FIG. 1.

Regression of transplanted melanomas following extinction of H-RAS^V12G expression. (A) Soft-agar assay of early passage R545 melanoma cells expressing H-RAS^V12G (on) or not expressing H-RAS^V12G (off), showing that colony formation is dependent on H-RAS^V12G activation. (B) Impact of doxycycline withdrawal on tumor size. Early passage melanoma cell lines were injected subcutaneously into SCID mice given doxycycline in the drinking water. After 14 days, doxycycline was removed from the water, and tumors were isolated at the indicated time points following doxycycline withdrawal. Melanomas with an inducible H-RAS^V12G transgene regress, while the C590 line that constitutively expresses H-RAS^V12G continues to grow. The data shown are from a representative experiment consisting of four tumors per time point. Comparable results were obtained in multiple repeat experiments. (C) Apoptotic and proliferative indices of tumors from R545 cells at 0 and 72 h following doxycycline withdrawal. (D) Impact of doxycyline withdrawal on tumor histopathology. Tumors from mice on doxycycline (left panels) or 72 h following doxycycline withdrawal (right panels) were analyzed by RNA in situ hybridization for H-RAS^V12G expression, H&E staining, CD-31 immunostaining to detect vasculature, TUNEL staining to detect apoptosis, and Ki-67 immunostaining to measure proliferation.