Partial purification of a liver-derived tumor cell growth inhibitor that differentially inhibits poorly-liver metastasizing cell lines: identification as an active subunit of arginase.

Abstract

Organ specific tumor metastasis is thought in part to require the ability of metastatic cells to respond to target-organ-associated growth factors or to avoid the effects of target organ associated growth inhibitors. We previously found that murine and rat liver-conditioned media inhibited the growth of the poorly-liver metastasizing murine RAW117-P large-cell lymphoma cells more than their highly liver-metastasizing RAW117-H10 counterparts. Using a six step chromatographic procedure, the major RAW117-P cell proliferation inhibitor from a rat liver extract was purified. The factor displayed a Mr of approximately 35,000 and an isoelectric point > 8.5. This material inhibited the growth of many cells at high concentration; however, in dose-response studies it displayed a higher IC50 for highly-liver metastatic murine RAW117-H10 lymphoma and human KM12SM colon carcinoma cells than for their poorly-liver metastatic counterparts. Attempts to identify the growth inhibitor led to the supplementation of tissue culture inhibitor assays with various components, including excess amino acids, and this was found to completely abrogate the factor's activity. Specifically, the addition of excess arginine resulted in the complete cellular recovery from inhibitor exposure. This tentatively identified the liver growth inhibitor as the enzyme arginase, a Mr approximately 10,000 multisubunit protein. A microtiter plate-based assay for arginase was developed and the purification repeated using human liver as a source of activity and the human KM12C colon carcinoma line as a target. The growth inhibitory and arginase activities were found to co-purify, identifying the factor as arginase. Highly-metastatic cells displayed no ability to preferentially inactivate or inhibit the activity of arginase, but they did they display slightly greater amounts of intracellular arginine. The liver is a major site of arginase localization as the enzyme is required for the functioning of the urea cycle. The results indicate that certain liver-colonizing tumor cells can escape, to a degree, the proliferation-damping effects of arginine depletion.