RESUMO
The modulation of the production of collagenase by an epithelial cell line derived from a spontaneously arising rat mammary carcinoma has been studied. The cell line, BC1, was grown permanently under defined serum-free conditions, thus avoiding the poorly characterized and variable effects of serum on collagenase production. Piperazine-N,N'-bis-(2-ethanesulfonic acid) (Pipes), retinoic acid and cytochalasin B all stimulated collagenase secretion, while dexamethasone inhibited it and progesterone, prolactin, prostaglandin E2, and estrogen had no effect. This profile of response to exogenous compounds was distinct from that of cells of mesenchymal origin and from human keratinocytes. For the production of large quantities of collagenase, culture medium was supplemented with Pipes (30 mM, pH 6.8), and retinoic acid (1 microM, on alternate feeds). The collagenase secreted by BC1 cells grown under these conditions was latent and had a molecular mass of 59 kDa. Treatment of the 59 kDa form with trypsin or APMA caused a progressive decrease in molecular mass via 54 kDa and 52 kDa intermediates, to a 48 kDa form. This form was purified to electrophoretic homogeneity by heparin-Sepharose, zinc-chelate-Sepharose, and Sephacryl S-200 chromatography. Five milligrams of purified collagenase were recovered per litre of culture medium.
Assuntos
Neoplasias Mamárias Experimentais/enzimologia , Colagenase Microbiana/metabolismo , Células Tumorais Cultivadas/enzimologia , Animais , Carcinoma , Linhagem Celular , Citocalasina B/farmacologia , Colagenase Microbiana/isolamento & purificação , Peso Molecular , Ratos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Five carcinomas and 5 sarcomas were investigated in relation to their production of neutral proteases capable of digesting polymeric collagen. The carcinomas were far more active than the sarcomas but all the malignant tumours produced enzymes which were capable of causing collagenolysis in vitro. collagenolytic enzymes were recovered from extracts of neoplastic cells from long-term culture, from the media in which these cells were cultured, from the media of mixed cell cultures (neoplastic, stromal and inflammatory cells from minced tumours), and from normal fibroblasts cultures. In contrast to the cultures of non-neoplastic fibroblasts, the tumour cells produced active enzymes, since limited proteolysis with trypsin or treatment with p-aminophenyl-mercuric acetate (APMA) caused no increase in enzyme activity. These tumours possess collagenolytic ability in vitro which may be partly responsible for their invasive nature in vivo.