Expression of activated MEK1 or MEK2 enhances the proliferation of IEC-6 cells and induces the formation of intestinal tumors. (A) IEC-6 cells infected with the indicated constructs were plated in triplicate wells of 24-well plates in complete medium containing 10% serum. Cell proliferation was measured each day using the MTT assay. (B) Anchorage-independent proliferation was assayed by measuring the formation of cell colonies in soft agar. Results indicate the number of colonies after 21 days and are representative of 3 independent experiments. (C) IEC-6 cell populations expressing the indicated MEK constructs were either injected subcutaneously (subcutaneous tumors) into the flanks of athymic mice or orthotopically transplanted into the cecal wall (cecal tumors) of the mice as described in Methods. The mice were monitored for tumor development. (D) Growth rate of subcutaneous tumors. Values represent the average volume +/- SEM of 8 tumors (4 mice). Data are representative of 3 independent experiments. (E) Representative pictures of intestinal tumors generated by orthotopic implantation of MEK1DD- or MEK2DD-expressing IEC-6 cells in the ceacum. N, normal mucosa; T, tumor. Note the poorly differentiated morphology of tumor cells typical of high-grade adenocarcinomas. Inset, signet-ring cells.