Enhancement of transformed cell growth in agar by serine protease inhibitors

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.     

The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB 15)

Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum-free medium alone, so serum-free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phospho-diesterase inhibitor R020–1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin

Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.     

Autocrine growth factors for human tumor clonogenic cells

A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.     

Induction of differentiation in human myeloid leukemic cells by proteolytic enzymes

Exogenous serine proteases were found to induce differentiation in human myeloid leukemic cells from either in vitro established long-term cell lines or in primary cultures of cells derived directly from patients with acute myeloid leukemia. Exposure of the human promyelocytic cell line HL-60 to trypsin, chymotrypsin, or elastase induced the appearance, within 3-6 days, of neutrophilic granulocytes defined by their morphology, their ability to reduce nitroblue tetrazolium, and their efficient phagocytosis of latex particles. Upon further incubation monocyte-like cells appeared. While these cells developed into fully mature macrophages other types of cells disappeared and on day 12 the culture consisted of a pure macrophage population. The inducing effect could be observed when the enzyme was presented alone, whereas a synergistic effect was noted when the protease was added in the presence of subthreshold concentrations of chemicals known to induce differentiation in this cell line such as dimethylsulfoxide, retinoic acid, butyric acid, or hexamethylene bisacetamide. Optimal induction of differentiation by trypsin required a 48 hr continuous exposure to the enzyme. When the protease was removed earlier no appreciable differentiation was noticed. The protease-induced differentiation involved a direct interaction with the cells and was not due to a proteolytic cleavage of a serum component because it could be obtained in serum-free cultures. The enzymatic activity of the protease was needed for its effect on cell maturation: Addition of protease inhibitors such as soybean-trypsin inhibitor or trasylol completely blocked differentiation induced by the proteases but had no effect on differentiation induced by the other inducers. It is still to be determined whether a proteolytic process is a general molecular event in cell differentiation or induction by chemicals involves a mechanism different from that initiated by exogenous proteases.     

Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells

Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.     

Evidence that protease action is not specifically involved in the hatching of rabbit blastocysts caused by commercial bovine serum albumin in culture

Commercial samples of bovine serum albumin (BSA) in a complex medium caused growth of 1-cell rabbit embryos to completely hatched blastocysts. Heat treatment of the BSA at 65 or 80 degrees C significantly decreased blastocyst formation and expansion and destroyed the ability to cause blastocyst hatching. Addition of trypsin at levels down to 20 ng/ml caused the formation of hatched blastocysts which degenerated rapidly. The effects of 5 protease inhibitors (ovomucoid trypsin inhibitor, alpha-1-antitrypsin, TAME, TLCK and soybean) were tested. Ovomucoid trypsin inhibitor, TAME and TLCK significantly inhibited blastocyst hatching but only at the highest concentration used. These inhibitors also reduced blastocyst formation and expansion, indicating that their effect was not specifically on blastocyst hatching in vitro. It is concluded that hatching of rabbit blastocysts is probably not dependent on protease action.     

Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.     

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.     

Studies of long-range density effects on the proliferation of 3T3 and RLCW cells in recirculated medium.

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by 3H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Studies of Long-Range Density Effects on the Proliferation of 3T3 and RLCW Cells in Recirculated Medium

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by ³H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Proteases stimulate proliferation of human fibroblasts.

Incubation of primary human fibroblasts in serum-free medium with small concentrations of thrombin, trypsin or plasmin resulted in manyfold increase in total DNA synthesis and in the number of 3H-thymidine labelled cells. Rise in the frequency of mitoses indicates that the proteases stimulated also cell division. Because proteases induced only a fraction of cells to proliferate increase in the total number of cells remained moderate. Calf, horse and rabbit serum inhibited the growth stimulating effect of trypsin but chicken, dog and monkey serum were permissive. Specific inhibitors of proteases prevented the stimulation of cell proliferation suggesting strongly that proteases act in virtue of their enzymatic activity.     

A novel protease obtained from FBS-containing culture supernatant,that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Evaluation of assays for detecting alpha-1-protease inhibitor during purification from rat serum

We purified the R1 alpha-1-protease inhibitor from rat serum and developed a convenient assay for its detection during purification procedures. Purification was accomplished by desalting, DEAE-Sephacel, zinc chelate, and reactive green-agarose columns. The resultant antiprotease had a molecular weight of 54,000 and inhibited elastase, chymotrypsin, and trypsin. By isoelectric focusing, five bands were produced with pI values from 4.3 to 4.7. Functional assays utilizing protease substrates imbedded in agarose plates were evaluated for the ability to distinguish the R1 alpha-1-protease inhibitor from the other serum antiproteases eluted in column chromatography fractions. This technique of screening for anti-protease activity was compared to conventional spectrophotometric methods and was found to correlate well when quantifying inhibition of elastase and chymotrypsin, but not trypsin. The presence of alpha-1-protease inhibitor was most reliably detected by testing for anti-elastase activity. Technician time and expense were saved by employing protease substrate plates to test chromatogrpahy fractions. This technique may facilitate purification of other protease inhibitors.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.