Figure 1.
Figure 1. Coadministration of UCN-01 and farnesyltransferase inhibitors dramatically induces apoptosis and loss of clonogenicity in human leukemia cells. (A) U937, Jurkat, HL-60, and Raji cells were exposed for 18 hours to 10 μM L744832 (L) with or without UCN-01 (UCN or U, U937, and HL-60, 100 nM; Jurkat and Raji, 150 nM), respectively, after which the percentage of cells exhibiting annexin V positivity was determined by annexin V-FITC/PI staining and flow cytometry as described in “Materials and methods.” (B) U937 cells were exposed to 20 μM FTI-277 with or without 100 nM UCN-01 for 18 hours, after which the percentage of apoptotic cells was determined as described above. (C) U937, Jurkat, HL-60, and Raji cells were treated as in panel A, after which TUNEL staining was performed to document apoptosis. Original microscope magnification, × 300. The results of a representative experiment are shown; an additional study yielded equivalent results. (D) U937 and Jurkat cells were treated as in panel A, after which cells were washed free of drug and plated in soft agar as described in the text. After 12 days of incubation, colonies (more than 50 cells) were scored and colony formation for each condition expressed relative to untreated control cells. (E-F) Blasts from 3 patients with acute myeloid leukemia (AML; FAB classification M2) were isolated as described in “Materials and methods.” Mononuclear cells were isolated from the bone marrow (BM/MC) and peripheral blood (PB/MC) of patients with nonmalignant, nonmyeloid hematopoietic disorders or healthy volunteers, respectively, as described in “Materials and methods.” Primary rodent hepatocytes (HC) were isolated as described in “Materials and methods.” Cells were treated with 10 μM L744832 with or without 150 nM UCN-01 for 18 hours (AML, BM/MC, and PB/MC) or 10 μM L744832 with or without 100 nM UCN-01 for 24 hours (HC), respectively, after which the percentage of dead cells (E) was determined by evaluating Wright-Giemsa-stained (AML, BM/MC, and PB/MC) or Hoechst 33342-stained (HC) cytospin preparations. Alternatively, the percentage of annexin V-positive cells in AML blast samples (F, upper panels) and BM/MC and PB/MC (F, lower panels) was assessed by flow cytometry. The results of representative experiments are shown; additional studies yielded equivalent results. For panels A, B, D, and E, results represent the means ± SD for 3 separate experiments performed in triplicate.

Coadministration of UCN-01 and farnesyltransferase inhibitors dramatically induces apoptosis and loss of clonogenicity in human leukemia cells. (A) U937, Jurkat, HL-60, and Raji cells were exposed for 18 hours to 10 μM L744832 (L) with or without UCN-01 (UCN or U, U937, and HL-60, 100 nM; Jurkat and Raji, 150 nM), respectively, after which the percentage of cells exhibiting annexin V positivity was determined by annexin V-FITC/PI staining and flow cytometry as described in “Materials and methods.” (B) U937 cells were exposed to 20 μM FTI-277 with or without 100 nM UCN-01 for 18 hours, after which the percentage of apoptotic cells was determined as described above. (C) U937, Jurkat, HL-60, and Raji cells were treated as in panel A, after which TUNEL staining was performed to document apoptosis. Original microscope magnification, × 300. The results of a representative experiment are shown; an additional study yielded equivalent results. (D) U937 and Jurkat cells were treated as in panel A, after which cells were washed free of drug and plated in soft agar as described in the text. After 12 days of incubation, colonies (more than 50 cells) were scored and colony formation for each condition expressed relative to untreated control cells. (E-F) Blasts from 3 patients with acute myeloid leukemia (AML; FAB classification M2) were isolated as described in “Materials and methods.” Mononuclear cells were isolated from the bone marrow (BM/MC) and peripheral blood (PB/MC) of patients with nonmalignant, nonmyeloid hematopoietic disorders or healthy volunteers, respectively, as described in “Materials and methods.” Primary rodent hepatocytes (HC) were isolated as described in “Materials and methods.” Cells were treated with 10 μM L744832 with or without 150 nM UCN-01 for 18 hours (AML, BM/MC, and PB/MC) or 10 μM L744832 with or without 100 nM UCN-01 for 24 hours (HC), respectively, after which the percentage of dead cells (E) was determined by evaluating Wright-Giemsa-stained (AML, BM/MC, and PB/MC) or Hoechst 33342-stained (HC) cytospin preparations. Alternatively, the percentage of annexin V-positive cells in AML blast samples (F, upper panels) and BM/MC and PB/MC (F, lower panels) was assessed by flow cytometry. The results of representative experiments are shown; additional studies yielded equivalent results. For panels A, B, D, and E, results represent the means ± SD for 3 separate experiments performed in triplicate.

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