Temperature-sensitive synthesis of a metalloproteinase in ts110-MSV-M-transformed NRK cells

Previously, we reported that transformation associated protein (TAP) was over-expressed in the 6m2 line, but not in their normal counterparts (1,2). 6m2 is a culture of NRK cells transformed by the ts-110 mutant of MSV-M. The synthesis of TAP and the expression of transformation properties in the 6m2 cells are all temperature-sensitive (2; 3; 4). TAP is secreted as two polypeptides of 64 kD and 68 kD (P64 and P68) (2). Experiments were carried out to determine whether any metalloproteinase (MP) activity was associated with TAP. Results of zymograms indicated that the two forms of purified TAP (P64 and P68) had MP activity, using gelatin or collagen type IV as substrates. Serum-free medium (SFM) of 6m2 cells incubated at 33 degrees C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 39 degrees C lacked such MP activity, indicating that the synthesis of MP was temperature-sensitive. The association of MP activity with the P64 and P68 bands of TAP (purified or in SFM) was confirmed by simultaneous Western blot analysis, which showed the reactivity of the two MP bands with monoclonal or polyclonal antibodies to TAP. Accordingly, what we previously designated as TAP is apparently one form of MP, which are known to be involved in tumor cell metastasis.