Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Significant mRNA overexpression of t-DARPP and ERBB2 in adenocarcinomas of the esophagus and stomach (141 tumors and 51 normal tissue samples) (p<0.001). B) Spearman’s correlation coefficient and correlation test where the cutoff gene expression is ≥log(5,2)=2.32, show that t-DARPP and ERBB2 overexpression levels are significantly correlated in tumors (r=0.58, p=0.003). C) The multivariate regression model analysis indicates that tumor stage has a significant effect on t-DARPP mRNA gene expression levels (p=0.02). D) Left panel, cell viability of OE19 and OE33 cells in response to trastuzumab treatment was evaluated by Trypan blue staining. OE19 cells were two-fold more sensitive to trastuzumab than OE33 cells (p<0.001). Right panel, Western blot analysis demonstrates higher protein expression of ERBB2 in OE19 cells than OE33 cells. In contrast, t-DARPP expression was undetectable in OE19 cells but highly expressed in OE33 cells.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Over Expression, Expressing, Staining, Western Blot

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) ERBB2 protein stability in OE19 cells stably expressing t-DARPP or pcDNA3 empty vector was evaluated by Western blot analysis after treatment with 80 μg/ml CHX to block new protein synthesis for the indicated times. The protein degradation data indicate that t-DARPP expression extended the protein half-life of ERBB2 from 30.8 h to 42.2 h relative to control (lower panel). B) ERBB2 protein stability in parental and trastuzumab resistant OE19 cells was assessed by Western blot analysis after treatment with CHX (80 μg/ml) for the indicated times. The protein degradation data show that endogenous t-DARPP expression in resistant cells was associated with increased ERBB2 protein half-life (60.8 h) relative to parental cells (31.3 h) (lower panel). C) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The data indicate that transient expression of t-DARPP increased p-ERBB2(Y1248) and p-AKT(S473) basal protein levels, and blocked trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. D) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells stably expressing t-DARPP or pcDNA3 vector after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results show that stable expression of t-DARPP increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and inhibited trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. E) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in parental or trastuzumab resistant OE19 cells following treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results indicate that endogenous t-DARPP expression was associated with increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and suppression of trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Blocking Assay, Infection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in OE33 cells transfected with control siRNA or t-DARPP siRNA and treated with vehicle or trastuzumab (20 μg/ml) for 48 h. The data indicate that knockdown of endogenous t-DARPP increased trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. B) Cell viability of OE33 cells transfected with control siRNA or t-DARPP siRNA in response to treatment with vehicle or trastuzumab (20 μg/ml) for 48 h, was evaluated by CellTiter-Glo Luminescent CellViability Assay. The results revealed that knockdown of endogenous t-DARPP with treatment induced a significant decrease in cell survival (p<0.01).

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Transfection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of co-immunoprecipitated exogenous t-DARPP and endogenous ERBB2 proteins with M2-flag or trastuzumab antibodies in OE19 cells infected with t-DARPP-flag adenovirus (10 MOI). The data demonstrate protein association of ERBB2 with t-DARPP. B) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses. Pulled-down ERBB2 band intensity was depicted as a ratio relative to input ERBB2 protein. The results show that exogenous t-DARPP expression blocked binding of trastuzumab to ERBB2 receptor relative to control. C) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in parental or trastuzumab resistant OE19 cells. The band intensity of immunoprecipitated ERBB2 protein was shown as a ratio relative to input ERBB2. The data indicate that endogenous t-DARPP expression in trastuzumab-resistant cells was associated with a significant decrease in trastuzumab/ERBB2 protein interaction relative to control.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Immunoprecipitation, Infection, Expressing, Binding Assay

Journal: Cancer gene therapy

Article Title: Treatment with targeted Vesicular Stomatitis Virus generates therapeutic multifunctional anti-tumor memory CD4 T-cells

doi: 10.1038/cgt.2011.90

Figure Lengend Snippet: Re-challenge with either erbb2 expressing D2F2/E2 or parent tumor D2F2 in animals cured by T-cell transfer

Article Snippet: A vector expressing a single chain antibody (SCA) based on the 4D5 anti-erbb2 antibody was a generous gift by Genentech Inc. As previously described, we used PCR to create a chimeric Sindbis gp which consisted of the first 71 amino acids of the Sindbis E2 gp followed in order by a poly-glycine linker, SCA to erbb2, CH1 linker, the remainder of the E2 gp and the entire E1 Sindbis gp.

Techniques: Expressing

Journal: Cancer gene therapy

Article Title: Treatment with targeted Vesicular Stomatitis Virus generates therapeutic multifunctional anti-tumor memory CD4 T-cells

doi: 10.1038/cgt.2011.90

Figure Lengend Snippet: Cytokine production by CD4 T-cells harvested from lymph nodes in animals receiving various treatment. A. Implanted with D2F2/E2. Treated with rrVSV and anti-CTLA4 MAb. Challenged >100 days later with D2F2/E2 (erbb2 expressing). B. Implanted with D2F2/E2. Treated with rrVSV and anti-CTLA4 MAb. Challenged >100 days later with D2F2 (non-erbb2 expressing). C. No tumor implanted. Treated with rrVSV and anti-CTLA4 MAb. Challenged >60-100 days later with D2F2/E2 (erbb2 expressing). D. No tumor. No treatment. Challenged with D2F2/E2 (erbb2 expressing). Values are concentration of cytokine in supernatant, pg/ml. Means of 4 experiments.

Article Snippet: A vector expressing a single chain antibody (SCA) based on the 4D5 anti-erbb2 antibody was a generous gift by Genentech Inc. As previously described, we used PCR to create a chimeric Sindbis gp which consisted of the first 71 amino acids of the Sindbis E2 gp followed in order by a poly-glycine linker, SCA to erbb2, CH1 linker, the remainder of the E2 gp and the entire E1 Sindbis gp.

Techniques: Expressing, Concentration Assay, Virus

Journal: Cancer gene therapy

Article Title: Treatment with targeted Vesicular Stomatitis Virus generates therapeutic multifunctional anti-tumor memory CD4 T-cells

doi: 10.1038/cgt.2011.90

Figure Lengend Snippet: Number of IFNγ secreting CD4 T-cells harvested from lymph nodes in animals receiving various treatments. Means of 3 experiments with standard error bars. A. Implanted with D2F2/E2. Treated with rrVSV and anti-CTLA4 MAb. Challenged >100 days later with D2F2/E2 (erbb2 expressing). B. Implanted with D2F2/E2. Treated with rrVSV and anti-CTLA4 MAb. Challenged >100 days later with D2F2 (non-erbb2 expressing). C. No tumor implanted. Treated with rrVSV and anti-CTLA4 MAb. Challenged >60-100 days later with D2F2/E2 (erbb2 expressing). D. No tumor. No treatment. Challenged with D2F2/E2 (erbb2 expressing).

Article Snippet: A vector expressing a single chain antibody (SCA) based on the 4D5 anti-erbb2 antibody was a generous gift by Genentech Inc. As previously described, we used PCR to create a chimeric Sindbis gp which consisted of the first 71 amino acids of the Sindbis E2 gp followed in order by a poly-glycine linker, SCA to erbb2, CH1 linker, the remainder of the E2 gp and the entire E1 Sindbis gp.

Techniques: Expressing