Evaluation of IGF-1R and InsR signalling pathway components. LoVo, A549, DU145 and MCF-7 cells were grown in DCCM-1 with 0.5% serum for 7 days. (A) Protein (75 μg) of cell lysate was electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total IGF-1R and InsR. (B) Cells were harvested for RNA, RT–PCR was performed and the resulting cDNA was amplified using primer sets for IGF-II. Fragments were resolved on agarose gels and densitometric scores were normalised to β-actin. Data represents mean values of three experiments and error bars indicating 95% CIs. ^*Shows significant differences assessed between LoVo cells and each of the other cell lines at the P<0.05 level. (C) Amplified InsR-A and InsR-B cDNA was resolved by PAGE (15%) and normalised to β-actin. (D) Following PCR amplification for InsR-A and InsR-B, the PCR products were digested with Ban I restriction enzyme, before resolution by PAGE. Data illustrates nondigested (ND) and digested (D) cDNA fragments in LoVo and DU145 samples. (E) Cells were grown in DCCM-1 with 0.5% serum in the absence and presence of 1 μM gefitinib for 7 days. Protein (75 μg) of cell lysate was electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total and phosphorylated InsR and Akt.

Modulation of pEGFR activity by insulin and IGF-II and the InsR/IGF-1R inhibitor ABDP. (A) LoVo cells were grown in routine culture medium until 70% confluent and after 24 h in serum-free DCCM-1 were challenged with IGF-II (10 ng ml⁻¹) and insulin (10 μg ml⁻¹) for 5 min. Cell lysates (75 μg) were electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total InsR, pInsR Tyr1146, total EGFR, pEGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173. Densitometric analysis was performed and results were normalised to β-actin levels. The data illustrated are representative of three separate experiments. (B) LoVo cells were grown in the absence and presence of 1 μM ABDP in DCCM-1 with 0.5% serum for 7 days and electrophoresed, immunoblotted for pEGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173 and analysed as described in (A). (C) LoVo cells were grown in routine culture medium until 70% confluent and after 24 h in DCCM-1, were challenged with insulin (10 μg ml⁻¹) and EGF (10 ng ml⁻¹) with and without 1 μM ABDP for 5 min. Cells exposed to ABDP were preincubated with this inhibitor for 6 h before electrophoresis, immunoblotting for total InsR, pInsR Tyr1146, pEGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173 and analysis as described in (A).

LoVo-ABDP-R cells have gained sensitivity to gefitinib. (A) LoVo-ABDP-R cells were challenged with EGF (10 ng ml⁻¹), gefitinib (1 μM) and EGF/gefitinib together for 7 days in DCCM-1 serum-free medium containing 1 μM ABDP. Data are mean values of three independent experiments (every point in each individual experiment also being evaluated in triplicate) and error bars represent 95% CIs. ^*Significant differences were assessed at the P<0.05 level. (B) LoVo cells were grown in DCCM-1 with 0.5% serum for 7 days and LoVo-ABDP-R cells were cultured in the absence and presence of 1 μM gefitinib for 7 days in DCCM-1 supplemented with 0.5% serum and 1 μM ABDP for 7 days. Protein (75 μg) of cell lysate was electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total EGFR, pEGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173, total ERK1/2, pERK1/2, total Akt and pAkt. Densitometric analysis was performed and results were normalised to total expression levels. The data illustrated are representative of three separate experiments.

Response to gefitinib and basal EGFR expression and phosphorylation status. (A) LoVo cells were grown in DCCM-1 containing varying serum concentrations (0–10%) in the absence and presence of 1 μM gefitinib. Data are mean values of three independent experiments (every point in each individual experiment also being evaluated in triplicate) and error bars represent 95% CIs. Significant differences were assessed at the P<0.05 level between the growth inhibition value obtained in the presence of 10% serum and gefitinib and the growth inhibitory values seen with each of the other serum concentrations in the presence of gefitinib. (B) Cells were grown in DCCM-1 with 0.5% serum in the absence and presence of 1 μM gefitinib for 7 days. Protein (75 μg) of cell lysate was electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total EGFR, phospho (p)-EGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173 and pERK1/2. Densitometric analysis was performed and results were normalised to β-actin levels. The data illustrated are representative of three separate experiments. (C) After growing to 70% confluency and serum starvation for 24 h, cells were challenged with EGF (5 min). Samples were electrophoresed and immunoblotted for total EGFR, pEGFR Tyr1068, pEGFR Tyr1173, total Akt, pAkt, total ERK1/2 and pERK1/2 and analysed as detailed in (B). (D) Cells were harvested for RNA, RT–PCR was performed and the resulting cDNA was amplified using primer sets for TGF-α. Fragments were resolved on agarose gels and densitometric scores were normalised to β-actin. Data represents mean values of three experiments and error bars indicating 95% CIs.

Growth responses of LoVo cells to mitogenic growth factors and the InsR/IGF-1R inhibitor ABDP. (A) LoVo cells were challenged with EGF, IGF-II (both at 10 ng ml⁻¹) and insulin (10 μg ml⁻¹) for 7 days in DCCM-1 serum-free medium. Data are mean values of three independent experiments (every point in each individual experiment also being evaluated in triplicate) and error bars represent 95% CIs. ^*Significant differences were assessed at the P<0.05 level. (B) LoVo cells were grown in the absence and presence of varying concentrations of ABDP (0–5 μM) in DCCM-1 with 0.5% serum for 7 days. Data are mean values of three independent experiments (every point in each individual experiment also being evaluated in triplicate) and error bars represent 95% CIs. ^*Significant differences were assessed at the P<0.05 level.

Gefitinib and ABDP in combination are more effective than either agent alone. (A) LoVo cells were cultured in varying concentrations of ABDP (0–1 μM) in the absence (filled bar) or presence (spotted bar) of 1 μM gefitinib in DCCM-1 with 0.5% serum for 7 days. Data are mean values of three independent experiments (every point in each individual experiment also being evaluated in triplicate) and error bars represent 95% CIs. ^*Significant differences were assessed at the P<0.05 level. (B) LoVo cells were grown in DCCM-1 with 0.5% serum containing 1 μM ABDP for 4 days and subsequently challenged with ABDP and gefitinib in combination for 24 h. Cell lysate (75 μg) was electrophoresed by SDS–PAGE (7.5%) and immunoblotted for total EGFR, pEGFR Tyr845, pEGFR Tyr1068, pEGFR Tyr1173, pAkt and pERK1/2. Data was assessed by densitometry and are representative of three separate experiments. (C) LoVo cells were chronically exposed to control media i.e. DCCM-1 with 0.5% serum (-♦-), 1 μM gefitinib (^…□^…), 1 μM ABDP (-▴-) or gefitinib/ABDP dual treatment (-•-) until cell loss occurred or resistance developed. The data demonstrates the total passage number with respect to time (weeks).