The Collagenase Produced by Neoplastic Rat Epithelial Cells: Modulation of Secretion, Molecular Weight Characteristics, and Purification

Lyons, J. Guy; Nethery, Andrew; O'Grady, Robert L.; Harrop, P.

doi:10.1016/s0934-8832(89)80012-5

Cited by 9 publications

(6 citation statements)

References 5 publications

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“…although not extensively characterized in the culture medium of several cell lines of neoplastic origin (Yamagata et al, 1988;Ballin ct al., 1988). We have shown recently that the highly invasive and metastatic rat mammary carcinoma cell line BC1 is capable of secreting high levels of interstitial collagenase (MMP-1) (O' Grady ct al., 1981;Lyons et al, 1989a). In this paper, wc demonstrate that BC1 cells also express high levels of a type IV and V collagen degrading the Mt 95 000 MMP that appears to be regulated in a manner different from interstitial collagenase.…”

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confidence: 68%

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Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

Lyons¹,

Birkedal-Hansen²,

Moore³

et al. 1991

Biochemistry

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A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.

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confidence: 68%

“…Large-scale cultures of BC1 and cocultures of BC1-E2 and BC1-M3 were grown in basal medium supplemented with 30 mM Pipes and I /^M retinoic acid (Lyons et al, 1989a).…”

Section: Methodsmentioning

confidence: 99%

Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

Lyons¹,

Birkedal-Hansen²,

Moore³

et al. 1991

Biochemistry

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“…The presence of active gelatinases was assayed by zymographic analyses. Rat collagenase was purified from rat mammary carcinoma cells as described previously (18) and supplied by Ms. A. Martorana from the University of Technology, Sydney. It was shown to degrade collagen using a dried collagen film assay (19) and gelatin by zymographic analysis.…”

Section: Methodsmentioning

confidence: 99%

The Degradation of Human Endothelial Cell-derived Perlecan and Release of Bound Basic Fibroblast Growth Factor by Stromelysin, Collagenase, Plasmin, and Heparanases

Whitelock

Murdoch

Iozzo

et al. 1996

Journal of Biological Chemistry

565

381

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Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the ϳ450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of ϳ300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

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“…Up until the mid-1980s, the Institute was responsible for the Hospital's pathology service, though the functions of the clinical pathologist were changed in the mid-1970s to facilitate a greater involvement in research. Over the next 10 years, Bob O'Grady developed a program on the control of connective tissue breakdown, particularly the role of collagenase (Lyons et al, 1989), which led to collaborative studies with Henning Birkedal-Hansen in Alabama (Lyons et al, 1991). Such collaborative studies, frequently with Visiting Fellows, have proved their value, as shown, for example, by the studies with Bob Fitzgerald (Jacques et al, 1980;Hardy et al, 1986;Willcox et al, 1993).…”

Section: Caries Prevention Programsmentioning

confidence: 99%

Neil Goldsworthy and His Legacy: Sydney's Institute of Dental Research

1999

J Dent Res

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Dr. Neil Goldsworthy had a profound effect on the development of dental research in the biological sciences in Australia, including measures for the prevention of dental caries. He played a major role in the establishment of the Institute of Dental Research in Sydney in 1946 and was Director until his sudden death in 1960. Reviewing the Institute's activities provides the opportunity for his achievements to receive due recognition and to show how they influenced its subsequent development. As one who has been associated with the Institute for most of the last 50 years, it is an honor for me to undertake this task.

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The Collagenase Produced by Neoplastic Rat Epithelial Cells: Modulation of Secretion, Molecular Weight Characteristics, and Purification

Cited by 9 publications

References 5 publications

Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

The Degradation of Human Endothelial Cell-derived Perlecan and Release of Bound Basic Fibroblast Growth Factor by Stromelysin, Collagenase, Plasmin, and Heparanases

Neil Goldsworthy and His Legacy: Sydney's Institute of Dental Research

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