Degradation of type IV collagen by neoplastic human skin fibroblasts

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Abstract
An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labeled by reductive alkylation. Both collagens were in the native form as shown by their low sensitivity to degradation by trypsin. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. The normal cells released a collagenase into the medium which after activation by trypsin or oxidized glutathione degraded type I collagen. Hut-11A cells also produced a collagenolytic activity that degraded type I collagen; no activation of the medium was required for this activity. Type IV collagen was not degraded by medium conditioned with the normal (KD) cells with or without activation. Hut-11A cells secreted an active collagenase into the medium that degraded type IV collagen extensively. Treatment of the medium from Hut-11A cells with trypsin resulted in only a loss of activity while treatment with oxidized glutathione was without effect. The degradation of type IV collagen by Hut-11A conditioned medium was linear for up to 1 h and the extent of degradation increased linearly with increasing amounts of conditioned medium. A cell associated assay was performed by growing either normal or transformed cells in the presence of radioactively labeled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen; Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity; protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. Apparently, the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion.