Targeted expression of hMet in mouse hepatocytes. (a) TRE-MET transgene construct. (b) Transgenic hMet is expressed specifically and conditionally in hepatocytes. Immunohistochemistry of liver sections using antibodies to hMet. Left, liver from LAP-tTA mouse. Middle, liver of a littermate doubly transgenic for LAP-tTA and TRE-MET. Inset, Met staining at higher magnification. Right, liver from a double transgenic mouse treated with doxycycline (Dox).

Apoptosis and cessation of proliferation were involved in tumor regression. Liver paraffin sections from a tumor-bearing mouse (a and c) and a tumor-bearing mouse after 3 d of doxycycline treatment (b and d) were stained for BrdU (a and b) or fragmented DNA (c and d). Cells that have incorporated BrdU stained brown. Apoptotic cells were detected by TUNEL assay, and the fragmented DNA was labeled with fluorescein.

Activation of transgenic hMet is dependent on cell attachment. (a) Transgenic hMet was phosphorylated in livers. Livers from mice transgenic for both TRE-MET and LAP-tTA (lane 1) or LAP-tTA as control (lanes 2 and 2′) were lysed and immunoprecipitated (IP) with antibodies to hMet (lanes 1 and 2) or to murine Met (lane 2′) and immunoblotted with the antibodies indicated. (b) Activation of the transgenic hMet requires cell attachment. Hepatocytes isolated from a tumor-bearing double transgenic mouse were lysed as single cell suspension (lanes 1 and 2) or after attachment to collagen-coated plates (lanes 3 and 4). Before lysing, cells were treated in the absence (lane 1 and 3) or presence of human HGF (lanes 2 and 4). Cell lysates were immunoprecipitated with antibodies to hMet and immunoblotted with antibodies to either phosphotyrosine or hMet.

Met in NCI-H441 cells is normal and can elicit signaling in response to cell attachment. (a) Both α and β subunits of Met in NCI-H441 cells are expressed on the cell surface. Cell surface proteins of NCI-H441 cells (lanes 2 and 2′) and the control A459 cells (lanes 1 and 1′), in which Met does not respond to cell attachment, were labeled with biotin and lysed. Met was immunoprecipitated (IP) with antibodies to hMet and resolved under reducing (lanes 1 and 2) or nonreducing (lanes 1′ and 2′) conditions. Both P140^met and P190^met were detected with Streptavidin–horseradish peroxidase. (b) Activation of Met by cell adhesion elicits Grb2 recruitment to the cell surface. NCI-H441 cells were lysed while in suspension (Det) or while attached (Att) and immunoprecipitated with antibodies to hMet. The immunocomplexes were resolved by SDS-PAGE and immunoblotted with antibodies to phosphotyrosine, Grb2, or hMet.

Overexpression of Met enables receptor activation by cell attachment. (a) Wild-type Met responds to cell attachment when overexpressed. HeLa cells were stably transfected with a plasmid containing mMet (lanes 5–8) or with the vector alone as a control (Co; lanes 1–4) and examined for Met phosphorylation in response to HGF or cell attachment. Cells in suspension (lanes 1, 2, 5, and 6) or on plates (lanes 3, 4, 7, and 8) were treated with either HGF (lanes 2, 4, 6, and 8) or control media (lanes 1, 3, 5, and 7). Cell lysates were immunoprecipitated (IP) with antibodies to mMet. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with antibodies either to phosphotyrosine or to mMet. (b) Trk/Met hybrid protein responds to cell attachment when overexpressed. HeLa cells were transiently transfected with vector alone (lane 1) or vector containing wild-type murine met (lane 2) or trk/met (lane 3). Cells were lysed on plates, and cell lysates were immunoprecipitated with antibodies to mMet. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with antibodies to phosphotyrosine.

Cell attachment activates Met in tumor cells but not in normal cells. Immunoprecipitation and immunoblotting analyses of Met phosphorylation in NHEMs and in human lung carcinoma cells (NCI-H441). NHEMs (lanes 1–4) and NCI-H441 (lanes 5–8) were treated differentially by cell attachment or HGF as described in Materials and Methods. NHEM and NCI-H441 cells were detached from culture dishes with EDTA and held in suspension before being lysed (lanes 3, 4, 7, and 8) or lysed on the plates (lanes 1, 2, 5, and 6). Cells were incubated in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of HGF for 15 min, lysed, and immunoprecipitated (IP) with antibodies to hMet. The immunocomplexes were resolved by SDS-PAGE under reducing conditions and subjected to immunoblotting with antibodies to either phosphotyrosine or hMet as indicated.

Expression of hMet in mouse hepatocytes induces HCC. (a and b) Survival and tumor incidences of TRE-MET mice in LAP-tTA background. For lines 1 and 2, doxycycline (Dox) was administered throughout pregnancy and after birth until pups were 4 wk old. For lines 3 and 4, doxycycline treatment was started when tumor-bearing mice were moribund. (c–f) Histopathology of tumor progression. (c) A 2-mo-old animal developed rare foci (black arrow around central veins). (d) By 4 mo of age, abnormal hepatocytes around the central vein grew into larger hyperplastic nodules (black arrowheads). (e) At 6 mo of age, less differentiated dysplatic nodules (white arrowheads) arise within hyperplastic liver parenchyma, resembling the progression of human HCC. (f) Diffuse trabecular growth pattern typical of HCC developed in the same liver that developed dysplatic foci in panel d.

Inactivation of Met results in the sustained regression of HCC. (a and b) Doxycycline dramatically reduces tumor burden within 1 mo. A moribund tumor-bearing mouse (lower) with enlarged abdomen before doxycycline treatment and the control littermate (upper; panel a). Substantial shrinkage of the tumor was evident after 1-mo treatment with doxycycline (b). (c–f) Gross pathology of HCC regression. Moribund tumor-bearing mice together with their wild-type littermates received doxycycline in their diet. Normal adult liver (c), HCC-containing liver before doxycycline treatment (d), HCC-containing liver after 20 d of doxycycline treatment (e), and liver of a previously HCC-bearing mouse after 4 mo of doxycycline treatment (f). The white arrows indicate normal liver tissue and black arrows point to the involuting residual mass. (g–j) Histology of liver with regressed tumors. Light microscopy of hematoxylin and eosin–stained liver sections from a previously tumor-bearing mouse treated for 4 mo with doxycycline. The majority of liver appeared normal (g), although a residual nodule was evident (h–j). The center of the residual mass was composed mainly of necrotic and calcified tissues (h). The border of normal liver (upper) and the involuted residue (lower) is marked by the white arrowheads in panel i. The border at higher magnification showed extensive infiltration of inflammatory cells (j). The arrows point to neutrophils and the arrowheads point to brown pigmented macrophages.