the cells were trypsinized from routine cell culture washed once with dmem f12 medium treated with soybean trypsin inhibitor washed again with dmem f12 medium and resuspended in growth medium dmem f12 supplemented with insulin 250 ng ml boehringer mannheim indianapolis ind 10 u03bcg ml transferrin sigma 2 6 ng ml sodium selenite collaborative research 1 4 u00d7 10 u2212 6 m hydrocortisone collaborative research 1 u00d7 10 u2212 10 m u03b2 estradiol sigma 5 u03bcg ml prolactin sigma the cells were counted and 100 000 cells were resuspended in 0 3 0 5 mls of growth factor reduced matrigel collaborative research this mixture was placed into a 1 5 cm diameter cell culture well and incubated at 37 degrees celsius for 30 minutes to induce gelling of the matrigel subsequently the gelled cell bm protein mixture was overlayed with growth medium with or without the addition of protease inhibitors or control substances the cells were cultured at 37 degrees celsius in a cell culture incubator at 5 carbon dioxide the growth medium and additives were changed every two days the additives included gm6001 at concentrations varying from 2 u03bcm to 40 u03bcm tapi immunex corp seattle wash at concentrations from 1 to 40 u03bcm and an inactive control analog of gm6001 named c1004 used at 40 u03bcm gm6001 n 2 r 2 hydroxamidocarbonylmethyl 4 methylpentanoyl l tryptophane methylamide is a noncytotoxic synthetic inhibitor that is specific for mmps the hydroxamic group of gm6001 binds to the critical active site zinc atom present in mmps in addition the isobutyl group and tryptophan side chain of gm6001 also binds to subsites on mmps which normally bind.