Tipifarnib preferentially inhibits signaling downstream of mTOR. (A) HL-60 cells treated with 0, 62.5, 125, 250, 500, or 1000 nM tipifarnib (lanes 1-6, respectively) were washed and examined for topoisomerase IIα content by immunoblotting. Numbers at right represent migration of molecular markers in kilodaltons. The same blot was probed with anti-Hsp90 as a loading control. (B) Alkaline elution to evaluate the possibility that tipifarnib enhances etoposide uptake and/or trapping of covalent topoisomerase II-DNA complexes. After log-phase HL-60 cells were treated for 24 hours with 1 μM tipifarnib or diluent, etoposide was added for 30 minutes. The ability of etoposide to stabilize covalent protein-DNA covalent complexes was quantitated as indicated.⁶²  (C) HL-60 cells were treated with diluent (lane 1), 62.5 (lane 2), 125 (lane 3), 250 (lane 4), 500 (lane 5), or 1000 nM (lane 6) tipifarnib for 24 hours. Whole-cell lysates were blotted with antibodies to phospho-Thr³⁸⁹-p70S6 kinase, phospho-Ser^235/236-S6, p70S6 kinase, and total S6 protein. (D) U937 cells were treated with diluent (lane 1), 125 (lane 2), 250 (lane 3), 500 (lane 4), or 1000 nM (lane 5) tipifarnib for 24 hours. Whole-cell lysates were blotted with antibodies to phospho-Ser^235/236-S6, total S6, the farnesylated protein HDJ-2 and, as a loading control, heat shock protein 90.