<div>Abstract<p>Microtubule inhibitors such as vinblastine cause mitotic arrest and subsequent apoptosis through the intrinsic mitochondrial pathway. However, although Bcl-2 family proteins have been implicated as distal mediators, their precise role is largely unknown. In this study, we investigated the role of Bak in vinblastine-induced apoptosis. Bak was mainly monomeric in untreated KB-3 cells, and multimers corresponding to dimer, trimer, and higher oligomers were observed after vinblastine treatment. The oligomeric Bak species were strongly diminished in cells stably overexpressing Bcl-xL. Immunoprecipitation with a conformation-dependent Bak antibody revealed that vinblastine induced Bak activation. Reciprocal immunoprecipitations indicated that vinblastine induced the interaction of active Bak with active Bax. Furthermore, Bcl-xL overexpression prevented Bak and Bax interaction and strongly inhibited apoptosis, whereas Bcl-2 overexpression did not prevent Bak-Bax interaction and only weakly inhibited apoptosis. The relative contributions of Bak and Bax were investigated using fibroblasts deficient in one or both of these proteins; double knockouts were highly resistant compared with single knockouts, with vinblastine sensitivities in the order of Bak<sup>+</sup>/Bax<sup>+</sup> > Bak<sup>+</sup>/Bax<sup>-</sup> > Bak<sup>-</sup>/Bax<sup>+</sup> > Bak<sup>-</sup>/Bax<sup>-</sup>. These results highlight Bak as a key mediator of vinblastine-induced apoptosis and show for the first time activation and oligomerization of Bak by an antimitotic agent. In addition, our results suggest that the interaction of the activated forms of Bak and Bax represents a key distal step in the apoptotic response to this important chemotherapeutic drug. [Mol Cancer Ther 2008;7(7):2224–32]</p></div>
The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-a isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.
Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs. Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs. Microtubule targeting agents (MTAs) including the taxanes and vinca alkaloids are widely used in cancer chemotherapy (1Steinmetz M.O. Prota A.E. Microtubule-targeting agents: Strategies to Hijack the cytoskeleton.Trends Cell Biol. 2018; 28: 776-792Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 2Dumontet C. Jordan M.A. Microtubule-binding agents: A dynamic field of cancer therapeutics.Nat. Rev. Drug Discov. 2010; 9: 790-803Crossref PubMed Scopus (1217) Google Scholar). These drugs bind to microtubules or tubulin and induce mitotic arrest and intrinsic apoptosis (3Weaver B.A. Cleveland D.W. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death.Cancer Cell. 2005; 8: 7-12Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 4Manchado E. Guillamot M. Malumbres M. Killing cells by targeting mitosis.Cell Death Differ. 2012; 19: 369-377Crossref PubMed Scopus (173) Google Scholar, 5Topham C.H. Taylor S.S. Mitosis and apoptosis: How is the balance set?.Curr. Opin. Cell Biol. 2013; 25: 780-785Crossref PubMed Scopus (135) Google Scholar). Intrinsic (or mitochondrial) apoptosis is regulated by the Bcl-2 protein family which exhibit either prosurvival or proapoptotic properties (6Czabotar P.E. Lessene G. Strasser A. Adams J.M. Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy.Nat. Rev. Mol. Cell Biol. 2014; 15: 49-63Crossref PubMed Scopus (1968) Google Scholar, 7Singh R. Letai A. Sarosiek K. Regulation of apoptosis in health and disease: The balancing act of BCL-2 family proteins.Nat. Rev. Mol. Cell Biol. 2019; 20: 175-193Crossref PubMed Scopus (561) Google Scholar, 8Chipuk J.E. Moldoveanu T. Llambi F. Parsons M.J. Green D.R. The BCL-2 family reunion.Mol. Cell. 2010; 37: 299-310Abstract Full Text Full Text PDF PubMed Scopus (1116) Google Scholar). Prosurvival members include Bcl-2, Bcl-xL, Mcl-1, Bcl-w, and Bfl-1/A1, and proapoptotic members include the multidomain effectors Bax, Bak, and Bok, and the BH3-only subfamily comprised of activators (Bim and Bid) and sensitizers (Bad, Noxa, Bik, Bmf, Hrk, and others). Bcl-2 proteins play an important role in mitotic-arrest–induced apoptosis (9Whitaker R.H. Placzek W.J. Regulating the BCL2 family to improve sensitivity to microtubule targeting agents.Cells. 2019; 8: 346Crossref Scopus (28) Google Scholar). Our studies using HeLa and several other cell lines, for example, have shown that MTAs cause Bax and Bak activation, leading to cytochrome c release from the mitochondria and caspase-3 activation, and that cells lacking Bax and Bak, or overexpressing Bcl-2 or Bcl-xL, are highly resistant to MTAs (10Upreti M. Lyle C.S. Skaug B. Du L. Chambers T.C. Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members.J. Biol. Chem. 2006; 281: 15941-15950Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Upreti M. Chu R. Galitovskaya E. Smart S.K. Chambers T.C. Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine.Mol. Cancer Ther. 2008; 7: 2224-2232Crossref PubMed Scopus (31) Google Scholar, 12Upreti M. Galitovskaya E.N. Chu R. Tackett A.J. Terrano D.T. Granell S. Chambers T.C. Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.J. Biol. Chem. 2008; 283: 35517-35525Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Eichhorn J.M. Sakurikar N. Alford S.E. Chu R. Chambers T.C. Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death.Cell Death Dis. 2013; 4: e834Crossref PubMed Scopus (41) Google Scholar). MTAs also induce phosphorylation of prosurvival members Bcl-2, Bcl-xL, and Mcl-1 during mitotic arrest. Phosphorylation of Bcl-2 and Bcl-xL disables prosurvival function by reducing affinity for select proapoptotic Bcl-2–binding partners (12Upreti M. Galitovskaya E.N. Chu R. Tackett A.J. Terrano D.T. Granell S. Chambers T.C. Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.J. Biol. Chem. 2008; 283: 35517-35525Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14Bah N. Maillet L. Ryan J. Dubreil S. Gautier F. Letai A. Juin P. Barille-Nion S. Bcl-xL controls a switch between cell death modes during mitotic arrest.Cell Death Dis. 2014; 5e1291PubMed Google Scholar), and phosphorylation of Mcl-1 is associated with its degradation (11Upreti M. Chu R. Galitovskaya E. Smart S.K. Chambers T.C. Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine.Mol. Cancer Ther. 2008; 7: 2224-2232Crossref PubMed Scopus (31) Google Scholar, 15Wertz I.E. Kusam S. Lam C. Okamoto T. Sandoval W. Anderson D.J. Helgason E. Ernst J.A. Eby M. Liu J. Belmont L.D. Kaminker J.S. O'Rourke K.M. Pujara K. Kohli P.B. et al.Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7.Nature. 2011; 471: 110-114Crossref PubMed Scopus (578) Google Scholar), a process that regulates the kinetics of mitotic death (16Sloss O. Topham C. Diez M. Taylor S. Mcl-1 dynamics influence mitotic slippage and death in mitosis.Oncotarget. 2016; 7: 5176-5192Crossref PubMed Scopus (41) Google Scholar, 17Clarke P.R. Allan L.A. Skowyra A. Timed degradation of Mcl-1 controls mitotic cell death.Mol. Cell Oncol. 2018; 5e1516450PubMed Google Scholar). Whether MTAs strictly target mitotic cells in clinical settings has been under debate (18Komlodi-Pasztor E. Sackett D. Wilkerson J. Fojo T. Mitosis is not a key target of microtubule agents in patient tumors.Nat. Rev. Clin. Oncol. 2011; 8: 244-250Crossref PubMed Scopus (1) Google Scholar, 19Komlodi-Pasztor E. Sackett D.L. Fojo A.T. Inhibitors targeting mitosis: Tales of how great drugs against a promising target were brought down by a flawed rationale.Clin. Cancer Res. 2012; 18: 51-63Crossref PubMed Scopus (153) Google Scholar, 20Kitagawa K. Too early to say, "no targeting of mitosis.Nat. Rev. Clin. Oncol. 2011; 8 (author reply 444): 444Crossref PubMed Scopus (4) Google Scholar, 21Tunquist B.J. Wood K.W. Walker D.H. Tales of how great drugs were brought down by a flawed rationale--letter.Clin. Cancer Res. 2013; 19: 1302Crossref PubMed Scopus (4) Google Scholar, 22Wissing M.D. Carducci M.A. Gelderblom H. van Diest P.J. Tales of how great drugs were brought down by a flawed rationale--letter.Clin. Cancer Res. 2013; 19: 1303Crossref PubMed Scopus (5) Google Scholar, 23Komlodi-Pasztor E. Sackett D.L. Fojo T. Tales of how great drugs were brought down by a flawed rationale--response.Clin. Cancer Res. 2013; 19: 1304Crossref PubMed Scopus (5) Google Scholar). The lack of clinical efficacy of drugs that target mitotic kinases and mitotic motor proteins, together with the extended doubling time and low mitotic indices of tumors that are susceptible to MTAs, has led to the hypothesis that MTAs may primarily target interphase microtubules rather than mitotic spindles (18Komlodi-Pasztor E. Sackett D. Wilkerson J. Fojo T. Mitosis is not a key target of microtubule agents in patient tumors.Nat. Rev. Clin. Oncol. 2011; 8: 244-250Crossref PubMed Scopus (1) Google Scholar, 19Komlodi-Pasztor E. Sackett D.L. Fojo A.T. Inhibitors targeting mitosis: Tales of how great drugs against a promising target were brought down by a flawed rationale.Clin. Cancer Res. 2012; 18: 51-63Crossref PubMed Scopus (153) Google Scholar, 24Mitchison T.J. The proliferation rate paradox in antimitotic chemotherapy.Mol. Biol. Cell. 2012; 23: 1-6Crossref PubMed Scopus (230) Google Scholar). However, since cultured cancer cell lines are typically only susceptible to MTAs in mitosis, relevant laboratory models are lacking. We therefore screened a large selection of cultured cancer cells and discovered that primary adult acute lymphoblastic leukemia (ALL) cells are susceptible to clinically relevant concentrations of MTAs not only in M phase but also in G1 phase (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar). These studies were facilitated by the use of centrifugal elutriation which enabled the isolation of cells highly enriched in either G1 or G2/M phases (26Delgado M. Kothari A. Hittelman W.N. Chambers T.C. Preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation.J. Vis. Exp. 2017; 129: 56418Google Scholar). Subsequent investigation showed that death directly in G1 phase required active cell cycle advance and did not occur in G1-arrested cells (27Delgado M. Chambers T.C. Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase.Cell Cycle. 2018; 17: 1784-1796Crossref PubMed Scopus (7) Google Scholar). Furthermore, G1 phase death of primary ALL cells was found to occur only in response to microtubule destabilizing drugs under conditions of complete microtubule depolymerization and was not observed after treatment with microtubule stabilizing drugs where polymerized microtubule structures were retained (28Delgado M. Urbaniak A. Chambers T.C. Contrasting effects of microtubule destabilizers versus stabilizers on induction of death in G1 phase of the cell cycle.Biochem. Pharmacol. 2018; 162: 213-223Crossref PubMed Scopus (6) Google Scholar). In addition, of cells in interphase, only those in G1 phase were susceptible to microtubule depolymerization; cells in S or G2 phases were not, instead progressing to M phase where they arrested and died (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar). From these observations, we concluded that functional microtubules are essential for successful G1 phase advance of primary ALL cells, possibly due to their role in trafficking key proteins needed for S phase to the nucleus, but are dispensable for G1-arrested cells that are not preparing for S phase (27Delgado M. Chambers T.C. Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase.Cell Cycle. 2018; 17: 1784-1796Crossref PubMed Scopus (7) Google Scholar). The availability of primary ALL cells enriched in either G1 or G2/M phases provided an opportunity to investigate and compare the death pathway(s) activated in response to microtubule destabilization. Numerous mechanisms of cell death have been recognized including apoptosis, necrosis, necroptosis, ferroptosis, parthanatos, autophagy-dependent, and many others (29Galluzzi L. Vitale I. Aaronson S.A. Abrams J.M. Adam D. Agostinis P. Alnemri E.S. Altucci L. Amelio I. Andrews D.W. Annicchiarico-Petruzzelli M. Antonov A.V. Arama E. Baehrecke E.H. Barlev N.A. et al.Molecular mechanisms of cell death: Recommendations of the nomenclature committee on cell death 2018.Cell Death Differ. 2018; 25: 486-541Crossref PubMed Scopus (2074) Google Scholar). Here we report the results of such a study which shows that while certain features are common to both, distinct pathways are responsible for death of G1 versus M phase primary ALL cells in response to vincristine (VCR). To the best of our knowledge, these results are the first to demonstrate that a specific drug can enter a single cell type and induce two different forms of cell death. The findings provide a basis for advancing our understanding of clinical mechanisms of MTAs. Primary ALL cells were subjected to centrifugal elutriation as described in Experimental procedures and fractions corresponding to cells in G1 phase or G2/M phases were prepared and stained with propidium iodide to determine DNA content and verify purity. Representative data are shown in Figure 1, together with that of the initial asynchronous population. The majority of cells in asynchronous culture were in G1 phase containing 2N DNA, with much smaller proportions in S and G2/M phases with >2N-4N DNA content (Fig. 1A). Asynchronous cultures also routinely contained a small proportion (∼2.5%) of dead or dying cells indicated by sub-G1 DNA content. The G1 phase pool was highly enriched in cells with 2N DNA content (97%), with extremely low contamination of cells in other cell cycle phases (Fig. 1B). The G2/M pool was also greatly enriched in cells with 4N DNA content (>80%), with relatively low levels of cells in other phases (Fig. 1C). Importantly, primary ALL cells in S or G2 phases when treated with VCR or other microtubule destabilizers transit to M phase where they arrest and die (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar, 28Delgado M. Urbaniak A. Chambers T.C. Contrasting effects of microtubule destabilizers versus stabilizers on induction of death in G1 phase of the cell cycle.Biochem. Pharmacol. 2018; 162: 213-223Crossref PubMed Scopus (6) Google Scholar). Thus, over 96% of cells in the designated G2/M fraction are destined to undergo mitotic death upon drug treatment. Conversely, the majority of cells in the G1 phase fraction, when treated with VCR or other microtubule destabilizers at concentrations of ≥30 nM, die directly in G1 (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar, 28Delgado M. Urbaniak A. Chambers T.C. Contrasting effects of microtubule destabilizers versus stabilizers on induction of death in G1 phase of the cell cycle.Biochem. Pharmacol. 2018; 162: 213-223Crossref PubMed Scopus (6) Google Scholar). Thus, the two fractions generated by centrifugal elutriation are ideally suited to the purpose of this study to compare the respective phase-specific death pathways. Previous studies by us and others have shown that Bcl-2 proteins play a key role in mitotic death (10Upreti M. Lyle C.S. Skaug B. Du L. Chambers T.C. Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members.J. Biol. Chem. 2006; 281: 15941-15950Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Upreti M. Chu R. Galitovskaya E. Smart S.K. Chambers T.C. Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine.Mol. Cancer Ther. 2008; 7: 2224-2232Crossref PubMed Scopus (31) Google Scholar, 12Upreti M. Galitovskaya E.N. Chu R. Tackett A.J. Terrano D.T. Granell S. Chambers T.C. Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.J. Biol. Chem. 2008; 283: 35517-35525Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Eichhorn J.M. Sakurikar N. Alford S.E. Chu R. Chambers T.C. Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death.Cell Death Dis. 2013; 4: e834Crossref PubMed Scopus (41) Google Scholar, 14Bah N. Maillet L. Ryan J. Dubreil S. Gautier F. Letai A. Juin P. Barille-Nion S. Bcl-xL controls a switch between cell death modes during mitotic arrest.Cell Death Dis. 2014; 5e1291PubMed Google Scholar, 15Wertz I.E. Kusam S. Lam C. Okamoto T. Sandoval W. Anderson D.J. Helgason E. Ernst J.A. Eby M. Liu J. Belmont L.D. Kaminker J.S. O'Rourke K.M. Pujara K. Kohli P.B. et al.Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7.Nature. 2011; 471: 110-114Crossref PubMed Scopus (578) Google Scholar, 16Sloss O. Topham C. Diez M. Taylor S. Mcl-1 dynamics influence mitotic slippage and death in mitosis.Oncotarget. 2016; 7: 5176-5192Crossref PubMed Scopus (41) Google Scholar, 17Clarke P.R. Allan L.A. Skowyra A. Timed degradation of Mcl-1 controls mitotic cell death.Mol. Cell Oncol. 2018; 5e1516450PubMed Google Scholar), consistent with observations that cell death in response to mitotic arrest occurs largely via intrinsic apoptosis (3Weaver B.A. Cleveland D.W. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death.Cancer Cell. 2005; 8: 7-12Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 4Manchado E. Guillamot M. Malumbres M. Killing cells by targeting mitosis.Cell Death Differ. 2012; 19: 369-377Crossref PubMed Scopus (173) Google Scholar, 5Topham C.H. Taylor S.S. Mitosis and apoptosis: How is the balance set?.Curr. Opin. Cell Biol. 2013; 25: 780-785Crossref PubMed Scopus (135) Google Scholar). We therefore first examined the expression, phosphorylation, and activation status of members of the Bcl-2 protein family. G1 or G2/M phase ALL cells were prepared and treated with vehicle (0.1% DMSO) or 100 nM VCR. Previously, it was shown that death of cells in G2/M phases in response to VCR, based on assessment of poly (ADP)-ribose polymerase (PARP) cleavage, occurred more rapidly than that of cells in G1 phase (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar). Therefore, drug exposure times differed, with periods up to 48 h for G1 cells and up to 24 h for G2/M cells. KB-3 cells, a HeLa subline, were used as a control for mitotic death because we have shown previously that in response to MTAs, they exhibit a well-defined mitotic arrest followed by intrinsic apoptosis (30Terrano D.T. Upreti M. Chambers T.C. Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis.Mol. Cell Biol. 2010; 30: 640-656Crossref PubMed Scopus (173) Google Scholar, 31Du L. Lyle C.S. Chambers T.C. Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: Evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction.Oncogene. 2005; 24: 107-117Crossref PubMed Scopus (69) Google Scholar). As shown in Figure 2, left panel, VCR induced time-dependent cleavage of PARP in KB-3 cells, while PARP remained intact in vehicle-treated cells. VCR also induced phosphorylation of both Mcl-1 and Bcl-xL at 24 h, indicated by mobility shifts of these proteins on immunoblots employing phosphorylation-independent antibodies, as well as by detection using phospho-specific antibodies (Fig. 2). In the case of Bcl-xL, phosphorylation was reversed by 48 h, whereas in the case of Mcl-1, phosphorylation resulted in loss of Mcl-1 expression, determined previously to be due to proteosome-mediated degradation (32Chu R. Terrano D.T. Chambers T.C. Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration.Biochem. Pharmacol. 2012; 83: 199-206Crossref PubMed Scopus (49) Google Scholar). While Mcl-1 levels were decreased after VCR treatment as evaluated by use of a phosphorylation-independent antibody, strong immunoreactivity with the phospho-specific Mcl-1 antibody was retained. This is due to the high level of Mcl-1 phosphorylation, at up to nine sites, as we reported previously (33Chu R. Alford S.E. Hart K. Kothari A. Mackintosh S.G. Kovak M.R. Chambers T.C. Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: Nine phosphorylation sites identified.Oncotarget. 2016; 7: 78958-78970Crossref PubMed Scopus (7) Google Scholar). Bcl-2 is only weakly expressed in KB-3 cells but nonetheless VCR induced a transient mobility shift, previously determined to be due to phosphorylation (31Du L. Lyle C.S. Chambers T.C. Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: Evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction.Oncogene. 2005; 24: 107-117Crossref PubMed Scopus (69) Google Scholar). Overall similar changes were noted when G2/M phase ALL cells were treated with VCR (Fig. 2, right panel). Thus, PARP underwent cleavage, and phosphorylation of Mcl-1, Bcl-xL, and Bcl-2 was observed, together with loss of Mcl-1 expression. In contrast, when ALL cells in G1 phase were treated with VCR (Fig. 2, center panel), PARP cleavage was not accompanied by phosphorylation of prosurvival Bcl-2 proteins, and while Mcl-1 expression was reduced, it occurred in a phosphorylation-independent manner. Next, Bax activation was examined, as described in Experimental procedures. ALL cells in G1 or G2/M phases were prepared and treated with vehicle or 100 nM VCR. Extracts were made under native conditions and subjected to immunoprecipitation with 6A7 active Bax antibody, followed by immunoblotting for Bax. Asynchronous ALL cells treated with the Bcl-2/Bcl-xL inhibitor ABT-263, which induces rapid intrinsic apoptosis by disrupting Bcl-2/Bcl-xL suppression of prodeath Bcl-2 proteins (34Shoemaker A.R. Mitten M.J. Adickes J. Ackler S. Refici M. Ferguson D. Oleksijew A. O'Connor J.M. Wang B. Frost D.J. Bauch J. Marsh K. Tahir S.K. Yang X. Tse C. et al.Activity of the Bcl-2 family inhibitor ABT-263 in a panel of small cell lung cancer xenograft models.Clin. Cancer Res. 2008; 14: 3268-3277Crossref PubMed Scopus (166) Google Scholar), was used as a positive control. As shown in Figure 3, ABT-263 induced strong Bax activation in asynchronous cells as expected, and VCR also induced strong Bax activation in G2/M phase cells, and this occurred in concert with PARP cleavage. However, only weak Bax activation was associated with PARP cleavage in G1 phase cells treated with VCR for 24 or 48 h. Total Bax levels in whole cell extracts remained relatively unchanged under all conditions (Fig. 3). Our earlier studies showed that while the majority of G1 phase ALL cells treated with VCR died directly from G1 phase, a small proportion that varied from 5 to 15% escaped direct death and advanced in the cell cycle and died via mitotic death (25Kothari A. Hittelman W.N. Chambers T.C. Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells.Cancer Res. 2016; 76: 3553-3561Crossref PubMed Scopus (40) Google Scholar). Since cyclin B expression is a characteristic marker of mitosis (35Malumbres M. Barbacid M. Mammalian cyclin-dependent kinases.Trends Biochem. Sci. 2005; 30: 630-641Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar), the same extracts were probed for cyclin B. Strong cyclin B expression was observed in G2/M phase cells treated with VCR as anticipated, and weak cyclin B expression was observed in VCR-treated G1 cells (Fig. 3). Band intensities were quantified by Image J analysis, and the level of Bax activation after VCR treatment in G1 phase cells was determined to be about 20% that of G2/M cells. Similarly, the level of cyclin B expression after VCR treatment of G1 cells was found to be about 20% that of G2/M cells. These results strongly suggest that the low level of Bax activation observed in drug-treated G1 phase cells derives from the small proportion of cells that escape direct death and advance in the cell cycle and undergo mitotic death and strengthen the notion that G1 phase death is Bax-independent. Intrinsic apoptosis is associated with activation of initiator and executioner caspases including caspase-3 (36Shalini S. Dorstyn L. Dawar S. Kumar S. Old, new and emerging functions of caspases.Cell Death Differ. 2015; 22: 526-539Crossref PubMed Scopus (687) Google Scholar). Cell extracts prepared after VCR treatment of G1 or G2/M phase ALL-5 cells were subjected to caspase-3 assay, as described in Experimental procedures. Asynchronous ALL-5 cells treated with the Bcl-2 inhibitor ABT-263 were used as a positive control. ABT-263 treatment strongly and rapidly activated caspase-3, and this was completely inhibited by cotreatment with the pan caspase inhibitor Z-VAD-FMK (Fig. S1). VCR strongly activated caspase-3 in G2/M phase cells after 24 h treatment but only weakly activated caspase-3 in G1 phase cells at 24 h (Fig. 4). To exclude the possibility that caspase-3 activation was delayed in G1 phase cells, assays were also conducted at 48 h of VCR treatment, and levels considerably lower than G2/M phase cells were still observed (Fig. 4). A highly comparable profile of results were obtained when an independent culture of primary ALL cells, ALL-2, which were derived from a different patient (37Alford S.E. Kothari A. Loeff F.C. Eichhorn J.M. Sakurikar N. Goselink H.M. Saylors R.L. Jedema I. Falkenburg J.H. Chambers T.C. BH3 inhibitor sensitivity and Bcl-2 dependence in primary acute lymphoblastic leukemia cells.Cancer Res. 2015; 75: 1366-1375Crossref PubMed Scopus (56) Google Scholar), were used (Fig. S2). DNA fragmentation during intrinsic apoptosis is in part caspase-dependent, through a mechanism whereby caspases degrade the inhibitor of the responsible nuclease thus activating it (38Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.Nature. 1998; 391: 43-50Crossref PubMed Scopus (2770) Google Scholar, 39Larsen B.D. Sorensen C.S. The caspase-activated DNase: Apoptosis and beyond.FEBS J. 2017; 284: 1160-1170Crossref PubMed Scopus (93) Google Scholar). Since caspase-3 was differentially activated by VCR in G1 versus G2/M phase ALL cells, we sought to determine whether differences existed with respect to caspase dependence of DNA fragmentation. Asynchronous ALL-5 cells treated with the Bcl-2 inhibitor ABT-263 were used as a positive control, and DNA fragmentation was assessed by sub-G1 DNA content after propidium iodide staining and flow cytometry. As shown in Fig. S3, the caspase inhibitor Z-VAD-FMK completely inhibited DNA f
