagents promote the in vitro growth of murine granulocyte/macrophage progenitor cells by neutralizing endogenous inhibitor(s)

Growth of murine hemopoietic cells in culture requires the presence of a stimulator of stem cell proliferation, "colony stimulating factor" (CSF). A widely used source of CSF is lung conditioned medium (LCM). We have earlier shown that the great variability of CSF activities in different batches of LCM is due to varying amounts of inhibitor(s). The present study expands the observation that the addition of ascorbic acid to the murine bone marrow soft agar assay system removes the inhibitory activity. The vitamin probably acts as an antioxidant or free radical scavenger, since addition of reduced (but not oxidized) glutathione, cysteine, dithiothreitol or 2-mercaptoethanol to the cultures also inactivates the endogeneous inhibitor. Cysteine and glutathione gave the highest colony numbers, were active at concentrations present in body fluids and did not inhibit colony growth even at concentrations ten times higher than optimum. No synergistic effects could be observed between the different antioxidants. At optimum concentration (usually 0.45 mmol/l) the otherwise bell-shaped dose-response curve for conditioned medium changed to a sigmoid curve. Antioxidants had no growth promoting effect in the absence of CSF. The presence of cysteine or vitamin C revealed CSF-like activity in conditioned media of tissues not considered to be potent producers of such factors. It has been reported that individual batches of foetal calf serum contain different levels of reduced glutathione, and we suggest that one of the batch variable growth regulators in foetal calf serum may be reduced glutathione. The results indicate a possible physiological role of antioxidants in granulopoiesis and suggest that cysteine or reduced glutathione should be freshly added to culture systems assaying CSF and/or granulocyte macrophage progenitor cells.     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. IV. The release of inhibitor from cultured cells.

Previous reports from our laboratory have shown that a culture medium, conditioned by the growth of isolated cells of Xenopus laevis blastulae, contains a low-molecular-weight substance which selectively inhibits 18 and 28S ribosomal RNA (rRNA) synthesis. Although the occurrence of an inhibitor in an acid-soluble fraction of blastulae has recently been demonstrated, our observation of an inhibitor in a conditioned medium has not been confirmed by other laboratories. To resolve this discrepancy, we have reexamined the effects of conditioned media and acid-soluble extracts on rRNA synthesis by neurula cells. (1) The inhibitory activity for rRNA synthesis can consistently be observed in blastula-conditioned media, provided some of the cells have been broken down during conditioning. If cell rupture is avoided, an inactive conditioned medium is obtained. (2) A homogenate of blastulae inhibits total RNA synthesis and shows no selective inhibition of rRNA synthesis. (3) Charcoal treatment of the conditioned medium and homogenate enhances their specificity for rRNA synthesis. It is then likely that cell breakdown may be involved in the release of the inhibitor into the medium and that some differences in the methods of preparation of conditioned medium may account for the above discrepancy.     

Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles

Isolated morphologically normal bovine preantral follicles (40 to 70 microm) were cultured for 8 d in collagen gel in control medium or in 1 of 3 conditioned media from the murine granulosa cell lines GRMO1L, GRMO2 and GE2. The percentages of follicles at Day 1 that remained nomal at Day 8 were similar for follicles cultured in the conditioned and control media (84 to 90%). A significantly higher percentage of follicles cultured in each of 3 conditioned media started to grow (89%; P < 0.05) and their increase in diameter was greater than that of follicles cultured in control medium (72%; P < 0.05). The mitotic activity of the granulosa cells from follicles cultured in conditioned media was increased (P < 0.05) indicated by a higher percentage of nuclei that incorporated BrdU compared with that of follicles cultured in control medium. Follicular viability was measured by the presence of nonspecific esterase activity, active mitochondria and dead cells in cultured follicles using the fluorescent probes calcein-AM, rhodamine 123 and ethidium homodimer-1 in combination with confocal laser scanning microscopy. The percentages of follicles with esterase activity and active mitochondria present in their granulosa were similar for follicles in all groups. Culturing in GRMO2 or GE2 tended to lower the number of granulosa with dead cells. The percentage of follicles with oocytes without esterase activity and active mitochondria was lower (P < 0.05) in follicles cultured in GRMO2 or GE2 compared with those cultured in control medium. Moreover, the percentages of dead oocytes tended to be higher in follicles cultured in GRMO1L and GE2 compared with oocytes of follicles incubated in control medium. Taken together, the conditioned media stimulated follicular growth and granulosa viability as well as enhance mitotic activity of the granulosa cells. However, they negatively affected oocyte viability.     

Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor, which was identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human carcinoma cell line. HGF activator is a blood coagulation factor XII-like serine protease that is responsible for proteolytic activation of the inactive single chain precursor of HGF in injured tissues. The predicted sequence of the primary translation product of HAI-1, which has a hydrophobic sequence in its COOH-terminal region, suggested that HAI-1 is first produced in a membrane-associated form. In this study, we identified a transmembrane form of HAI-1 integrated in the plasma membrane of cultured cells using a monoclonal antibody against HAI-1. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGF activator. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of HAI-1.     

Characterization of immunotrap assays for urokinase plasminogen activator and its inhibitors and measurements of these molecules in human plasma and mouse macrophage in culture

• 1.1. Immunotrap assays that can measure the activities of urokinase-type plasminogen activator (uPA) and its inhibitors (PAIs) were characterized.
• 2.2. Both human plasma and mouse macrophages in culture were found to contain much higher inhibitor activity than uPA-like activity.
• 3.3. The balance between pro- and anti-flbrinolytic activities was quantitatively changed in the murine macrophages after the injection of thioglycollate. uPA-like materials were synthesized by the macrophages and secreted to the conditioned medium continuously, while PAI activity was unchanged during the same time period.

     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

Murine recombinant leukemia inhibitory factor modulates inhibitory effect of 1,25 dihydroxyvitamin D3 on alkaline phosphatase activity in MC3T3-E1 cells

We demonstrated murine leukemia inhibitory factor (mLIF) mRNA in osteoblastic MC3T3-E1 cells, but not mLIF in their conditioned medium. Recombinant mLIF had an inhibitory effect on alkaline phosphatase (ALP) activity, but not on DNA synthesis, in these mLIF-free cells. This inhibitory effect was not prostaglandin E2 dependent. mLIF also modulated the inhibitory effect of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] on ALP activity, partly via down regulation of 1,25(OH)2D3 binding sites. These results suggest that LIF may play a role in regulating osteoblast differentiation.     

Glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney cells, but not in their polyoma virus-transformed analogs

Glycopeptides isolated from mouse cerebral cortex cell surfaces (BCSG) were shown to inhibit cell growth and protein synthesis in baby hamster kidney (BHK)-21 cells, whereas polyoma virus-transformed BHK-21 cells (pyBHK-21) were refractory to the inhibitory activity of the glycopeptides. Growth inhibition was shown to be reversible and non-lethal to BHK-21 cells. Despite that difference in sensitivity to the action of the glycopeptides, both cell lines could bind the inhibitor in a saturable fashion and in similar quantities. After trypsinization, BHK-21 cells appeared refractory to the inhibitor, whereas pyBHK-21 cells became sensitive. The data suggested the presence of a receptor for BCSG on the cell surface of both cell lines. Incubating BCSG with conditioned medium from pyBHK-21 cells resulted in loss of the glycopeptide's inhibitory activity. In contrast, medium conditioned by BHK-21 cells had no effect on the inhibitory activity of BCSG. We hypothesize that the refractoriness of pyBHK-21 cells to BCSG is related to their autonomous growth characteristics and failure to respond to topo-inhibitory growth control. BCSG may be a naturally occurring growth regulator whose function can be explored by use of the BHK-21/ pyBHK-21 model system.     

The production of factors regulating the proliferation of haemopoietic spleen colony-forming cells by bone marrow macrophages

Media conditioned by normal murine bone marrow cells contain an inhibitor of haemopoietic spleen colony-forming cell proliferation that is concentrated in a nominal 50-100K fraction. Media conditioned by regenerating marrow cells contain a proliferation-stimulatory activity that is concentrated in a nominal 30-50K fraction. Cell separation experiments demonstrated that the activities are produced by adherent, phagocytic, radioresistant, Thy 1.2- Fc+, F4/80+ cells. Cultured macrophages, obtained from long-term marrow cultures or derived from progenitor cells in methyl cellulose cultures are also capable of producing inhibitory and stimulatory activities. The results are consistent with macrophages being an important source of stem cell proliferation regulators in the bone marrow.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.