Erlotinib Increases Efficacy of 5-Azacitidine (AZA) by Inhibiting Drug Efflux Via ABC-Transporters

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 974-974
Author(s):  
Elodie Lainey ◽  
Marie Sebert ◽  
Sylvain Thepot ◽  
Maximilien Tailler ◽  
Lionel Ades ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Surface Expression ◽  
Drug Efflux ◽  
Spontaneous Apoptosis ◽  
Induced Apoptosis ◽  
Over Time

Abstract Abstract 974 Background: We and others showed that the TKI erlotinib (Erlo) has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008) and clinical studies defining more closely its therapeutic benefit are underway in MDS, including by our group (NCT00977548, NCT01085838). We tried to define in this preclinical study the potential interest of combining Erlo to hypomethylating agents, which have become reference treatments in MDS. Methods: Erlo (10mM) was combined to AZA, (2mM) or decitabine (DAC, 2mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining in MDS- and AML-derived cell lines (MOLM-13, KG-1, HL-60) and ex vivo patient (pt) MDS and AML cells. To fully evaluate the extent of Erlo-induced sensitization, efficacy was compared to apoptosis induced by AraC (100nM), doxorubicine (Dox, 100nM), VP-16 (1mM) +/− Erlo. Single drug dosages had been chosen since they caused little to no apoptosis. Efflux via P-gp (by staining with DioC23), MRP (by calcein) and was quantified by FACS, specific biochemical efflux inhibitors (CSA, MK-571, KO143) served as controls, intracellular retention of mitoxantrone (MTZ) assessed overall functionality of ABC-transporters. Surface expression of P-gp was quantified by FACS. Results: Whereas co-incubation of Erlo with DAC did not increase apoptosis in any of the myeloid cell lines, combination of Erlo with AZA exhibited synergistic effects already observed at 24h (HL-60: Erlo: 3%, AZA: 10%, Erlo+ AZA: 30%), and increasing over-time (72h: Erlo: 6%, AZA: 15%, Erlo+ AZA: 40%). Noteworthy, the degree of apoptosis observed by combining Erlo+ AZA was comparable to cell death induced by 1mM VP-16, and largely exceeded apoptosis observed with 100nM Dox and 100nM AraC in the respective cell lines. To determine if Erlo also impacts on apoptosis in MDS- and AML-derived cells, we first screened 5 pt samples (with MDS or AML) for apoptosis observed under AZA and DAC, and demonstrated that, in all samples, AZA-induced apoptosis exceeded DAC-induced apoptosis (by up to 40% at 24h). Noteworthy, the “peak” of apoptosis in sensitive pt cells occurred at 24h and – under single agents - did not increase over-time. Co-incubation with Erlo and AZA was able to induce synergistic effects already at 24h (AML post MDS: Erlo: 10%, AZA: 33%, Erlo+ AZA: 55%) and apoptosis increased over-time. In addition, whereas malignant CD34- cells already showed a high rate of “spontaneous” apoptosis (that is in the absence of any drug, about 50% of apoptosis at 48h), CD34+ blasts not only exhibited a lower rate of spontaneous apoptosis (remaining below 15% at 72h), but also a considerable degree of chemoresistance (48h: no increase of apoptosis under AraC, Dox or VP-16). Testing if the combination of Erlo+ AZA also targets this resistant population, we showed that it is particularly efficient in CD34+ progenitors (inducing 80% cell death at 72h), exceeding efficiency of all other tested agents/combinations (AraC, Dox, VP-16, Dec+/−Erlo). Hypothesizing that this pattern of sensitization might be due to a decreased drug efflux via ABC-transporters (overexpressed on malignant myeloid progenitors), we tested this concept on cell lines and ex vivo pt cells. We found that erlotinib can inhibit drug efflux via P-gp (as evidenced by increased intracellular retention of DioC23) and MRP (increased retention of calcein). Notably, Erlo increased intracellular accumulation of MTZ in MDS- and AML-derived CD34- and CD34+ blasts, and exhibited - as single agent - a comparable degree of efflux inhibition as the combination of all three biochemical inhibitors. To more closely define the advantageous effect of Erlo+AZA in pt cells, we quantified drug-induced changes in P-gp expression separately in CD34+CD38dim and CD34dimCD38+ blasts. We found that Erlo down-regulates P-gp surface expression (decrease of 50% at 48h), and that this down-regulation is more pronounced in the immature CD34+CD38dim population than in the CD34dimCD38+ blasts. Conclusions: Our study shows erlotinib's ability to reverse the drug-resistance phenotype in malignant blasts and its interesting combination with AZA. We also provide novel evidence that Erlo (combined to AZA) can target chemoresistant myeloid progenitors by diminishing functionality as well as surface expression of ABC-transporters. Those results suggest a potential clinical interest of combining erlotinib to AZA in MDS Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

Erlotinib Inhibits ABC Transporters of AML Progenitors with Stem Cell Features and Increases Chemosensitivity to Current AML Drugs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2163-2163
Author(s):  
Marie Sebert ◽  
Elodie Lainey ◽  
Sylvain Thepot ◽  
Maximilien Tailler ◽  
Lionel Ades ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Surface ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Myeloid Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Induced Apoptosis ◽  
Over Time

Abstract Abstract 2163 Background: Treatment failure in AML is attributed to the persistence of AML progenitors able, among others, to efflux chemotherapeutic drugs via ABC-transporters. Increased efflux capacity is considered a stem cell feature, and therapeutic inhibition may increase chemosensitivity and help eradicate this progenitor population. Nevertheless, clinical studies assessing a potential benefit of ABC-inhibitors in AML treatment showed no significant survival advantage, possibly because AML cells express different ABC-transporters and classical inhibitors target only a restricted type of efflux channels. We assessed the efficacy of the TKI erlotinib (Erlo) to antagonize drug efflux via most important AML-associated efflux channels, ie P-gp, MRP and BCRP. Methods: Overall drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ), and specific efflux via P-gp (substrates: DioC23 and rhodamine-123), MRP (substrates: calcein and CDCFDA) and BCRP (substrate: Hoechst 33342) were quantified by FACS at 1h and 6h following incubation with 10mM Erlo. Biochemical inhibitors of the respective ABC-transporters (CSA, verapamil, MK-571, KO143) served as controls. Surface expression of P-gp, MRP and BCRP was quantified by FACS. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), doxorubicine (Dox, 100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assays were carried out in myeloid cell lines (KG-1, MOLM-13, HL-60) and ex vivo AML cells (n=3). Immaturity of AML cells was determined in 2 samples by comparing CD34+ versus CD34- cells, and in one pt by co-staining for CD34, CD38, CD123 and CD133. Results: We found that I) Erlo inhibited efflux via P-gp and MRP as demonstrated by increased intracellular retention of DioC23/Rho-123, and calcein/CDCFDA, respectively; II) this degree of inhibition was higher in KG-1 cells than in MOLM-13 or HL-60 cells; III) inhibition of drug efflux was observed already at 1h of incubation, increased over time (6h); IV) Erlo increased intracellular retention of MTZ faster (at 1h with a further increase at 6h) and at least to the same extent than a combination of all three biochemical efflux inhibitors, showing that Erlo's capacity to hinder drug efflux is not restricted to a single ABC-transporter: V) surface expression of P-gp, MRP and BCRP was strongest on KG-1 cells and not altered upon 1h and 6h of Erlo incubation VI) Erlo increased Dox- and VP16-induced apoptosis (48h KG-1: Erlo alone 20%, Dox alone 10%, VP-16 alone 20%, Erlo+Dox: 40%, VP-16+Erlo: 70%), while having no impact on AraC-induced apoptosis; VI) this pattern of chemosensitization was observed in all myeloid cell lines, but once more most pronounced in KG-1 cells. To test the hypothesis that Erlo has comparable effects in pt-derived AML cells ex vivo, we showed by concomitant cell surface staining that I) immature AML subpopulations had a higher efflux capacity (notably via P-gp) than their more mature counterparts (i.e. in one pt with chemoresistant AML: DioC23/Rho-123 fluorescence twice as high in the CD34-/CD38+, CD123+, CD133- than in the CD34+/CD38dim, CD123-, CD133+ subpopulation); II) cell surface expression of P-gp is twice as high in this more immature population (CD34+/CD38dim, CD123-, CD133+) than in CD34-/CD38+, CD123+, CD133+ cells; III) Erlo antagonizes drug efflux via P-gp and MRP at 1h (increasing further at 6h) of incubation; IV) this effect is most pronounced in the immature progenitor cells (1h: decrease of DioC23/Rho-123 efflux in CD34-/CD38+, CD123+, CD133- cells by about 50% and in the more immature CD34-/CD38+, CD123-, CD133+ cells by about 70%); V) Erlo diminishes cell surface expression of P-gp (48h), most effectively in the progenitor populations (by 30% in the CD34-/CD38+, CD123+, CD133- cells versus 50% in CD34-/CD38+, CD123+, CD133- cells); VI) Erlo is able to retain MTZ in both CD34- and CD34+ AML-subpopulations; VII) these effects are accompanied by an increased sensitivity towards Dox and VP-16; VIII) Erlo-induced chemosensitization is higher in the CD34+ than in CD34- AML cells. Conclusions: We here provide novel evidence that erlotinib is able to overcome the stem cell features of increased expression and functionality of ABC-transporters thereby antagonizing the intrinsic chemoresistance of (immature) AML cells. Those results suggest a potential clinical interest of combining erlotinib to chemotherapy in AML Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

Erlotinib Antagonizes Efflux Via ABC Transporters and Decreases P-Gp Cell Surface Expression by Inhibiting SRC Kinase and mTOR Pathways in Acute Myeloid Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2564-2564 ◽  
Author(s):  
Elodie Lainey ◽  
Marie Sebert ◽  
Cyrielle Bouteloup ◽  
Carole Leroy ◽  
Sylvain Thepot ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Atpase Activity ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Hoechst 33342

Abstract Abstract 2564 Background: Erlotinib (Erlo) was originally developed as an epidermal growth factor receptor inhibitor, yet it also exerts antileukemic “off-target” effects, in vitro and in vivo in MDS and AML (Boehrer et al., Blood, 2008). In a preliminary pre-clinical study, we observed that Erlo increased chemosensitivity to current AML drugs in different AML cell lines and in ex vivo AML patient cells (n=3) (ASH 2010, 2163). Those first results suggested an implication of ABC-transporters in the potentiation of apoptosis. Here, we bring direct evidence for Erlo's ability to hinder efflux pumps and to decrease their expression on AML cells. Methods: Drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ or doxorubicin-Dox), and specific efflux via P-gp (substrates: DioC23 and Rho-123), MRP (s: Calcein and CDCFDA) and BCRP (s: Hoechst 33342) were quantified by FACS following incubation with 10mM Erlo. Intracellular VP-16) content was quantified by Rapid Resolution Liquid Chromatography (RRLC). Biochemical inhibitors of the respective ABC-transporters (CSA (1μM), verapamil (Vera-10μM), MK571 (10μM), KO143 (500nM) served as positive controls. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), Dox (100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assessment of sensitivity to the drug combinations listed above were carried out in KG-1 cells, and its more immature variant KG-1a and in ex vivo CD34+ marrow cells from AML patients (AML post MDS n=5, de novo AML n=5). P-gp's ATPase activity was quantified with the luminescence-based Pgp-Gloä Assay System. Surface expression of P-gp was determined by FACS analysis and total protein expression of MRP, BCRP and P-gp by immunoblot analysis. Functional relevance of signaling pathways was tested using the SRC inhibitor PP2 (10μM) and the mTOR inhibitor Rapamicin (10nM). Results: We found that I) Erlo inhibited efflux via P-gp, MRP and BCRP as demonstrated by increased intracellular retention of DioC23/Rho-123, Calcein/CDCFDA and Hoechst 33342, respectively, andby its ability to retain MTX (300nM) and Dox (200nM) intracellularly II) Inhibition of drug efflux was higher in KG-1 than in KG-1a cellss, in agreement with a lower expression of P-gp and BCRP on KG-1a as compared to KG-1 cells; III) Quantification of VP-16 by RRLC after incubation with or without Erlo showed the ability of Erlo to increase intracellular VP-16 contents by approximately 60%; IV) Erlo increased ATPase activity in a dose-dependant manner, supporting the notion that Erlo is a competitive inhibitor of P-gp; IV) Erlo combined to VP-16 induced synergistic effects on apoptosis in KG-1 cells, and to a lesser extent in KG-1a (48h KG-1: Erlo 20%, VP-16 38%, Erlo+VP16 78%, KG-1a 48h: Erlo 10%, VP-16: 12%, Erlo+VP16: 35%); V) 48h of incubation with Erlo reduced cell surface expression of P-gp in KG-1 cells by 50%, whereas total P-gp protein expression remained unchanged, suggesting that Erlo interferes exclusively with the protein form expressed on the cell surface, VI) Decrease of P-gp cell surface expression was recapitulated upon incubation with PP2 (10μM) or Rapamicin (10nM); VII) the combination of Erlo+VP-16 in 10 AML-patient samples induced synergistic effects on apoptosis in 5 of them and additive effects in 3 of them. Conclusions: We here confirm that Erlo increases sensitivity towards chemotherapeutic agents subjected to drug efflux via ABC-transporters and delineate the molecular pathways conveying these effects. Disclosures: Fenaux: Celgene: Honoraria, Research Funding.

MTH1 Inhibitor-Induced Cytotoxicity in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1273-1273
Author(s):  
Mithun Vinod Shah ◽  
Karen S. Flatten ◽  
B. Douglas Smith ◽  
Allan D. Hess ◽  
Scott H. Kaufmann
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Myeloid Cell ◽  
Vehicle Control ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML. Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known. METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control. With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control. RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing. DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Disclosures No relevant conflicts of interest to declare.

scholarly journals Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3699
Author(s):  
Marya Kozinova ◽  
Shalina Joshi ◽  
Shuai Ye ◽  
Martin G. Belinsky ◽  
Dinara Sharipova ◽  
...  
Keyword(s):  
Cell Death ◽  
Gastrointestinal Stromal Tumor ◽  
Cell Lines ◽  
Synergistic Effects ◽  
Single Agent ◽  
Xenograft Model ◽  
Stromal Tumor ◽  
In Vivo Studies ◽  
Preclinical Model ◽  
Kit Mutation

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

scholarly journals 2',3'-Dideoxyadenosine is selectively toxic for TdT-positive cells

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1601-1608
Author(s):  
Z Spigelman ◽  
R Duff ◽  
GP Beardsley ◽  
S Broder ◽  
D Cooney ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Ex Vivo ◽  
Exposure Period ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Parental Line ◽  
Continuous Exposure ◽  
Hiv Replication

The 2′,3′-dideoxynucleosides (ddNs) are currently undergoing clinical evaluation as antiretroviral agents in HIV-infected individuals. When phosphorylated, the ddNs (ddNTPs) function as chain-terminating substrate analogues with reverse transcriptase, thereby inhibiting HIV replication. These nucleoside analogues can also inhibit, by chain- terminating additions, the primitive lymphoid DNA polymerase, terminal deoxynucleotidyl transferase (TdT). To determine the effect of possible intracellular chain-terminating additions of ddNMPs by TdT, we exposed a series of TdT-positive and TdT-negative cell lines to 2′,3′- dideoxyadenosine (ddA), a representative ddN. At ddA concentrations 25- fold higher than required for inhibition of HIV replication, progressive dose-related cytotoxicity was observed in the TdT-positive cell lines. This was accentuated by the adenosine deaminase inhibitor Coformycin (CF), presumably by enhancing the intracellular generation of ddATP from ddA. A central role of TdT in mediating the ddA/CF cytotoxicity was suggested by studies in a pre-B-cell line rendered TdT positive by infection with a TdT cDNA-containing retroviral vector. After a 48-hour continuous exposure period to 250 mumol/L ddA and 30 mumol/L CF, 30% cell death was observed in the TdT-negative parental line, whereas 90% cell death was observed in the TdT-positive daughter line. Exposure of fresh TdT-positive leukemic cells to ddA/CF for 72 hours ex vivo resulted in cytotoxicity (six cases of acute lymphocytic leukemia [ALL]) while not affecting TdT-negative acute leukemic cells (six cases). We conclude that ddA/CF selectively damages TdT-positive cells, presumably by chain-terminating additions of ddAMP, and that this may have therapeutic relevance in TdT-positive malignant disease.

scholarly journals Upstream Regulatory Role for XIAP in Receptor-Mediated Apoptosis

2004 ◽  
Vol 24 (16) ◽  
pp. 7003-7014 ◽  
Author(s):  
John C. Wilkinson ◽  
Enrique Cepero ◽  
Lawrence H. Boise ◽  
Colin S. Duckett
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Death Receptor ◽  
Full Length ◽  
Proliferative Capacity ◽  
Term Survival ◽  
Truncation Mutant ◽  
Short And Long Term ◽  
Induced Apoptosis

ABSTRACT X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-xL. Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-xL-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.

Distinct Apoptotic Responses Imparted by c-myc and max

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1003-1010 ◽  
Author(s):  
Chadd E. Nesbit ◽  
Saijun Fan ◽  
Hong Zhang ◽  
Edward V. Prochownik
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Drug Induced ◽  
Enhanced Sensitivity ◽  
Contrast Enhanced ◽  
Apoptotic Pathways ◽  
American Society ◽  
Induced Apoptosis ◽  
Cytokine Deprivation

Abstract The c-myc oncoprotein accelerates programmed cell death (apoptosis) after growth factor deprivation or pharmacological insult in many cell lines. We have shown that max, the obligate c-myc heterodimeric partner protein, also promotes apoptosis after serum withdrawal in NIH3T3 fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. We now show that c-myc– and max-overexpressing 32D cells differ in the nature of their apoptotic responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin (Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max overexpression increases sensitivity only to Camptothecin. Drug treatment of c-myc–overexpressing cells in the absence of IL-3 did not alter the spectrum of drug sensitivity other than to additively accelerate cell death. In contrast, enhanced sensitivity to Adriamycin, Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was revealed in max-overexpressing cells concurrently deprived of IL-3. Differential rates of apoptosis were not strictly correlated with the ability of the drugs to promote G1 or G2/M arrest. Ectopic expression of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc–overexpressing cells. These results provide mechanistic underpinnings for the idea that c-myc and max modulate distinct apoptotic pathways. © 1998 by The American Society of Hematology.

scholarly journals Involvement of Glutathione as a Mechanism of Indirect Protection against Spontaneous Ex Vivo Apoptosis Associated with Bovine Leukemia Virus

2004 ◽  
Vol 78 (12) ◽  
pp. 6180-6189 ◽  
Author(s):  
Teresa Sanchez Alcaraz ◽  
Pierre Kerkhofs ◽  
Michal Reichert ◽  
Richard Kettmann ◽  
Luc Willems
Keyword(s):  
Cell Death ◽  
B Lymphocytes ◽  
Bovine Leukemia Virus ◽  
Mononuclear Cells ◽  
Phorbol Esters ◽  
Ex Vivo ◽  
Leukemia Virus ◽  
Infected Sheep ◽  
Host Cells ◽  
Spontaneous Apoptosis

ABSTRACT Viruses have developed strategies to counteract the apoptotic response of the infected host cells. Modulation of apoptosis is also thought to be a major component of viral persistence and progression to leukemia induced by retroviruses like human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Here, we analyzed the mechanism of ex vivo apoptosis occurring after isolation of peripheral blood mononuclear cells from BLV-infected sheep. We show that spontaneous apoptosis of ovine B lymphocytes requires at least in part a caspase 8-dependent pathway regardless of viral infection. Cell death is independent of cytotoxic response and does not involve the tumor necrosis factor alpha/NF-κB/nitric oxide synthase/cyclooxygenase pathway. In contrast, pharmaceutical depletion of reduced glutathione (namely, γ-glutamyl-l-cysteinyl-glycine [GSH]) by using ethacrynic acid or 1-pyrrolidinecarbodithioic acid specifically reverts inhibition of spontaneous apoptosis conferred indirectly by protective BLV-conditioned media; inversely, exogenously provided membrane-permeable GSH-monoethyl ester restores cell viability in B lymphocytes of BLV-infected sheep. Most importantly, intracellular GSH levels correlate with virus-associated protection against apoptosis but not with general inhibition of cell death induced by polyclonal activators, such as phorbol esters and ionomycin. Finally, inhibition of apoptosis does not correlate with the activities of GSH peroxidase and GSH reductase. In summary, our data fit into a model in which modulation of the glutathione system is a key event involved in indirect inhibition of apoptosis associated with BLV. These observations could have decisive effects during therapeutic treatment of δ-retroviral pathogenesis.

scholarly journals Side-by-side comparison of BH3-mimetics identifies MCL-1 as a key therapeutic target in AML

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Larissa Ewald ◽  
Jessica Dittmann ◽  
Meike Vogler ◽  
Simone Fulda
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Treatment Options ◽  
B Cell Lymphoma ◽  
Bh3 Mimetics ◽  
Domain 3 ◽  
Functional Relevance ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Agents

AbstractDespite advances in the treatment of acute myeloid leukemia (AML), prognosis of AML patients is still dismal and better treatment options are required. B-cell Lymphoma 2 (BCL-2) homology domain 3 (BH3)-mimetics are emerging as a novel class of apoptosis-inducing agents that are currently being tested for the treatment of different hematological malignancies including AML. Particularly, the selective BCL-2 inhibitor ABT-199/Venetoclax is demonstrating clinical responses and has recently been approved in combination for the treatment of AML. Compounds targeting the related protein MCL-1 have recently entered clinical trials, highlighting the urgency to compare the different BH3-mimetics and identify the most promising antiapoptotic target in AML. We performed a side-by-side comparison of different highly selective and potent BH3-mimetics targeting BCL-2 (ABT-199), MCL-1 (S63845) or BCL-xL (A1331852) in a panel of AML cell lines and primary patient cells. Gene knockdown using siRNAs was utilized to investigate the functional relevance of BCL-2 proteins. Western blotting and immunoprecipitations were used to explore the influence of BH3-mimetics on interactions between pro- and antiapoptotic BCL-2 proteins. A1331852 induced apoptosis only in selected cases, indicating that BCL-xL is not a very promising therapeutic target in AML. However, S63845 displayed higher potency than ABT-199, with more cell lines and primary cells responding to S63845 than to ABT-199. MCL-1 dependency in AML cells was confirmed by siRNA-mediated knockdown of MCL-1, which was sufficient to induce apoptosis. S63845-induced cell death was accompanied by a displacement of the BH3-only protein BIM as well as BAK, resulting in BAK-dependent apoptosis. In contrast, ABT-199-induced cell death was mediated by BAX rather than BAK, indicating distinct non-redundant molecular functions of BCL-2 and MCL-1 in AML. Our study reveals that MCL-1 may be a more prevalent therapeutic target than BCL-2 in AML and identifies BIM and BAK as important mediators of S63845-induced apoptosis in AML.

scholarly journals Cross-Resistance of CD95- and Drug-Induced Apoptosis as a Consequence of Deficient Activation of Caspases (ICE/Ced-3 Proteases)

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3118-3129 ◽  
Author(s):  
Marek Los ◽  
Ingrid Herr ◽  
Claudia Friesen ◽  
Simone Fulda ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Cell Death ◽  
Drug Treatment ◽  
Tumor Cells ◽  
Anticancer Drugs ◽  
Ex Vivo ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Strong Increase ◽  
Drug Induced ◽  
Induced Apoptosis

Abstract The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. We report here that tumor cells resistant to CD95 (APO-1/Fas) -mediated apoptosis were cross-resistant to apoptosis-induced by anticancer drugs. Apoptosis induced in tumor cells by cytarabine, doxorubicin, and methotrexate required the activation of ICE/Ced-3 proteases (caspases), similarly to the CD95 system. After drug treatment, a strong increase of caspase activity was found that preceded cell death. Drug-induced activation of caspases was also found in ex vivo-derived T-cell leukemia cells. Resistance to cell death was conferred by a peptide caspase inhibitor and CrmA, a poxvirus-derived serpin. The peptide inhibitor was effective even if added several hours after drug treatment, indicating a direct involvement of caspases in the execution and not in the trigger phase of drug action. Drug-induced apoptosis was also strongly inhibited by antisense approaches targeting caspase-1 and -3, indicating that several members of this protease family were involved. CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis. Our data strongly support the concept that sensitivity for drug-induced cell death depends on intact apoptosis pathways leading to activation of caspases. The identification of defects in caspase activation may provide molecular targets to overcome drug resistance in tumor cells.

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