Establishment and Characterization of Bortezomib-Resistant Myeloma Cell Lines: A Possible Role of Mutated Proteasome β5 Subunit in Preventing the Accumulation of Unfolded Protein, Which Is Otherwise Followed by Catastrophic Endoplasmic Reticulum (ER) Stress.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3772-3772
Author(s):  
Masaki Ri ◽  
Shinsuke Iida ◽  
Takayuki Nakashima ◽  
Hideyuki Miyazaki ◽  
Fumiko Mori ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Point Mutation ◽  
Cell Lines ◽  
Binding Pocket ◽  
Protein Accumulation ◽  
Misfolded Protein ◽  
Unique Point ◽  
Resistant Cells

Abstract Abstract 3772 Poster Board III-708 [Purpose] Bortezomib (BTZ), a proteasome inhibitor, has been introduced into the treatment of multiple myeloma (MM). It shows remarkable response against both relapsed/refractory and newly diagnosed MM. However, it is often encountered that BTZ treatment achieves very short duration of response and permits early drug resistance. Therefore, understanding the mechanisms underlying this drug resistance is necessary to develop novel treatments to overcome this problem. [Materials & Methods] We established two stable BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, whose IC50 values were respectively 24.7- and 16.6-fold higher than their parental cell lines, under continuous exposure to BTZ. Using these resistant cells, we investigated on their proteasome activity, the alteration of proteasome β5 subunit (PSMB5) gene, misfolded protein accumulation, endoplasmic reticulum (ER) stress, and apoptosis signals including BH3 only proteins in comparison with their parental cells at clinically achievable concentration of BTZ treatment. [Results & Discussion] No activation of caspase -3,-8, and -9 and BH3 only protein, Noxa, which were initially up-regulated in BTZ-treated cells, were noted in BTZ-resistant cells even in the presence of BTZ. These results indicate avoidance of fatal intracellular stress may block transcriptional activation of Noxa in resistant cells at an early phase after BTZ exposure. In gel shift assay detecting NF-kB-DNA complexes, BTZ-resistant cells maintained constitutive NF-kB activation, whereas their parental cells lost its activity in the presence of BTZ. In addition, cellular proteasome activities including chymotrypsin-like and caspase-like activity were markedly inhibited by BTZ treatment in parental cells, and moderately also in BTZ-resistant cells, when detected by fluorogenic substrates specific for each proteasome activity. While time-dependent accumulation of ubiquitinated proteins was prevented only in BTZ-resistant cells, but not in their parental cells after BTZ exposure. Resistance was partly explained by the presence of a unique point mutation, G322A, in the gene encoding PSMB5 in both BTZ-resistant cell lines, which substituted Thr for Ala at the codon 49 in amino acid level. This constitution has been reported to gives rise to the conformational change of BTZ-binding pocket in β5 subunit, which results in partial disruption of the contact between BTZ and chymotrypsin-like active site. Furthermore, BTZ-resistant and parental MM cells had nearly equal expression of cytoplasmic and ER chaperons, however, only BTZ-resistant cells could prevent misfolded protein accumulation and therefore avoid fatal ER stress represented as activation of CHOP and of caspase-4, -12 after BTZ treatment. [Conclusion] Two kinds of stable BTZ-resistant MM cell lines were established, which acquired the unique point mutation (G322A) in BTZ-binding pocket of PSMB5, prevented the accumulation of misfolded proteins probably via reduced affinity of 26S proteasome to BTZ and avoided the development of catastrophic ER stress unlike their parental cells. These cell lines will provide better understanding of the underlying mechanisms of the BTZ-resistance, and will lead to the development of novel treatment strategies for overcoming BTZ-resistance in the patients with MM. Disclosures: Iida: JANSSEN PHARMACEUTICAL: Honoraria; KYOWA KIRIN: Research Funding. Nakashima:KYOWA KIRIN: Employment. Miyazaki:KYOWA KIRIN: Employment. Shiotsu:KYOWA KIRIN: Employment.

scholarly journals Arginine Depletion in Combination with Canavanine Supplementation Induces Massive Cell Death in Myeloma Cells By Interfering with Their Protein Metabolism and Bypassing Potential Rescue Mechanisms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3205-3205
Author(s):  
Johannes Windschmitt ◽  
Björn Jacobi ◽  
Yagmur Bülbül ◽  
Lilli Sester ◽  
Janine Tappe ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Er Stress ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Protein Translation ◽  
Protein Accumulation ◽  
Misfolded Protein ◽  
Myeloma Cells ◽  
Aggresome Formation

Abstract Introduction Although the therapeutic armamentarium against multiple myeloma has tremendously increased in recent years, it still remains an incurable disease. A highly promising novel anti-tumoral treatment strategy is to target specific non-redundant metabolic achilles heels of individual cancer entities. The semi-essential amino acid arginine can be synthesized from citrulline in most physiological tissues due to expression of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1). Various tumor entities do not express ASS1, therefore depend on the exogenous availability of arginine and pharmacological approaches to systemically deplete arginine are in phase I-III clinical development for such arginine-auxotrophic cancers. Cell death induction by arginine depletion can be dramatically enhanced by co-application of the arginine analogue canavanine. Canavanine can be used by the respective aminoacyl tRNA synthetase instead of arginine during protein translation and this leads to a highly toxic intracellular accumulation of misfolded proteins. In preliminary work we have seen that myeloma cells are largely arginine-auxotrophic and can be killed by arginine depletion and canavanine supplementation within hours, while ASS1 expressing cells are completely protected by their endogenous arginine rescue capability. Encouraging results of tumor control have already been seen in a murine myeloma model. Methods Human myeloma cell lines (NCI-H929_A2 and FD50, developed in our laboratory) were cultured and treated in RPMI-1640 medium with or without arginine. Protein levels were determinded by western blot analysis. Cell viability was measured by propidium iodide staining and flow cytometry analysis. RNA quantification was done by qRT-PCR. For autophagosome and aggresome quantification we used immunofluorescence staining (IF) and laser scanning microscopy (LSM). Results Arginine depletion and canavanine supplementation led to misfolded protein accumulation which was followed by massive apoptotic cell death. Both processes were further enhanced by co-treatment with the proteasome inhibitor bortezomib, indicated by an increase in intracellular polyubiquitinated proteins as well as higher cleaved caspase 3 levels and propidium-iodide positive cells after only 8-12 h in both tested cell lines. Unexpectedly, the endoplasmic reticulum (ER)-stress response was activated only very moderately. Expression of CHOP, a pro-apoptotic transcription factor that is highly translated under toxic ER stress, was not altered compared to control conditions. Tunicamycin-mediated induction of enhanced ER stress significantly improved the viability of arginine-starved and canavanine treated cells. This suggests that protein accumulation mainly takes place in the cytoplasm rather than the ER and tunicamycin might alleviate cell death by reduction of total protein translation. Despite severe arginine deficiency and induction of misfolded protein stress, the cells were not able to respond by an adequate upregulation of macroautophagy, as determined by an altered LC3 metabolism. The autophagic flux was significantly reduced compared to control conditions after 4-8 h of treatment. There was a strong induction of BAG3 and p62 proteins, which are both associated with chaperone-assisted autophagy as well as aggresome formation and are normally cleared via macroautophagy. Cytoplasmic aggresome formation was not detectable until onset of apoptosis. Also, no relevant modulation of phosphorylation of the autophagy inducer mTORC and the downstream kinase p70S6K1 was noted upon arginine depletion and canavanine co-treatment. Finally, ER stress induction via tunicamycin did not improve autophagic protein turnover, as determined by IF staining, LSM and western blot. Conclusions Arginine starvation in combination with canavanine supplementation induces fast and highly efficient cell death in arginine-auxotrophic myeloma cells. This novel strategy interferes with myeloma cellular metabolism by induction of misfolded protein accumulation. A relevant upregulation of potentially protective cellular strategies like ER stress responses, aggresome formation and autophagy are either not detectable or they remain insufficient. We hypothesize that our novel metabolic anti-tumor strategy is either too potent or too fast for the tumor cells to cope with its consequences. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of apoptosis may lead to the development of bortezomib resistance in multiple myeloma cancer cells

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Emine Öksüzoğlu ◽  
Gül Kozalak
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Expression Levels ◽  
Diphenyltetrazolium Bromide ◽  
Apoptotic Pathways ◽  
Resistant Cells

AbstractBackgroundMultiple myeloma (MM), a malignancy of plasma cells, is the second most prevalent hematological cancer. Bortezomib is the most effective chemotherapeutic drug used in treatment. However, drug-resistance prevents success of chemotherapy. One of the factors causing drug-resistance is dysfunction of apoptotic-pathways. This study aimed to evaluate the relationship between expression levels of Bcl-2, Bax, caspase-3 and p-53 genes involved in apoptosis and the development of bortezomib-resistance in MM cell lines.Materials and methodsMultiple myeloma KMS20 (bortezomib-resistant) and KMS28 (bortezomib-sensitive) cell lines were used. 3-[4,5-Dimethylthiazol-2-yl] 1-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine IC50 values of bortezomib. RNAs were isolated from bortezomib-treated cell lines, followed by cDNA synthesis. Expression levels of the genes were analyzed by using q-Realtime-PCR.ResultsAs a result, Bcl-2/Bax ratio was higher in KMS20 (resistant) cells than in KMS28 (sensitive) cells. Expression of caspase-3 decreased in KMS20-cells, whereas increased in KMS28-cells. The results indicate that apoptosis was suppressed in resistant cells.ConclusionThese findings will enable us to understand the molecular mechanisms leading to drug-resistance in MM cells and to develop new methods to prevent the resistance. Consequently, preventing the development of bortezomib resistance by eliminating the factors which suppress apoptosis may be a new hope for MM treatment.

Activation of the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) in Aggressive B-Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2038-2038
Author(s):  
Olga Balague ◽  
Luis Colomo ◽  
Armando Lopez-Guillermo ◽  
Elias Campo ◽  
Antonio Martinez
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factors ◽  
Western Blot ◽  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Lymphoid Tissues ◽  
B Cell Lymphomas

Abstract BACKGROUND The UPR is a prosurvival pathway activated in cells under ER stress induced by the accumulation of unfolded proteins. UPR activation in B cells normally occurs during the differentiation to antibody secreting plasma cells and requires XBP1activation. XBP-1 is a member of the TREB family of transcription factors that exists in the endoplasmic reticulum (ER) as a 33kDa protein, and in the nucleus as an active 50kDa transcription factor. The UPR stimulates two different ER proteins, ATF-6 and Ire-1, to increase XBP-1 transcription and XBP-1 mRNA splicing resulting in the accumulation of the active 50kDa nuclear protein. Moreover XBP1 is a target of proteosome inhibitors and is related to the aggressive behaviour of some carcinomas. The role of the activation of XBP-1 in lymphomas is still unknown. DESIGN: Reactive lymphoid tissues and 25 neoplastic human B-cell lines representing different stages of B-cell development were studied for XBP-1 expression by western blot and XBP-1, PAX-5, Blimp-1/prdm1, MUM-1/IRF-4 and ICSBP1/IRF-8 by immunohistochemistry. XBP-1 activation was assessed in 225 B-cell lymphomas from the archives of the laboratory of pathology by western blot, RT-PCR and immunohistochemistry . To further evaluate whether XBP-1 activation was related to the plasmacytic program or to ER stress signals we analyzed the cell lines by Western blot for XBP-1 and ATF-6 expression. RESULTS We characterize XBP-1 expression in reactive lymphoid tissues, 25 human cell lines and 225 B-cell tumors. In nearly all tonsillar lymphoid cells XBP-1 was detected as a cytoplasmic protein with a paranuclear dot pattern. Nuclear positivity was observed only in scattered centrocytes in the light zone of the germinal centers and in plasma cells, always coexpressed with plasma cell related transcription factors as MUM-1/IRF-4 and Blimp1/prdm1. Active p50XBP-1 was found in 24/25 cell lines by western blot regardless ATF-6 expression and confirmed by immunohistochemistry . Moreover p50XBP1 was found in 27/31(87%) plasmacytomas, 36/64(56%) DLBCL-ABC and in 3/10(30%) DLBCL-GCB and 22/43(51%) plasmablastic lymphomas. Intriguingly, p50XBP1 was detected also in 2/11(18%)BL and 4/25(16%)MCL with blastic features. CONCLUSIONS.XBP-1 is activated in a subset of follicular centre cells committed to plasma cell differentiation and in plasma cells.UPR prosurvival pathways in the neoplastic cell lines are activated independently of the extent of the ATF-6 activation.p50XBP1 is mostly activated in aggressive B-cell lymphomas regardless to the plasmacytic differentiation of the tumours. Thus, p50XBP-1 may be a new molecular target in the treatment of aggressive B-cell malignancies.

scholarly journals Induction of Paraptotic Cell Death in Breast Cancer Cells by a Novel Pyrazolo[3,4-h]quinoline Derivative through ROS Production and Endoplasmic Reticulum Stress

Antioxidants ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 117
Author(s):  
Phuong Linh Nguyen ◽  
Chang Hoon Lee ◽  
Heesoon Lee ◽  
Jungsook Cho
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Quinoline Derivative ◽  
Ros Generation ◽  
Protein Accumulation ◽  
Potential Candidate ◽  
Development Of Resistance

Chemotherapy has been a standard intervention for a variety of cancers to impede tumor growth, mainly by inducing apoptosis. However, development of resistance to this regimen has led to a growing interest and demand for drugs targeting alternative cell death modes, such as paraptosis. Here, we designed and synthesized a novel derivative of a pyrazolo[3,4-h]quinoline scaffold (YRL1091), evaluated its cytotoxic effect, and elucidated the underlying molecular mechanisms of cell death in MDA-MB-231 and MCF-7 breast cancer (BC) cells. We found that YRL1091 induced cytotoxicity in these cells with numerous cytoplasmic vacuoles, one of the distinct characteristics of paraptosis. YRL1091-treated BC cells displayed several other distinguishing features of paraptosis, excluding autophagy or apoptosis. Briefly, YRL1091-induced cell death was associated with upregulation of microtubule-associated protein 1 light chain 3B, downregulation of multifunctional adapter protein Alix, and activation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase. Furthermore, the production of reactive oxygen species (ROS) and newly synthesized proteins were also observed, subsequently causing ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress. Collectively, these results indicate that YRL1091 induces paraptosis in BC cells through ROS generation and ER stress. Therefore, YRL1091 can serve as a potential candidate for the development of a novel anticancer drug triggering paraptosis, which may provide benefit for the treatment of cancers resistant to conventional chemotherapy.

scholarly journals Macrolide Antibiotics Block Autophagy Flux and Sensitize to Bortezomib Via Endoplasmic Reticulum-Stress-Mediated CHOP Induction in Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4992-4992
Author(s):  
Shota Moriya ◽  
Xiao-Fang Che ◽  
Seiichiro Komatsu ◽  
Akihisa Abe ◽  
Tomohiro Kawaguchi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Lines ◽  
Macrolide Antibiotic ◽  
Combined Treatment ◽  
Ubiquitin Proteasome System ◽  
Macrolide Antibiotics ◽  
Autophagy Flux ◽  
Selective Degradation ◽  
Ubiquitin Proteasome

Abstract Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved cellular process of self-degradation in eukaryotes. Intracellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intracellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) cell lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM cell lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins, and induced the proapoptotic transcription factor CHOP. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. BIM, BAX, DR5, and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and up-regulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER-stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chloroquine induces apoptosis in pancreatic neuroendocrine neoplasms via endoplasmic reticulum stress

2020 ◽  
Vol 27 (7) ◽  
pp. 431-439
Author(s):  
Kenzo Nakano ◽  
Toshihiko Masui ◽  
Akitada Yogo ◽  
Yuichiro Uchida ◽  
Asahi Sato ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Tumour Size ◽  
Neuroendocrine Neoplasms ◽  
Low Grade ◽  
Pancreatic Neuroendocrine Neoplasms ◽  
Dismal Prognosis ◽  
Well Differentiated

Although pancreatic neuroendocrine neoplasms (PanNENs) are generally indolent, patients with distant metastasis have a dismal prognosis. Recently, the autophagy inhibitor chloroquine (CQ) has been shown to suppress the tumour growth of PanNENs, but the detailed mechanisms have not been elucidated. Furthermore, these results were obtained from poorly differentiated cell lines rather than well-differentiated cell lines, which is the most prevalent type in this tumour. To explore the mechanism and efficacy of CQ on PanNENs, we applied CQ to cell lines and evaluated the resulting apoptosis and endoplasmic reticulum (ER) stress. CQ treatment induced ER stress, and an unfolded protein response was activated through the PERK-eIF2α-ATF4 pathway, resulting in the expression of the pro-apoptotic protein C/EBP homologous protein (CHOP), which reflects ER-stress-mediated apoptotic cell death. Furthermore, hydroxychloroquine (HCQ) was effective in Men1 heterozygous-deficient (Men1+/ΔN3-8) mice, a mouse PanNEN model that is considered to correspond to human low-grade PanNEN. HCQ administration decreased tumour size in Men1+/ΔN3-8 mice. In the HCQ group, histological analyses revealed that proliferative activity was unchanged, but apoptosis was accelerated, accompanied by CHOP expression. These results suggest that autophagy inhibition by CQ/HCQ could be used for the treatment of PanNEN, including the well-differentiated type.

scholarly journals MLN4924, a Protein Neddylation Inhibitor, Suppresses the Growth of Human Chondrosarcoma through Inhibiting Cell Proliferation and Inducing Endoplasmic Reticulum Stress-Related Apoptosis

2018 ◽  
Vol 20 (1) ◽  
pp. 72 ◽  
Author(s):  
Meng-Huang Wu ◽  
Ching-Yu Lee ◽  
Tsung-Jen Huang ◽  
Kuo-Yuan Huang ◽  
Chih-Hsin Tang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Mouse Model ◽  
Cell Viability ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Cycles ◽  
Chondrosarcoma Cell ◽  
Xenograft Mouse Model ◽  
Human Chondrosarcoma

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stress–related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stress–related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stress–related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma.

A GPI-Anchored Co-Receptor for TFPI Controls Its Intracellular Trafficking and Endothelial Surface Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1927-1927
Author(s):  
Anna C. Cunningham ◽  
Charles M. Mansbach ◽  
Alan E. Mast
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Tissue Factor ◽  
Cell Lines ◽  
Surface Expression ◽  
Resistant Cell ◽  
Wild Type ◽  
Endothelial Surface ◽  
Cellular Processing ◽  
Resistant Cells

Abstract Tissue factor pathway inhibitor (TFPI) is the major endogenous inhibitor of tissue factor initiated blood coagulation and a is key regulator of the development of intravascular thrombosis. TFPI indirectly binds to the endothelial surface through tight association with a GPI-anchored co-receptor. The location of recombinant TFPI expression in mammalian cells varies depending on the cell line used. In cell lines that do not produce endogenous TFPI, CHO and HEK293 cells, recombinant TFPI is secreted into the culture media. However, in a cell line that produces endogenous surface TFPI, EaHy926 cells, recombinant TFPI is expressed on the cell surface. These data suggest that TFPI is expressed on the surface of cells that produce the GPI-anchored co-receptor and is secreted by cells that do not. To further investigate the function of the co-receptor in TFPI cellular trafficking we developed aerolysin resistant ECV304 and EaHy926 cells lines. Both of these cell lines produce endogenous, surface associated TFPI. The cell lines were mutated with ethyl methanesulfonate and selected with aerolysin. Mutant cells lacking surface GPI-anchored proteins are resistant to the toxic effects of aerolysin and survive. The morphology and growth rate of the two aerolysin resistant cell lines are identical to that of the wild-type cells. They were first characterized to rule out the presence of a mutation that could directly alter cellular metabolism of TFPI. Sequencing of TFPI cDNA indicates that no mutations are present in the TFPI exons. Analysis of mRNA by real time PCR demonstrates that the aerolysin resistant cells make similar amounts of TFPI mRNA as their wild-type counterparts. Thus, transcription and translation of TFPI appear identical to the wild-type cells. In addition, the two independently derived cell lines have very similar phenotypes, as described below, indicating that the aerolysin resistant cell lines have a defect in GPI-anchor biosynthesis but not additional random mutations that could alter cellular processing of TFPI. Characterization of protein expression by flow cytometry indicates that the aerolysin resistant cell lines do not express GPI-anchored proteins (CD59, uPAR) or TFPI on the cell surface but do have wild-type surface expression of transmembrane proteins (CD9, tissue factor). Interestingly, instead of being secreted, western blot analysis of cellular lysates indicates that TFPI is degraded within the aerolysin resistant cells in a manner similar to that observed for GPI-anchored proteins. Intracellular degradation of TFPI is prevented by brefeldin A indicating that degradation takes place in a post endoplasmic reticulum compartment. Pepstatin A, but not MG-132, also prevents degradation, indicating that degradation is lysosomal rather than proteosomal. It appears that binding of TFPI to its co-receptor occurs early in cellular processing, likely within the endoplasmic reticulum. Cellular trafficking of TFPI is controlled by its co-receptor, which has not yet been identified. The co-receptor directs TFPI to the cell surface in wild-type endothelial cells or to be degraded in aerolysin resistant cells. In the absence if its co-receptor TFPI is secreted. Therefore, regulation of co-receptor expression provides a mechanism for the production of cell associated TFPI, as occurs in endothelial cells, versus soluble TFPI, as may occur in megakaryocytes/platelets.

Bortezomib Induces an Antioxidant and ER-Stress Response Gene Expression Signature in Mantle Cell Lymphoma: Implications for Response Prediction and Optimized Chemotherapy Regimens.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 830-830
Author(s):  
Edgar G. Rizzatti ◽  
Helena Mora-Jensen ◽  
Raymond Lai ◽  
Masanori Daibata ◽  
Therese White ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Er Stress ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Er Stress Response ◽  
Mantle Cell ◽  
Sensitive Group ◽  
Resistant Cells

Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell lymphoma for which new treatment options are needed. Recent phase II clinical trials reported response to the proteasome inhibitor bortezomib (BZM) in up to 50% of pre-treated patients. Despite the successful use of BZM in the clinic, the precise molecular mechanisms underlying sensitivity or resistance to BZM in MCL remain largely unknown. To address this issue, we used U133A 2.0 microarrays to analyze gene expression in MCL cells from peripheral blood of 5 patients with previously untreated leukemic MCL. Samples were collected immediately before (0h) and at 3, 6, 24, and 72 hours after administration of BZM (1.5 mg/m2). After the blood collection at 72 hours, a second dose of BZM was given, and cells were collected 24 hours later. Two patients had major reductions in peripheral ALC already at 24h from dose 2 and normalized their blood counts by day 21 (sensitive), 1 patient had no change over a full course of 4 injections (resistant), and 2 patients had some decrease in ALC (intermediate). Genes differentially expressed with treatment were ranked according to the degree of correlation with time (Pearson). We used gene set enrichment analysis (GSEA) to detect distinct functional gene expression signatures; the most consistently up-regulated of which was a signature composed by proteasome and chaperone genes. To confirm and expand these findings, we exposed 10 MCL cell lines (7 sensitive, IC50<10nM; 3 resistant IC50>10nM) to 10nM of BZM and analyzed gene expression at 1, 3, 6 and 24 hours. The proteasome signature was again dominant, and the majority of the up-regulated genes in both clinical and cell line samples shared binding motifs for the NRF, MAF, ATF and HSF families of transcription factors (TF). Thus genes up-regulated by BZM in vivo and in cell lines predominantly belonged to a functional response to oxidative and/or endoplasmic reticulum (ER) stress. Under physiologic conditions, this is thought to help restore homeostasis and protect from apoptosis. This response could therefore contribute to drug resistance or be a marker of an overwhelming insult before the cells undergo apoptosis. To address this issue, we investigated differences in response to BZM between sensitive and resistant cell lines. The proteasome signature was more strongly up-regulated in sensitive cells than in resistant cells, and the ER-stress response as measured by genes controlled by the NRF and MAF family of TFs was also more highly expressed in the sensitive group. Consistently, expression of HMOX1, which encodes a key enzyme in the antioxidant response, was increased by 32× at 24h in the sensitive group, but only by 4× in the resistant group; the expression of DDIT3, a transcription factor implicated in a pro-apoptotic response to ER-stress was 5.5-fold up-regulated in the sensitive cells but only 1.4-fold in the resistant cells. We conclude that in sensitive cells BZM induces an overwhelming ER-stress response with high expression of proteasome components and chaperone proteins that could serve as a predictor of response to BZM.

A New Form Of Therapeutic Resistance: Drug Glucuronidation Regulated By The Sonic Hedgehog Factor Gli1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 821-821
Author(s):  
Hiba A Zahreddine ◽  
Biljana Culjkovic-Kraljacic ◽  
Sarit Assouline ◽  
Abdellatif Amri ◽  
Patrick Gendron ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Phase Ii ◽  
Resistant Cell ◽  
Type I ◽  
Type Ii ◽  
Protein Levels ◽  
Gli 1 ◽  
Type Ii Resistance ◽  
Resistant Cells

Abstract Despite many recent successes in the treatment of cancer, the development of chemoresistance in many of the initially responding patients, and primary resistance in others, remains a major impediment in therapy development. Our studies provide evidence for a novel mechanism underlying drug resistance: Gli1 dependent drug glucuronidation. While carrying out a Phase II clinical trial of targeting the eukaryotic translation initiation factor eIF4E with ribavirin in M4/M5 subtypes of AML, we observed that all responding patients eventually became clinically and molecularly resistant. To understand the cause of this resistance, we generated ribavirin resistant cell lines. In these models, ribavirin no longer targeted eIF4E activity or impaired growth, and importantly, the ability of ribavirin to bind eIF4E was severely impaired. However, the eIF4E gene was not mutated and its protein levels were not altered. The cell lines could be divided into two groups: type I with a defect in drug uptake and type II with a normal uptake. In type I resistant cells, we observed a substantial reduction in levels of Adenosine Kinase (ADK) an enzyme that catalyzes the rate limiting step in the metabolic activation of ribavirin allowing its retention in the cells. We used RNA Sequencing to examine the molecular underpinnings of type II resistance. Our data revealed a drastic increase in the levels of Gli1. In stably overexpressing cells, Gli1 was sufficient to produce the same resistance phenotype that we observed for type II cell models, both molecularly and at the level of cell growth. In addition, Gli1 overexpression correlated with the loss of drug-to-target interaction, as observed by our eIF4E immunoprecipitation studies using 3H-Ribavirin, similarly to the resistant cell lines. Conversely, Gli1 knockdown in type II cells or its pharmacological inhibition with the FDA approved Gli1 inhibitor GDC0449/Vismodegib, restored the eIF4E-ribavirin interaction and re-sensitized these cells to ribavirin. Our subsequent studies revealed a close correlation between Gli1 expression and the protein levels of the UGT1A glucuronosyl transferase enzymes involved in phase II drug metabolism whereby xenobiotics or metabolites are modified by the addition of a sugar, glucuronic acid. Given these findings, we examined whether the loss of the eIF4E interaction in resistant cells was due to the glucuronidation of ribavirin. Using 13C/12C ribavirin and mass spectrometry, we observed glucuronidated forms of ribavirin in resistant cells and cells overexpressing Gli1 but not in parental cells and that ribavirin is glucuronidated on its triazole ring which binds eIF4E. Treatment of cells with the Gli1 inhibitor GDC0449 reduces UGT1A levels, and correlates with reduced levels of ribavirin-glucuronides and the re-emergence of ribavirin-eIF4E complexes. We further hypothesized that the type II resistant cells could be resistant to other drugs. We observe that our ribavirin resistant cell lines are also resistant to the cornerstone of AML therapy, cytarabine. GDC0449/Vismodegib treatment reverts resistance to cytarabine in these cells. Preliminary studies indicate that these cells are also resistant to azacytidine and cisplatin. This is particularly striking as these cells were never exposed to these compounds. Thus, this could represent a novel form of multi-drug resistance. To establish the clinical relevance of our findings to patients in our AML ribavirin trial, we examined features of type I and type II resistance. Out of 10 patient samples available for evaluation, all six responding patient specimens showed elevated Gli-1 mRNA levels, up to 26 fold, upon relapse relative to levels during response. For most, the ratio of Gli1 during response relative to at relapse was about 2-4 fold with some patients up to 10 fold. For the two patients examined that did not respond, both had highly elevated Gli-1 levels prior to treatment relative to healthy individuals, and this was not lowered after 28 days of ribavirin treatment. We also noted elevated UGT1A protein levels upon relapse in our patient population. Type I resistance was observed in only two patients whereas Gli1 and UGT1A were dysregulated at relapse in all patients examined. In summary, we identified a novel form of drug resistance: Gli1 dependent drug glucuronidation. Treatment with Gli1 inhibitors appears to be a promising avenue for overcoming this form of drug resistance. Disclosures: No relevant conflicts of interest to declare.

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