Article Figures & Data
Figures
- Figure 1.
Expression of H-RAS, K-RAS, and N-RAS in HCT116 cells transfected with empty vector or a mutant H-RAS plasmid. A, levels of mRNA for H-RAS, K-RAS, and N-RAS in HCT116 cell clones. mRNA levels of H-RAS, K-RAS, N-RAS, and β-actin were determined by RT-PCR using the specific primers as described in Materials and Methods. Densitometry values of each RAS isoform mRNA were normalized with respect to the total amount of β-actin mRNA in each sample and then expressed as percentages with the mRNA content for K-RAS in HCT116 (WT) cells being defined as a 100% value. Columns, mean of four independent experiments; bars, SE. *, P < 0.05 less than corresponding WT cell value. Inset, quantitative RT-PCR H-RAS, K-RAS, and N-RAS mRNA levels from a representative experiment showing data from WT, C2 (mutant K-RAS-deleted), C10 (mutant K-RAS-deleted expressing H-RAS V12), and C3 (mutant K-RAS-deleted expressing H-RAS V12). B, expression of K-RAS and H-RAS proteins determined by immunoblotting using RAS isoform-specific antibodies. Densitometry values were normalized with respect to the total amount of β-actin in each sample and then expressed as percentages with the value for K-RAS expression in WT cells being defined as a 100% value. Columns, mean of four independent experiments; bars, SE. *, P < 0.05 less than corresponding WT cell value; #, P < 0.05 greater than corresponding WT cell value. Inset, H-RAS and K-RAS protein levels from a representative experiment showing data from WT, C2, and C10. C, expression of ERBB1, ERBB2, and ERBB3 proteins determined by Western blot using receptor-specific antibodies. Densitometry values were normalized with respect to the total amount of β-actin in each sample and then expressed as percentages with the value for ERBB1 expression in WT cells being defined as a 100% value. Columns, mean of four independent experiments; bars, SE. #, P < 0.05 greater than corresponding WT cell value. Inset, ERBB1, ERBB2, and ERBB3 protein levels from a representative experiment showing data from WT, C2, and C10. D, levels of mRNA for ERBB family paracrine growth factors in WT, C2, or C10. Growth factor expression was determined by RT-PCR using specific primers as described in Materials and Methods. Densitometry values of the mRNAs for epiregulin (EPI) and heregulin (HER) were normalized with respect to the total amount of β-actin in each sample and then expressed as percentages with the value for epiregulin mRNA levels in WT cells being defined as a 100% value. Columns, mean of five experiments; bars, SE. *, P < 0.05 less than corresponding WT cell value; **, P < 0.05 less than mutant K-RAS deleted value; #, P < 0.05 greater than corresponding WT cell value. Inset, epiregulin and heregulin mRNA levels from a representative experiment. E and F, heregulin protein expression by Western blot (E) and heregulin expression by mRNA content (F) in WT, C2, and C10. Densitometry values were normalized with respect to the total amount of β-actin protein/mRNA in each sample and expressed as percentages of the protein or mRNA content in WT cells (defined as 100%). Columns, mean of five experiments; bars, SE. #, P < 0.05 greater than corresponding WT cell value. Inset, epiregulin and heregulin mRNA and protein levels from a representative experiment.
- Figure 2.
Phosphorylation of P-JNK1/2, P-ERK1/2, and P-AKT Ser473 in HCT116 cells after a 1 Gy radiation exposure. A, phosphorylation of protein kinases was determined by immunoblotting using antibodies for the phosphorylated forms of ERK1/2, AKT Ser473/Thr308, JNK1/2, and p38 in parental WT, C2, or C10. Total β-actin, ERK2, and AKT1/2 expression was blotted in the same membrane as a loading control. B, alteration of JNK1/2 phosphorylation 0–6 h after a 1 Gy exposure in WT, C2, or C10. Densitometry values were normalized with respect to total β-actin protein expression and are expressed as percentages of JNK1/2 phosphorylation in WT cells at t = 0. Points, mean of three experiments; bars, SE. C, alteration of JNK1/2 phosphorylation 6–24 h after a 1 Gy exposure in WT, C2, and C10. Densitometry values were normalized with respect to total β-actin protein expression and are expressed as percentages of JNK1/2 phosphorylation in WT cells at t = 0. Points, mean of three experiments; bars, SE. D, alteration of ERK1/2 phosphorylation 0–6 h after a 1 Gy exposure in WT, C2, or C10. Densitometry values were normalized with respect to total β-actin protein expression and are expressed as percentages of ERK1/2 phosphorylation in WT cells at t = 0. Points, mean of three experiments; bars, SE. E, alteration of ERK1/2 phosphorylation 6–24 h after a 1 Gy exposure in WT, C2, and C10. Densitometry values were normalized with respect to total β-actin protein expression and are expressed as percentages of ERK1/2 phosphorylation in WT cells at t = 0. Points, mean of three experiments; bars, SE. F, alteration of AKT Ser473 phosphorylation 0–6 h after a 1 Gy exposure in WT, C2, and C10. Densitometry values were normalized with respect to total β-actin protein expression and expressed as percentages of AKT phosphorylation in WT cells at t = 0. Points, mean of four independent experiments; bars, SE. G, alteration of AKT Ser473 phosphorylation 6–24 hours after a 1 Gy exposure in WT, C2, and C10. Densitometry values were normalized with respect to total β-actin protein expression and expressed as percentages of AKT phosphorylation in WT cells at t = 0. Points, mean of four independent experiments; bars, SE.
- Figure 3.
Expression of H-RAS V12 causes a greater radioprotective/survival effect than K-RAS D13 in HCT116 cells. A, plating efficiency (colonies formed per total number of cells plated) of WT, C2, or C10/C3 cells. Cells were plated as described in Materials and Methods, and the relative efficiency of cell plating (percentage colonies recovered per total number of cells plated) was calculated making the plating efficiency value for WT equal 1.00. Columns, means of six separate dishes from three separate experiments; bars, SE. *, P < 0.05 less than corresponding WT cell value; #, P < 0.05 greater than corresponding WT cell value. B, dose-response survival curve after exposure of HCT116 cell lines to ionizing radiation: WT cells, mutant K-RAS-deleted cells, and mutant K-RAS-deleted cells transfected with the empty vector (C2) or with the mutant H-RAS V12 plasmid (C3 and C10). Cells were serum starved for 24 h before exposure to the respective dose of radiation, medium containing serum was added 24 h after exposure, and 10– 14 d later colonies were counted as indicated in Materials and Methods. Numbers of colonies were expressed as a fraction of the respective mock irradiated cells and plotted in a semilog graph. Points, means of six separate dishes from three separate experiments; bars, SE. *, P < 0.05 less than corresponding WT cell value; #, P < 0.05 greater than corresponding WT cell value; ∼, P < 0.001 greater than mutant K-RAS-deleted cells value. C, HCT116 cells expressing H-RAS V12 (C10 cells) were transfected with vector control plasmid or plasmid to express dominant-negative AKT as described in Materials and Methods and in Fig. 5 legend. Pools of transfected cells were plated for colony formation assays and irradiated (1 Gy) as described in Materials and Methods. Columns, means of three separate dishes from three separate experiments; bars, SE. *, P < 0.05 less than corresponding C10 cell or C10 control plasmid cell value. Inset, expression of total AKT, total GSK3, and P-GSK3 S9 from a representative experiment (n = 3).
- Figure 4.
Neutralization of heregulin function in HCT116 H-RAS V12 (C10) cells inhibits radiation-induced AKT activation and causes H-RAS V12 cell radiosensitization. A, H-RAS V12 (C10) cells were serum starved for 24 h and 30 minutes before irradiation treated with an anti-heregulin antibody (αHer) 14 μg/mL (final in medium) or a nonspecific IgG of the same subtype. Cells were irradiated (1 Gy) or mock exposed, and homogenates were taken 0–24 h afterward to determine AKT Ser473 phosphorylation and total ERK2 expression as described in Materials and Methods. Data are from 6 and 12 h after irradiation. Representative experiment (n = 3). B, H-RAS V12 cells (C10) were serum starved for 24 h and either mock exposed or irradiated (1 Gy). Six hours after irradiation, medium was removed from the irradiated plates and incubated for 1 h with anti-heregulin antibody or a nonspecific IgG of the same subtype. After 1 h, medium was added to unirradiated plates of H-RAS V12 (C10) cells. Ten minutes after medium addition, cells were lysed and prepared to determine AKT Ser473 phosphorylation. Representative experiment (n = 3). C, effect of neutralizing antibody against heregulin on the survival of irradiated H-RAS V12 cells. Plated cells were serum starved for 24 h and treated with 14 μg/mL anti-heregulin antibody or an identical concentration of an unspecific IgG of the same isotype and subjected to a 1 Gy or a mock exposure as described in Materials and Methods. Colonies were counted 10–14 days later as described in Materials and Methods. Numbers of colonies were expressed as percentages of the respective mock exposed group. Columns, means of six separate dishes from three separate experiments; bars, SE. *, P < 0.05 less than corresponding unirradiated cell value; %, P < 0.05 less than corresponding WT cell value.
- Figure 5.
Neutralization of heregulin function promotes radiation-induced apoptosis by caspase 8– and caspase 9–dependent mechanisms. H-RAS V12 (C10) cells were serum starved for 24 h and 30 minutes before irradiation treated with an anti-heregulin antibody (α-HER) 14 μg/mL (final in medium) or a nonspecific IgG of the same subtype. Cells were irradiated (1 Gy) or mock exposed and cells were isolated 0–96 h afterward to determine the percentage apoptosis 96 h after exposure in the presence or absence of vehicle (DMSO), the caspase-8 inhibitor IETD (50 μmol/L), or the caspase-9 inhibitor LEHD (50 μmol/L) as described in Materials and Methods. Columns, mean of individual triplicate samples from two experiments; bars, SE. *, P < 0.05 greater than corresponding vehicle-treated value; #, P > 0.05 for value compared with corresponding irradiated value.
- Figure 6.
A neutralizing antibody against heregulin inhibits AKT activation in H-RAS V12 (C10) cells induced by medium from irradiated mutant K-RAS-deleted (C2) cells. A, mutant K-RAS deleted (C2) cells and H-RAS V12 (C10) cells were serum starved for 24 h and either mock exposed or irradiated (1 Gy). Six hours after exposure, medium was removed from the treated plates and incubated for 1 h with 14 μg/mL (final in medium) of anti-heregulin antibody or with an identical concentration of an unspecific IgG of the same subtype. After 1 h, medium was added to unirradiated plates of either mutant K-RAS-deleted cells (HCT116 C2) or H-RAS V12 (HCT116 C10) cells. Ten minutes after medium addition, cells were lysed and prepared to determine AKT Ser473 phosphorylation. Lane 1, C2 cells treated with control IgG + C2 unirradiated medium; lane 2, C2 cells treated with control IgG + C10 unirradiated medium; lane 3, C2 cells treated with α-Her IgG + C2 irradiated medium; lane 4, C2 cells treated with α-Her IgG + C10 irradiated medium; lane 5, C2 cells treated with control IgG + C2 irradiated medium; lane 6, C2 cells treated with control IgG + irradiated C10 medium; lane 7, C10 cells treated with control IgG + C2 unirradiated medium; lane 8, C10 cells treated with control IgG + C2 irradiated medium; lane 9, C10 cells treated with α-Her IgG + C2 irradiated medium. Representative experiment (n = 3). B, heregulin activates AKT in C2 and C10 cell lines. Mutant K-RAS-deleted (C2) cells and H-RAS V12 (C10) cells were serum starved for 24 h and either mock treated with vehicle (PBS) or treated with heregulin (10 ng/mL final). Cells were isolated at the indicated times after treatment. Cells were lysed and prepared to determine AKT Ser473 phosphorylation by immunoblotting. Representative experiment (n = 3).
- Figure 7.
AKT activation and radiation resistance is dependent on heregulin-stimulated association of membrane-associated PI3K with ERBB3. A, WT, C2, and C10 cells were plated in parallel and serum starved for 24 h. Cells were then either lysed to determine total ERBB3 and β-actin expression or lysed with hypotonic buffer before preparation of plasma membranes as described in Materials and Methods. Equal amounts of membrane protein were loaded onto SDS-PAGE and immunoblotting done against the indicated membrane-associated proteins. Representative experiment (n = 4). B, H-RAS V12 (C10) cells were serum starved for 24 h and treated with 14 μg/mL (final in medium) of anti-heregulin antibody or an identical concentration of an unspecific IgG of the same subtype. Cells were subjected to 1 Gy irradiation or mock exposed and cells were isolated 1–3 h after exposure. ERBB3 was immunoprecipitated from the cell lysates as described (38), and the immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting to determine the levels of ERBB3 (protein loading control), ERBB3 tyrosine phosphorylation, and presence/association of PI3K p85 and PI3K p110. Representative experiment (n = 3). C, H-RAS V12 (C10) cells were serum starved for 24 h in the presence of a farnesyltransferase inhibitor (FTI; 2 μmol/L FTI277). In parallel, serum-staved cells that were vehicle treated were exposed to a neutralizing anti-heregulin antibody or control antibody for 3 h. Cells under all treatment conditions were then lysed with hypotonic buffer before preparation of membranes as described in Materials and Methods. Equal amounts of membrane protein were loaded onto SDS-PAGE. Representative experiment (n = 3).
Volume 4, Issue 2
