FROG PNKT-4B CELLS EXPRESS SPECIFIC EXTRACELLULAR MATRIX-DEGRADING ENZYMES AND CYTOKINES CORRELATED WITH AN INVASIVE PHENOTYPE

A temperature-dependent metastatic phenotype reported for a frog cell line, PNKT-4B, provided a means for studying potential mediators of ce ll-matrix interaction involved in metastatic invasion. Zymography reve aled that these cells secreted enzyme species with properties and char acteristics of mammalian metalloproteinases: collagenase, stromelysin, gelatinase A, and gelatinase B. These enzymes were produced by PNKT-4 B cultures maintained at both invasive-permissive (28 degrees C), and invasion-restrictive (20 degrees C) temperatures. However, under the i nvasive-permissive culture condition cells produced more of the putati ve gelatinase B and A enzymes. In addition, an activated form of gelat inase A was produced only in invasion-permissive cultures. DNA synthes is bioassays (Mv1Lu cell line and mouse thymocytes) to detect growth p romoting and/or inhibitory cytokines, revealed that PNKT-4B cultures k ept at 28 degrees C released significantly higher levels of stimulator y (interleukin-1-like) and latent inhibitory (transforming growth fact or-beta-like) substances into the medium compared to 20 degrees C cult ures. Pre-absorption of media samples with heparin-sepharose indicated a second stimulatory cytokine as well. A corneal fibroblast bioassay that tests for mediators of collagenase synthesis, detected a stimulat ory substance whose activity was greatly reduced in the presence of in terleukin-1 receptor antagonist protein. Collagenase stimulatory activ ity present in 28 degrees C culture medium was significantly higher th an equal samples from 20 degrees C cultures. These studies provide a m olecular correlation between release of cytokines with properties of t he metastatic phenotype seen in vivo. They further provide some of the first characterizations of frog MMPs and cytokines, which are likely to be involved in other tissue remodeling events. (C) 1997 Wiley-Liss, Inc.