Cysteine proteinase inhibitors in human melanoma transplanted into nude mice

Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A.