Figure 6.
The myeloma cell growth factor activity of HB-EGF is inhibited by a pan-ErbB kinase inhibitor. (A) A431 cells were stimulated with HB-EGF, with or whithout PD169540, for 15 minutes prior to lysis and immunoprecipitation of ErbB1. Tyrosine phosphorylation of ErbB1 was assayed using an antiphosphotyrosine antibody (4G10; Upstate Biotechnology) and ErbB1 was quantified with an anti-ErB1 antibody (clone LA-22). (B) Myeloma cells were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with either 5 pg/mL IL-6 or 5 pg/mL IL-6 and 1 μg/mL HB-EGF, without or with 1 μM of the PD169540 ErbB inhibitor. Cells were cultured for 3 days and stained with FITC–annexin-V to determine the percentage of apoptotic cells. (C) Myeloma cell lines were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with 5 pg/mL IL-6 and 1 μg/mL HB-EGF without (□) or with (▪) the PD169540 ErbB inhibitor (1 μM). Cells were cultured for 5 to 7 days and pulsed with tritiated thymidine at the end of the culture. Mean tritiated thymidine incorporations were determined in sixplicate culture wells and results are expressed as percentages ± SDs of the mean proliferation without ErbB inhibitor. *Indicates that the mean value is statistically significantly different from that obtained with 5 pg/mL IL-6 and HB-EGF without inhibitor, using a Student t test (P ≤ .05). (D) XG-1, XG-6, and XG-7 cells were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with a high concentration of IL-6 (500 pg/mL), without (□) or with (▪) the PD169540 ErbB inhibitor (1 μM). Mean tritiated thymidine incorporations were determined in sixplicate culture wells and results are expressed as percentages ± SDs of the mean proliferation without ErbB inhibitor. *Indicates that the mean value is statistically significantly different from that obtained without inhibitor, using a Student t test (P ≤ .05). (E) XG-7 myeloma cells were starved overnight in culture medium without serum or cytokines with or without the PD169540 ErbB inhibitor. Cells were then stimulated with either no cytokine (Co), or IL-6 (20 ng/mL), or HB-EGF (5 μg/mL), or IGF-1 (1 μg/mL) for 15 minutes at 37°C. Lysates from unstimulated (Co) or stimulated myeloma cells were immunoblotted with anti–phospho-specific AKT antibody (top panel) and reprobed with anti-AKT antibody (bottom panel).

The myeloma cell growth factor activity of HB-EGF is inhibited by a pan-ErbB kinase inhibitor. (A) A431 cells were stimulated with HB-EGF, with or whithout PD169540, for 15 minutes prior to lysis and immunoprecipitation of ErbB1. Tyrosine phosphorylation of ErbB1 was assayed using an antiphosphotyrosine antibody (4G10; Upstate Biotechnology) and ErbB1 was quantified with an anti-ErB1 antibody (clone LA-22). (B) Myeloma cells were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with either 5 pg/mL IL-6 or 5 pg/mL IL-6 and 1 μg/mL HB-EGF, without or with 1 μM of the PD169540 ErbB inhibitor. Cells were cultured for 3 days and stained with FITC–annexin-V to determine the percentage of apoptotic cells. (C) Myeloma cell lines were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with 5 pg/mL IL-6 and 1 μg/mL HB-EGF without (□) or with (▪) the PD169540 ErbB inhibitor (1 μM). Cells were cultured for 5 to 7 days and pulsed with tritiated thymidine at the end of the culture. Mean tritiated thymidine incorporations were determined in sixplicate culture wells and results are expressed as percentages ± SDs of the mean proliferation without ErbB inhibitor. *Indicates that the mean value is statistically significantly different from that obtained with 5 pg/mL IL-6 and HB-EGF without inhibitor, using a Student t test (P ≤ .05). (D) XG-1, XG-6, and XG-7 cells were IL-6 starved for 3 hours and cultured in RPMI 1640 culture medium and 5% FCS with a high concentration of IL-6 (500 pg/mL), without (□) or with (▪) the PD169540 ErbB inhibitor (1 μM). Mean tritiated thymidine incorporations were determined in sixplicate culture wells and results are expressed as percentages ± SDs of the mean proliferation without ErbB inhibitor. *Indicates that the mean value is statistically significantly different from that obtained without inhibitor, using a Student t test (P ≤ .05). (E) XG-7 myeloma cells were starved overnight in culture medium without serum or cytokines with or without the PD169540 ErbB inhibitor. Cells were then stimulated with either no cytokine (Co), or IL-6 (20 ng/mL), or HB-EGF (5 μg/mL), or IGF-1 (1 μg/mL) for 15 minutes at 37°C. Lysates from unstimulated (Co) or stimulated myeloma cells were immunoblotted with anti–phospho-specific AKT antibody (top panel) and reprobed with anti-AKT antibody (bottom panel).

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