Article Figures & Data
Figures
- Fig. 1.
8226/R5 cells are highly resistant to R115777. A, degree of resistance was determined using the MTT reduction assay. 8226/S and 8226/R5 cells were treated with increasing concentrations of R115777 for 72 hours. Percentage surviving cells was calculated relative to cells treated with control media only (see Materials and Methods). IC50 determinations were done by linear regression analysis. B, cell cycle analysis after R115777 treatment. Sensitive and resistant 8226 lines were treated with increasing concentrations of R115777 for 24 hours. Cell cycle arrest was determined by flow cytometry after propidium iodide staining. Gating was on live cells only. C, evaluation of cell death after R115777 treatment. 8226/S, 8226/Dox40, 8226/LR5, and 8226/R5 cells were treated with increasing concentrations of R115777 for 72 hours. Cell death was determined by flow cytometry after Annexin V-FITC and propidium iodide staining. Specific cell death was calculated by subtracting background death in untreated samples. The presented data is representative of three independent experiments.
- Fig. 2.
Resistance in 8226/R5 cells is not linked to prenylation. A, 8226/R5 cells are resistant to the FTase-specific inhibitor FTI-277. 8226/S and 8226/R5 cells were treated with increasing concentrations of FTI-277 for 72 hours. Cell death was determined by flow cytometry after Annexin V-FITC and propidium iodide staining. Specific cell death was calculated by subtracting background death in untreated samples. B, 8226/R5 cells are resistant to perillic acid, an inhibitor of both FTase and GGTase I. Sensitive and resistant cells were treated with increasing concentrations of perillic acid for 72 hours. Samples were analyzed as in (A). C, K-Ras remains prenylated in sensitive and resistant cells after R115777 treatment. 8226/S and 8226/R5 cells were treated with control media or 5 μmol/L R115777 for 72 hours. Cell lysates were harvested and evaluated by Western blotting using the indicated antibodies. D, HDJ-2 farnesylation is inhibited in both 8226/S and 8226/R5 cell lines. Sensitive and resistant cells were treated and analyzed as in (C) using the indicated antibodies. u, unprocessed form; p, processed form. (A) and (B) are representative of three independent experiments; (C) and (D) are representative of two independent experiments.
- Fig. 3.
Resistance is not related to decreased influx or increased efflux of R115777. A, P-glycoprotein surface expression is not increased in 8226/R5 cells. 8226/S, 8226/Dox40, and 8226/R5 cells were stained with anti–P-glycoprotein (dashed line histograms) or isotype control antibody (solid line histograms) as described in Materials and Methods. Samples were analyzed by flow cytometry. Decreased accumulation and/or increased efflux of R115777 is not responsible for resistance in the 8226/R5 line (B-E). B, 8226/S and 8226/R5 cells were exposed to 5 μmol/L R115777 (1:2.5 dilution of [14C]R115777 to cold R115777) in supplemented media for increasing time periods. Cells were washed and accumulation of [14C]R115777 was quantitated by scintillation counting (see Materials and Methods). C, 8226/S and 8226/R5 cells were incubated in supplemented media containing 5 μmol/L R115777 at decreasing dilutions (1:10, 1:2.5, 1:1) of [14C]R115777 for 1 hour. Samples were processed and analyzed as in (B) to confirm a dose-dependent accumulation of [14C]R115777 in each line. D, sensitive and resistant lines were treated with 5 μmol/L R115777 (1:2.5 dilution of [14C]R115777 to cold R115777) for 1 hour, then washed and placed in R115777-free supplemented media for increasing time periods. Samples were processed and analyzed as in (B). E, 8226/S and 8226/R5 cells were treated with 5 μmol/L R115777 at increasing dilutions (1:1, 1:2.5, 1:10) of [14C]R115777 for 1 hour and then washed and placed in R115777 free media for an additional hour. Samples were processed and analyzed as in (B) to confirm a dose-dependent efflux of [14C]R115777. (A), (B), and (D) are representative of two independent experiments; (C) and (E) are representative of three independent experiments.
- Fig. 4.
8226/R5 cells harbor a multidrug-resistant phenotype and resistance does not correlate with the expression of heat shock proteins. 8226/R5 cells display multidrug resistance (A-E). 8226/S and 8226/R5 lines were treated with the indicated compounds for 48 hours (A and B) or 24 hours (C-E). Cell death was determined by flow cytometry after Annexin V-FITC and propidium iodide staining. Specific cell death was calculated by subtracting background death in untreated samples. The presented data is representative of three independent experiments. F, resistance in 8226/R5 cells is not associated with increased expression of heat shock proteins. 8226/S and 8226/R5 cells were treated with control media or 5 μmol/L R115777 for 72 hours. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies (see Materials and Methods). The presented data is representative of two independent experiments.
- Fig. 5.
8226/R5 cells are resistant to PS-341. A, degree of resistance was determined by the MTT reduction assay. 8226/S and 8226/R5 cells were treated with increasing concentrations of PS-341 for 48 hours. Percentage surviving cells and IC50 determinations were done as in Fig. 1. B, analysis of cell cycle after PS-341 treatment. Sensitive and resistant 8226 lines were treated with increasing concentrations of PS-341 for 24 hours. Cell cycle arrest was determined by flow cytometry after propidium iodide staining. Gating was on live cells only. C, evaluation of cell death after PS-341 treatment. 8226/S and 8226/R5 cells were treated with increasing concentrations of PS-341 for 48 hours. Cell death was determined by flow cytometry after Annexin V-FITC and propidium iodide staining. Specific cell death was calculated by subtracting background death in untreated samples. The presented data is representative of three independent experiments.
Tables
- Table 1.
Gene expression changes in 8226/R5 cells
Expression increased Expression decreased Janus kinase 2 Interleukin-6 receptor Signal transducer and activator of transcription 1 Insulin-like growth factor 2 receptor Bcl-XL Mcl-1 Phosphoinositide-3-kinase, catalytic, δ polypeptide Heat shock protein 27 Fibroblast growth factor receptor activating protein 1 Retinoblastoma 1 Fibroblast growth factor 20 Transforming growth factor α Mevalonate kinase Tumor protein p53 Rap 2A Farnesyl diphosphate synthetase Rab GGTase, β subunit Lamin A/C Rab 4A Ras protein activator like-1 Ras and Rab interactor 3 Ras and Rab interactor 2 Mitogen-activated protein kinase 1 Rab 36 Mitogen-activated protein kinase 9 Rho GTPase-activating protein 6 Mitogen-activated protein kinase 14 Mitogen-activated protein kinase phosphatase Cyclin D3 Cyclin D1 Fas apoptotic inhibitory molecule Syndecan 1 Calmodulin 3 X-box–binding protein 1 Calpain 2 Actin, β S100 calcium binding protein A13 and A14 Fibronectin-1 Collagen type XXI α Integrin β5 Collagen IV, α3 Integrin β7 Paxillin Intercellular adhesion molecule-1 Integrin α-4 Ubiquitin-conjugating enzymes Integrin α-6 Proteosome subunit B-4 type Ubiquitin-specific proteases 15 Proteosome 26S subunit Ubiquitin-specific proteases 24 S100 calcium binding protein (A11 pseudogene) Proteosome inhibitor subunit 1 S100 calcium-binding protein (A11 calgizzarin)
Volume 11, Issue 16
