Table 2.
Activity of mutationally activated Trk-Met in NIH 3T3 cells
| TRK-MET construct* | Focus formation† (no. foci/μg DNA)
|
Tumorigenicity‡
|
Metastasis§
|
||
|---|---|---|---|---|---|
| NGF (−) | NGF (+) | No. mice with tumors/ no. mice injected | Mean tumor size in mm2 | No. mice with lung metastasis/ no. mice injected | |
| Control | 8 | 5 | 0/5 | 0 | 0/5 |
| Wild type | 13 | >300 | 0/5 | 0 | 0/5 |
| M1268T | >300 | >300 | 5/5 | 245 | 4/4 |
| L1213V | 74 | >300 | 5/5 | 133 | 4/4 |
| Y1248H | 105 | >300 | 5/5 | 257 | 5/5 |
| D1246H | 68 | >300 | 5/5 | 99 | 4/4 |
The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met. Each Met molecule is in the pMex vector, which utilizes the Moloney murine sarcoma virus long terminal repeat promoter.
NIH 3T3 cells transfected with the indicated constructs were scored for focus formation after approximately 2 weeks. Samples receiving NGF were treated with this ligand at 100 ng/ml throughout the duration of the assay. Similar results were obtained in two independent experiments.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected s.c. into nude mice at 8 × 105 cells per animal. Tumors were measured after 17 days. Mice injected with cells expressing wild-type Trk-Met were tumor-free even after 2 months.
NIH 3T3 cells were cotransfected with the indicated construct plus pSV2 Neo, selected as pools of G418-resistant cells, and injected i.v. into the tail vein of nude mice at 8 × 105 cells per animal. Animals were sacrificed after 4 weeks (or earlier for animals in distress) and examined for lung metastasis. All animals positive for metastasis presented with severe metastatic burden. Mice injected with cells expressing wild-type Trk-Met were metastasis-free even after 2 months.