Figure 2.

p66^Shc suppresses lung metastasis in vivo. (a) LLC cells stably expressing p66^Shc or p66(S36E) were plated on normal or low attachment plates for 16 h and cell death assessed. Expression of wild-type p66^Shc increased detachment-induced cell death (*P < 0.001 from vector control), whereas p66(S36E) had a reduced effect (^†P < 0.001 from p66^Shc floating). Mean ± s.e.m. of four determinations is shown. (b) 5-bromodeoxyuridine (BrDU) uptake of attached LLC cells expressing p66^Shc (p66 wt) or p66(S36E) was not different from vector control. (c) LLC cells expressing p66^Shc or p66(S36E)were injected into the tail vein of 6-week-old female C57BL/6 mice. Kaplan–Meier survival curve is shown; survival between groups was different, P < 0.0001. n = 19 (vector), n = 21 (p66 wt) and n = 19 (p66(SE)). (d) Representative mice receiving vector-LLC or p66-LLC cells. Multiple GFP-positive lung metastases are apparent in the former but not in the latter group. (e) Representative hematoxylin and eosin stain of lungs from mice injected with vector control, p66^Shc or p66(S36E)-expressing LLC cells. Vector control and p66(S36E) lungs demonstrated dense interstitial replacement with tumor with perivascular tumor cuffing. (f) Wet lung weights were assessed, reflecting tumor burden. *P < 0.01 from vector control, ^†P < 0.01 from p66 (wt). (g) Immunoblot showing p66^Shc expression of cells injected into animals (top panel). Lung tumors were dissected, pooled and immunoblotted for p66^Shc from the single mouse in the p66^Shc group that had metastatic tumors (middle lane), and representative tumors extracted from lungs of mice receiving LLC cells with empty vector (left lane) or p66(S36E) (right lane).