Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Type-specific collagenolysis: a type V collagen-degrading enzyme from macrophages

An enzymatic activity capable of degrading type V collagen at neutral pH was found in the medium from cultured rabbit pulmonary alveolar macrophages which had been “activated” $\text{in}\text{vivo}$ by injection of complete Freund's Adjuvant. This enzyme was characterized as a metalloproteinase by virtue of its inhibition by EDTA but not by phenylmethylsulfonyl fluoride or N-ethyl maleimide. Ion-exchange chromatography on DEAE-cellulose was successful in separating the type V collagen-degrading activity from the type I collagenase which is also secreted by these cells. These observations suggest that the degradation of type V collagen is independent of the degradation of the interstitial collagens and may require the action of its own “specific collagenase”.     

Effect of ozone and nitrogen dioxide on the agglutination of rat alveolar macrophages by concanavalin A

$\text{In vitro}$ exposure of rat alveolar macrophages to 0.5 ppm ozone for 60 minutes results in a 76% decrease in agglutination by concanavalin A. A decrease in the agglutinability of rat alveolar macrophages by concanavalin A was also observed following inhalation of 0.5 or 1.0 ppm ozone for two hours. In contradistinction, $\text{in vitro}$ exposure of rat alveolar macrophages to 2.4 ppm nitrogen dioxide for 60 minutes produced a 64% increase in agglutination by concanavalin A; and increased agglutinability was also noted following inhalation of 12.1 ppm nitrogen dioxide for two hours. Agglutination was almost completely inhibited by alpha-methyl-mannose. Neither pollutant significantly altered the binding of ³H-concanavalin A to rat alveolar macrophages. These two air pollutants, both of which are known to potentiate respiratory tract infections, appear to affect the response of the alveolar macrophage membrane to concanavalin A in a dissimilar fashion.     

Dental pulp collagenase: initial demonstration and characterization.

A collagenase, active against native helical collagen, was initially found in the explant medium of bovine dental pulp. In contrast to the collagenases from other oral tissues, all the pulp enzyme released was in a latent form which was activated by trypsin treatment, 4-aminophenylmercuric acetate, and some chaotropic agents. The activated enzyme was inhibited by low concentrations of EDTA and calf serum. The molecular weight of activated enzyme was tentatively estimated at 45,000 daltons by gel filtration. The enzyme attacked undenatured collagen in solution at 20°C producing characteristic products $\text{α}{}^{\mathrm{A}}\text{(}\text{3}\text{4}\text{)}$ and $\text{α}{}^{\mathrm{B}}\text{(}\text{1}\text{4}\text{)}$.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Demonstration of collagenase activity in rat liver homogenate

Collagenase activity became detectable in rat liver homogenate by washing liver tissue repeatedly with buffered saline before homogenization. This enzyme activity was inhibited by adding minute quantities of serum. These data suggest that collagenase is active $\text{in situ}$ in the liver, but is made inactive during the homogenization by forming a complex with contaminating serum factors.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils

Collagenase released from rheumatoid synivial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24°C and 37°C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibril can be degraded. Binding was not inhibited by the presence of α₂ macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding the substrate, and that activation by proteases such as plasmin intiates collagen breakdown.     

Cyclic nucleotide metabolism and reactive oxygen production by macrophages

The production of reactive oxygen species by elicited rat peritoneal macrophages was assessed by $\text{in vitro}$ measurement of chemiluminescence in the presence of luminol. The divalent ion ionophore A23187 stimulated the production of reactive oxygen species. This action was inhibited by monobutyryl and dibutyryl derivatives of cyclic AMP but was not affected by derivates of cyclic GMP. Cyclic AMP and cyclic GMP concentrations increased rapidly in macrophages exposed to A23187 or zymosan. Indomethacin (20 μmol/1) inhibited the increase in cyclic AMP concentration but not the increase in cyclic GMP concentration. Neither A23187 nor zymosan stimulated adenylate cyclase activity in broken cell preparations of macrophages. The observations are consistent with the hypothesis that PGE produced by macrophages after phagocytotic stimuli may inhibit certain macrophage functions and perform a regulatory role in these cells. This action of PGE may be mediated by cyclic AMP.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Intracellular origin of hormone-sensitive lipolysis in the rat

The hormone-stimulated hydrolysis of endogenous triglycerides in heart and adipose tissue was found to be inhibited by chloroquine, which is known to accumulate in lysosomes and to inhibit the lysosomal degradation of protein and cholesterolesters.When the triglyceride depôt in heart cells was increased by feeding rats a diet enriched in erucic acid for three days prior to $\text{in vitro}$ perfusion of the heart, the spontaneous and the norepinephrine stimulated rates of lipolysis were both found to be increased. Both were inhibited by chloroquine. Analysis of trioleoylglycerol hydrolysis in heart homogenates after $\text{in vitro}$ heparin perfusion revealed the virtual absence of neutral lipase in contrast to an acid lipase activity. The results of this study suggest that lipolytic activity of lysosomal origin is the main source of hormone-sensitive endogenous triacylglycerol hydrolysis.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Choline acetyltransferase activity of spinal cord cell cultures is increased by diisopropylphosphorofluoridate.

Diisopropylphosphorofluoridate (DFP) increased the specific activity of choline acetyltransferase (ChAT) in mouse spinal cord cell cultures but paraoxon did not, though both toxicants inhibited cholinesterase activity to a comparable extent. This effect of DFP was observed after prolonged exposure but not after short-term application to the cultures. It is postulated that this delayed effect of DFP $\text{in vitro}$ may possibly be related tothe delayed neuropathy caused by DFP in certain species $\text{in vivo}$.     

The action of dibucaine in vitro on fat cell lipolysis and protein kinase activity.

Dibucaine at 0.1 and 0.25 mM markedly inhibited epinephrine-stimulated lipolysis in rat epididymal fat cells $\text{in}$$\text{vitro}$ but did not inhibit protein kinase activity. At 1.0 mM, dibucaine half-maximally stimulated protein kinase of fat cells under basal conditions but did not stimulate lipolysis. It is concluded that dibucaine inhibits lipolysis by a mechanism not involving inhibition of protein kinase.     

Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans.

Immunoreactive somatostatin is released from islets of Langerhans, isolated from rat pancreas by collagenase digestion, when incubated in an $\text{in vitro}$ system. The rate of somatostatin secretion is independent of extracellular glucose concentration, but is stimulated by addition of 8-Br-cyclic AMP or theophylline.     

Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase

Incorporation of ³²P from [γ-³²P]ATP into a homogeneous preparation of rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was catalyzed by a homogeneous preparation of the catalytic subunit of the cyclic AMP dependent protein kinase from rat liver. Approximately 2 mol of phosphate were incorporated per mol of the dimeric enzyme and this was associated with inhibition of the phosphotransferase activity and activation of the phosphohydrolase activity. Acid hydrolysis of the enzyme that was phosphorylated $\text{in}$,$\text{vitro}$ revealed that only seryl residues were labeled. Fructose 2,6-bisphosphate inhibited the initial rate of phosphorylation of the enzyme. It is concluded that both activities of this bifunctional enzyme are regulated in a reciprocal manner by cyclic AMP-dependent phosphorylation and that this phosphorylation can be modulated by fructose 2,6-bisphosphate.