Meera S. Paranjpe

Activation of human breast carcinoma collagenase through plasminogen activator

Abstract 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 3 4 − 1 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 ( 3 4 − 1 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 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 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.

Retinoids block ornithine decarboxylase induction in cells treated with the tumor promotor TPA or the peptide growth hormones, EGF and SGF.

Abstract A clone of rat kidney cells is described that responds to the tumor-promoting agent tetradecanoyl phorbol acetate (TPA) and to the polypeptide growth hormones, epidermal growth factor (EGF) and sarcoma growth factor (SGF) by increasing ornithine decarboxylase (ODC) activity. The peak with EGF and SGF is at 2–4 hours after exposure; with TPA the response is slower but more sustained. Retinoic acid pretreatment of the cells blocked both the TPA-induced response as well as the EGF- and SGF-induced response.

Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline

Abstract cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murine cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.

Selective growth of malignant cells by in vitro incubation on Teflon

Abstract The selective growth of malignant cells on non-adhesive Teflon substrates is reported. Neoplastically transformed cell lines of human and murine origin proliferated on Teflon whereas similarly seeded normal non-tumorigenic cells were unable to undergo cell division and subsequent growth. This substrate should be especially useful in establishing primary cultures from human tumor material.

Discussion paper: specific paralysis of the antitumor cellular immune response produced by growing tumors studied with a radioisotope footpad assay.

The kinetics of the antitumor cellular immune response of mice with progressively growing syngeneic tumors were determined in vivo using a quantitative radioisotopic footpad assay. A close correlation was found between the size of the tumor and the degree of the cellular immune response. An initial phase of cellular immune stimulation was followed by specific suppression and subsequent immunologic paralysis as the tumor grew larger. This immune paralysis was attributed to increased tumor load since a homogenate of an SV40 transformed fibrosarcoma injected intraperitoneally into tumor-immune mice specifically depressed their cellular immune response. The fraction of the tumor homogenate that brought about this depression was present in the high speed supernatant and pellet of a 3M KCl extract of the tumor. The specificity of the depression was determined in vivo by the radioisotopic footpad assay and in vitro by a 51Cr cytolysis assay. Unwashed spleen cells harvested from mice bearing large tumors were unreactive in a local adoptive footpad assay. However, reactivity could be restored by repeatedly washing the spleen cells.

Intermittent sphering of virus-transformed and other neoplastic cells observed by time lapse cinematography.

Abstract A number of mouse cell lines neoplastically transformed by SV40 virus, one by Herpes simplex type II virus, and one of five ‘spontaneously’ transformed neoplastic cell lines contained a significant fraction of cells that exhibited ‘intermittent sphering’ when observed by time lapse cinematography. That is, the cells repeatedly rounded up and became highly refractile for some minutes as though preparing to divide, then stretched out for minutes or hours before sphering again. The specific pattern of the frequency and duration of sphering periods was a heritable property. Occasional revertants to permanent non-sphering status were observed. Animal passage did not alter the fraction of intermittent sphering cells in the population. BALB/3T3 cells planted on paraffin, but not on plastic substrates also exhibited intermittent sphering, indicating that this property is related to a decrease in cell-substrate adhesivity.