Angiostatin generation by human tumor cell lines: Involvement of plasminogen activators

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an inte rnal fragment of plasminogen. Little is known about the production of angio statin by human tumors. In this study, we examined the in vitro angiostatin -generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determi ned by assessing the level of plasminogen digestion in conditioned medium b y Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed inter mediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were ab le to generate angiostatin at intermediate levers. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkabl y, angiostatin bands were not of equal size in all plasminogen digests. Sin ce reported data have indicated that plasminogen activators (uPA and tPA) w ere able to excise the angiostatin fragment from the plasminogen parent mol ecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiosta tin-generating capacity. Whereas prostate- and bladder-carcinoma lines capa ble of generating high levels of angiostatin showed high uPA revels, angios tatin generation in melanoma cell lines was correlated with tPA levels. Gen erally, angiostatin non-producers did not express uPA or tPA. In 6 out of 7 5 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alte rnative proteolytic pathways in the generation of angiostatin. (C) 2000 Wil ey-Liss, Inc.