Cytotoxicity of farnesyltransferase inhibitors in lymphoid cells mediated by MAPK pathway inhibition and Bim up-regulation

Abstract

The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.

Figure 1

Tipifarnib induces mitochondrial pathway-dependent apoptosis in Jurkat cells. (A-C) Aliquots containing 1 to 2 × 10⁵ Jurkat cells/mL or Jurkat derivatives were incubated for 48 hours with diluent (0.1% DMSO), 1000nM tipifarnib (A; tipibarnib), or the concentration of tipifarnib indicated in panels B and C. At the completion of the incubation, cells were stained with Hoechst 33258 and examined by fluorescence microscopy (A), stained with APC-coupled annexin V and examined for phosphatidylserine externalization (B), or permeabilized with 0.1% Triton X-100 in 0.1% (wt/vol) sodium citrate containing 50 μg/mL propidium iodide and subjected to flow microfluorometry (C). (D) Whole cell lysates were subjected to immunoblotting for the indicated antigens. (E-F) Results obtained when the indicated Jurkat variants were treated for 72 hours with 1000nM tipifarnib, for 6 hours with 25 ng/mL CH.11 agonistic anti-Fas antibody, or for 24 hours with 2μM etoposide (E) or with varying tipifarnib concentrations (F) and analyzed as indicated in panel C. Errors bars in panel E indicate mean ± SD of 3 independent experiments.

Figure 2

Role of Bim up-regulation in tipifarnib-induced apoptosis. (A-B) After Jurkat cells were treated for 72 hours with the indicated tipifarnib concentration in the presence of 5μM Q-VD-OPh, a broad-spectrum caspase inhibitor, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigens. Hsp90 and β-actin served as loading controls. The shift in mobility of the farnesyltransferase substrate HDJ-2 confirmed the inhibition of farnesylation. In this and subsequent figures, gray and black arrows indicate farnesylated and unfarnesylated species, respectively. Asterisk (*) represents nonspecific band present in all lanes. (C) After Jurkat cells were treated with diluent (0.1% DSMO, −) or 800nM tipifarnib (+) in the presence Q-VD-OPh for 72 hours, the indicated subcellular fractions were isolated and subjected to immunoblotting. GAPDH served as a marker for cytosol; and cytochrome c oxidase subunit IV (CoX IV) served as a marker for mitochondria. (D) After Jurkat cells were treated with the indicated concentration of tipifarnib for 72 hours in the presence 5μM Q-VD-OPh, cell lysates prepared in 1% CHAPS were subjected to immunoprecipitation with protein G-Sepharose cross-linked to antibodies that recognize Bcl-2, Bcl-x_L, or Mcl-1. Immunoprecipitates were washed, resolved by SDS-PAGE, and subjected to immunoblotting as indicated. In each immunoprecipitation experiment, tubes lacking primary antibody served as controls to assess the specificity of protein recovery with the protein G-Sepharose beads. (E) Twenty-four hours after transfection of control oligonucleotide or Bim siRNA along with plasmid encoding EGFP-histone H2B (to mark successfully transfected cells), cells were treated for 48 hours with tipifarnib before staining with APC-conjugated annexin V and analysis by 2-color flow cytometry. Error bars indicate mean ± SD of 3 experiments. (E) Inset: Immunoblots of whole cell lysates prepared from siRNA-treated cells incubated in drug-free medium in parallel with samples harvested for flow cytometry.

Figure 3

Prominent tipifarnib-induced inhibition of MAPK signaling. (A,C) after Jurkat cells were treated for 72 hours with the indicated tipifarnib concentration in the presence of 5μM Q-VD-OPh, whole cell lysates were subjected to immunoblotting with antibodies that recognize the indicate polypeptides. Ribosomal protein S6 and Hsp90 served as loading controls. (B) After Jurkat cells were treated for 72 hours with the indicated tipifarnib concentration in the presence of 5μM Q-VD-OPh, cDNA was prepared and RT-PCR was performed using primers specific for Bim.

Figure 4

Tipifarnib-induced Bim up-regulation reflects inhibition of MAPK signaling. (A) After Jurkat cells were treated for 72 hours with 800nM tipifarnib or 20μM U0126, in the presence of 5μM Q-VD-OPh, whole cell lysates were subjected to immunoblotting with antibodies that recognize the indicated polypeptides. (B) After Jurkat cells were treated for 72 hours with 800nM tipifarnib or 20μM U0126, samples were stained with propidium iodide and subjected to flow microfluorometry. (C) After Jurkat cells and constitutively active MEK1 clones 10 and 12 were treated for 72 hours with diluent (−) or 800nM tipifarnib (+) in the presence of 5μM Q-VD-OPh, whole cell lysates were subjected to immunoblotting with antibodies that recognize the indicated polypeptides. (D) After the indicated clones were treated for 72 hours with diluent or 800nM tipifarnib, samples were stained with propidium iodide and subjected to flow microfluorometry. Error bars in panels B and D indicate mean ± SD of at least 3 independent experiments.

Figure 5

Role of RasGRP1 in tipifarnib sensitivity. (A) Twenty-four hours after transfection with cDNA encoding H-Ras, cells were treated with the indicated tipifarnib concentration for 72 hours in the presence of 5μM Q-VD-OPh and prepared for immunoblotting. (B) Forty-eight hours after transfection with control siRNA (lane 1) or RasGRP1 siRNA (lane 2), whole cell lysates were prepared and subjected to immunoblotting with antibody to RasGRP1 or, as a control, poly(ADP-ribose) polymerase (PARP). (C) Beginning 24 hours after transfection with control siRNA or RasGRP1 siRNA, cells were treated for 48 hours with tipifarnib, stained with propidium iodide, and subjected to flow cytometry. Error bars, mean ± SD of 3 independent experiments.

Figure 6

Correlation between tipifarnib-induced Bim up-regulation and apoptosis. (A) After cells were treated for 72 hours with the indicated tipifarnib concentration, samples were stained with propidium iodide and subjected to flow microfluorometry. (B) After cells were treated with diluent (odd lanes) or 800nM tipifarnib (even lanes) in the presence of 5μM Q-VD-OPh, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies to the indicated antigen. For each antigen, all lanes were derived from corresponding signals on a single piece of x-ray film but were rearranged for clarity. (C-D) Hs445 (C) or SKW6.4 cells (D) were treated for 72 hours with the indicated tipifarnib concentration in the absence (open circles) or presence (closed circles) of 125nM ABT-263, stained with propidium iodide, and subjected to flow microfluorometry. Error bars in panels A, C, and D indicate mean ± SD of 3 independent experiments.

Figure 7

Pathways potentially contributing to tipifarnib-induced apoptosis in lymphoid cells. Experimental manipulations examined in the present study are indicated in gray. As described in “Results,” the Raf/MEK/ERK pathway plays the predominant role in Bim up-regulation in the cell lines examined, and the PI3 kinase/Akt/Foxo3a pathway plays at most a limited role.