CATHEPSIN-B FRACTION ACTIVE AT PHYSIOLOGICAL PH OF 7.5 IS OF PROGNOSTIC-SIGNIFICANCE IN SQUAMOUS-CELL CARCINOMA OF HUMAN LUNG

In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumou rs and normal lung tissue by means of fluorogenic assays with Z-Arg-Ar g-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With reaped to pH dependence of a ctivity, we found a deviation from a normal-shaped PH-activity curve. Besides the typical activity peak al pH 6.0, there were shoulders at p H 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumo ur and normal tissue. To test the stability of oath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82 -100% of residual cath B activity was found at pH 5.0-5.5, whereas act ivity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was stili 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 i s more abundant in tumour tissues compared. With the normal counterpar ts, we determined !his fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction ac tive at pH 7.5 in tumour tissue, although this fraction represented on ly a small part (about 16%) of the native, acidic (pH 6.0) cath B acti vity. However, in contrast to native cath B at 6.0, the cath B fractio n active at pH 7.5 was related to post-operative probability of surviv al in curatively operated patients, since activity Values higher than 292 (mu EU mg(-1) protein) were significantly associated with poor pro gnosis in patients with squamous cell carcinomas (n = 33, P = 0.04). I t is concluded that in lung tumour and in normal lung tissue, cath B a ctivity can be divided into at least three fractions with stability op tima at different pH values, indicating various forms of cath B. The c ath B fraction active at pH 7.5 provides prognostic information in pat ients with squamous cell carcinoma.