latter tumors were whitish exhibited a less anaplastic spindle cytology failed to express the h ras v12g transgene by western blot analyses and were trp 1 negative data not shown these data suggest that these doxycycline independent tumors are either a different cancer type such as fibrosarcoma or ras independent melanomas although these results and those of others 8 underscore a role for ras in tumorigenesis this novel model system permits an investigation of mechanisms underlying tumor regression in vivo to this end we first assessed the impact of doxycycline withdrawal in different tyr tet ras ink4a u2212 u2212 tumor derived cell lines in cell culture melanoma cell lines were established from primary tumors in doxycycline supplemented media and were shown to express h ras v12g mrna protein and activity in a doxycycline dependent manner fig4 a and data not shown the derivative melanoma cell lines remained doxycycline responsive as shown by their strict doxycycline dependency for tumorigenic potential in scid mice specifically subcutaneous injection of purified tyr tet ras ink4a u2212 u2212 melanoma cell lines yielded tumors in scid mice treated with doxycycline with 100 efficiency while those mice not receiving doxycycline remained tumor free after many months of observation fig3 a in cell culture the presence or absence of doxycycline did not significantly influence the subconfluent growth rates of multiple independently derived lines fig3 b similarly the presence of doxycycline failed to confer enhanced growth potential in low serum conditions 0 5 and 1 fcs data not shown however the presence of doxycycline modestly enhanced the growth in dense cultures suggesting that in vivo ras may provide a continued cell autonomous advantage for tumor growth on the other hand the data is also consistent with the possibility.