Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells

Abstract

Microtubule-targeting agents (MTA), such as the taxanes and vinca alkaloids, are used to treat a variety of cancers due to their ability to perturb microtubule dynamics. In cell culture, MTAs exert their anticancer effects primarily by causing mitotic arrest and cell death. However, accumulating indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. In this study, we sought to develop and characterize an experimental system in which to test the hypothesis that MTAs induce cell death during interphase. Primary adult acute lymphoblastic leukemia (ALL) cells treated with vincristine only weakly exhibited colocalization between mitotic and apoptotic markers and major characteristics of mitotic death, such as an increase in cells with 4N DNA content before the appearance of cells with <2N DNA content, suggesting a mixed response. Therefore, we separated ALL cells into distinct phases of the cell cycle by centrifugal elutriation, labeled cells with 5-ethynyl-2'-deoxyuridine (EdU), and then treated each population with vincristine. Cells isolated during G1 underwent cell death without evidence of EdU uptake, indicating that the cytotoxic effects of vincristine took place during G1 Conversely, cells isolated during S or G2-M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death programs in primary ALL cells depending on cell-cycle phase, and cells in G1 are particularly susceptible to perturbation of interphase microtubules. Primary ALL cells may therefore provide a powerful model system in which to study the multimodal mechanisms underlying MTA-induced cell death. Cancer Res; 76(12); 3553-61. ©2016 AACR.

Fig. 1. Vincristine induces cell death without prior robust mitotic arrest in primary ALL cultures

A. Flow cytometric analysis for cell cycle distribution of KB3, RS4;11, ALL-2 and ALL-5 cells, untreated or treated with 100 nM vincristine for the times indicated. Histograms are representative of three independent experiments. B. Quantitation of data from panel A showing the proportion of cells with <2N (sub-G1) or 4N (G2/M) DNA content with respect to time of vincristine treatment. Results are expressed as mean ± S.D. (n = 3). *p ≤ 0.05 (Student’s t test). C, D. Kinetics of PARP cleavage and relationship to the mitotic marker MPM2. Extracts were made from untreated or vincristine-treated (100 nM) KB3, ALL-2, or ALL-5 (C) or RS4;11 (D) cells at the times indicated and subjected to immunoblotting for PARP or MPM2. Intact and cleaved species of PARP are shown. GAPDH was used as a loading control.

Fig. 2. Distinct responses of primary ALL cells to vincristine in different phases of the cell cycle

A. ALL-2 cells separated by centrifugal elutriation and enriched for cells in either G1 (2N DNA, top two rows) or G2/M (4N DNA, bottom two rows) were treated with 0.1 % DMSO or 100 nM vincristine (VCR) for the times indicated. Cells were fixed and stained with propidium iodide and analyzed by flow cytometry. The histograms are representative of 3 independent experiments. Inset: Average 4N DNA content and selective <2N DNA content, expressed as percentage of total cells analyzed. B, C. ALL-2 or ALL-5 cells, as indicated, in G1 phase (panel B) or G2/M phase (panel C) were treated with 100 nM vincristine (VCR) for the times indicated and extracts subjected to immunoblotting for PARP or MPM2. Intact and cleaved species of PARP are shown. Untreated or VCR-treated KB3 cells (left two lanes) served as positive control. GAPDH was used as a loading control.

Fig. 3. Vincristine depolymerizes microtubules in both asynchronous and G1-phase ALL cells

Asynchronous (A) or G1-phase (B) ALL-5 cells were treated with 0.1 % DMSO (Ctrl) or 100 nM vincristine (VCR) for 1 h and harvested. Cells were also treated with 100 nM Taxol (TAX) for 1 h or 2mM CaCl₂ for 5 min to serve as polymerization and depolymerization controls, respectively. Soluble tubulin (S) and polymerized tubulin (P) were separated as described in Materials and Methods and subjected to immunoblotting for α-tubulin. Total tubulin, present in cell homogenates prior to centrifugation, was used as a loading control. The relative proportions of α-tubulin present in soluble and pellet fractions, determined as described in Materials and Methods and given as %S/%P, is as follows. Panel A: Cont., 42/58; TAX, 21/79; CaCl₂, 50/50; VCR, 56/44. Panel B: Cont., 60/40; TAX, 51/49; CaCl₂, 75/25; VCR, 76/24.

Fig. 4. Primary ALL cells in G1 phase can undergo death directly without transit to S phase

ALL-5 cells in G1 phase obtained by centrifugal elutriation were incubated with EdU and either 0.1% DMSO or 100 nM vincristine (VCR) for the times indicated. Cells were harvested, fixed and stained for EdU incorporation and with PI and analyzed by flow cytometry, with PI staining represented on the x-axis and EdU staining on the y-axis. B, C. Graphical representation of data from panel A. Panel B shows distribution between EdU-negative and EdU-positive for live cells (>2N DNA) and panel C shows distribution between EdU-negative and EdU-positive for dead cells (<2N DNA), expressed as percentage of total cells. Results are expressed as mean ± S.D. (n = 3).

Fig. 5. Vincristine does not affect EdU labeling of ALL cells

A. ALL-5 cells, separated by centrifugal elutriation and representing cells in both G1 (82.4%) and S (17.6%) phases (see Fig. S5B), were incubated with EdU and either 0.1% DMSO or 100 nM vincristine (VCR) for the time indicated, stained for incorporation of EdU and with PI, and subjected to flow cytometry, as in Fig. 4. B,C. Graphical representation of data from panel A. Panel B shows distribution between EdU-negative and EdU-positive for live cells (>2N DNA) and panel C shows distribution between EdU-negative and EdU-positive for dead cells (<2N DNA), expressed as percentage of total cells. Results are expressed as mean ± S.D. (n = 3).

Fig. 6. Proposed model for mode of action of vincristine in ALL cells

Cells in G1 phase are susceptible to death directly, whereas cells that have passed a putative “microtubule sensitivity checkpoint” in late G1/early S phase continue to cycle and die following mitotic arrest.