Vinblastine induces acute, cell cycle phase-independent apoptosis in some leukemias and lymphomas and can induce acute apoptosis in others when Mcl-1 is suppressed

Abstract

Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH(2)-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791-802. (c)2010 AACR.

Figures

Figure 1

Effect of MAPK signaling and protein synthesis on apoptosis induced by vinblastine in ML-1 cells. A: ML-1 cells were incubated with 2.2 μM vinblastine (VB), 50 μM PD98059 (PD), 30 μM SP600125 (SP) or a combination of each for the indicated time, and apoptotic cells were scored by chromatin condensation. B: ML-1 cells were incubated with 2.2 μM vinblastine alone or in combination with 50 μM PD98059, 30 μM SP600125, 42 μM z-VAD-fmk, or 10 μg/mL cycloheximide. Lysates were prepared at the indicated times and protein expression was analyzed by immunoblotting. C: ML-1 cells were incubated with 2.2 μM vinblastine, 10 μg/mL cycloheximide (CHX) or the combination, and apoptotic cells were scored by chromatin condensation. D: ML-1 cells were incubated for 6 h with 2.2 μM vinblastine followed by 50 μM PD98059 for 1–6 h. Lysates were prepared and analyzed by immunoblotting for the indicated proteins. E: ML-1 cells were incubated for 7 h with 2.2 μM vinblastine followed by 10 μg/mL cycloheximide for 1–6 h, and lysates were analyzed for protein expression.

Figure 2

Protection from apoptosis by inhibition of JNK. ML1 cells were incubated with 2.2 μM vinblastine in combination with either PD98059, SP600125 or JNK Inhibitor VIII as indicated. After 6–24 h, cells were harvested, stained and analyzed by flow cytometry for cell cycle distribution. Numbers reflect the percentage of sub-G1 cells and those in each cell cycle phase.

Figure 3

Suppression of Mcl-1 sensitizes ML-1 and OCI-AML4 cells to vinblastine. A. Cells were infected with four different Mcl-1 shRNA lentivirus constructs. After 48 h, the cells were incubated with 2.2 μM vinblastine (VB) and scored 6-h later for chromatin condensation (top panel) and protein expression (bottom panel). B. U937, THP-1 and Granta519 cells were incubated with 20 μM sorafenib (SF) for 6 h to suppress Mcl-1, then with 2μM vinblastine for a further 6 h. Lysates were analyzed by immunoblotting. The lack of cleavage of PARP demonstrates the lack of dependence on Mcl-1 for protecion of these cells from vinblastine.

Figure 4

Effect of PD98059 and cycloheximide on apoptosis induced by vinblastine in a panel of cell lines. A. The indicated cell lines were incubated with 2.2 μM vinblastine, 50 μM PD98059 or a combination of each, and apoptotic cells were scored by chromatin condensation. The inset shows protein expression in OCI-AML4 cells after a 6 h incubation as indicated. B. The same cell lines were incubated with 2.2 μM vinblastine, 10 μg/mL cycloheximide or a combination of each, and apoptotic cells were scored by chromatin condensation.

Figure 5

Expression of Bcl-2 family members and activation of MAPK proteins in the panel of cell lines. The indicated cell lines were either undamaged or incubated with 2.2 μM vinblastine for 4 h. Lysates were analyzed by immunoblotting for expression of the indicated proteins. In the second and third panels, ML-1 and THP-1 cells were reanalyzed to facilitate comparison between the various blots.

Figure 5

Expression of Bcl-2 family members and activation of MAPK proteins in the panel of cell lines. The indicated cell lines were either undamaged or incubated with 2.2 μM vinblastine for 4 h. Lysates were analyzed by immunoblotting for expression of the indicated proteins. In the second and third panels, ML-1 and THP-1 cells were reanalyzed to facilitate comparison between the various blots.

Figure 5

Expression of Bcl-2 family members and activation of MAPK proteins in the panel of cell lines. The indicated cell lines were either undamaged or incubated with 2.2 μM vinblastine for 4 h. Lysates were analyzed by immunoblotting for expression of the indicated proteins. In the second and third panels, ML-1 and THP-1 cells were reanalyzed to facilitate comparison between the various blots.

Figure 6

Acute apoptosis induced by vinblastine alone in mantle cell lymphoma and CLL cells. A. The three mantle cell lymphoma lines were incubated with 0–2 μM vinblastine for 6 h, then analyzed for expression of the indicated antigens. Parallel experiments included concurrent incubation with JNK inhibitor VIII. B. Cells from a CLL patient and peripheral lymphocytes from a normal individual were incubated with 0–2 μM vinblastine for 6 h and then analysed for expression of the indicated antigens. Cells from the CLL patient were also incubated concurrently with JNK inhibitor VIII. These results are similar to those observed in all 7 CLL samples.