Figure 3
Protease-resistant IGFBP4 inhibits tumour growth. 4T1.2 tumour cells were transfected with control plasmid (pCMV), plasmid expressing wild-type IGFBP4 (pCMV-BP4) or plasmid expressing protease-resistant IGFBP4 (pCMV-dBP4). (A) Western blot showing IGFBP4 expression by control cells (lane 1) and single cell clones expressing dBP4 (lanes 2–5) or BP4 (lanes 6–8). Clones marked with an asterisk were used for in vivo studies. (B) HEK293T cells, which do not express endogenous IGFBP4, were transfected with pCMV (lanes 1+2), pCMV-BP4 (lanes 3+4) or pCMV-dBP4 (lanes 5+6). Conditioned medium was treated with rhPAPP-A in the presence of IGF2 for 24 h (−; untreated, +; PAPP-A-digested). Intact IGFBP4 and cleavage fragments were identified by western blot. (C) Tumour growth curve. 4T1.2 tumour cells transfected with control plasmid (pCMV), plasmid expressing wild-type IGFBP4 (pCMV-BP4) or plasmid expressing protease-resistant IGFBP4 (pCMV-dBP4) were implanted into the mammary fat pad of BALB/c mice (n=7 per group). Tumour diameter was measured on alternate days. Data (n=7) expressed as mean±s.e.m. and analysed by ANOVA with LSD post hoc correction. *P<0.05 dBP4 vs pCMV or BP4. Data representative of two independent experiments. (D) Kaplan–Meier plot showing increased time to reach a mean tumour diameter (MTD) of 17 mm in mice with tumours expressing dBP4 relative to mice with tumours transfected with pCMV or BP4 (χ2=16.4, P<0.0001). Data representative of two independent experiments.
