A Novel Bcr-Abl Mediated Pro-Survival Pathway: Reduction of Releasable Calcium Levels in the Endoplasmic Reticulum Inhibits Calcium Dependent Apoptotic Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2621-2621
Author(s):  
Susanne Vejda ◽  
Katarzyna Piwocka ◽  
Thomas G. Cotter ◽  
Gerald C. O’Sullivan ◽  
Sharon L. McKenna
Keyword(s):  
Drug Resistance ◽  
Endoplasmic Reticulum ◽  
Disease Progression ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Transformed Cells ◽  
Apoptotic Signaling ◽  
Calcium Dependent ◽  
Transformed Cell Lines ◽  
Er Calcium

Abstract The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis (BC) and that this plays significant role in the achievement of drug resistance. We have established cell lines expressing low (C2 cells) and high levels (C4 cells) of Bcr-Abl to study the molecular mechanisms involved in disease progression especially with regard to drug resistance. Microarray analysis revealed that Bcr-Abl expressing cells exhibit increased expression of various endoplasmic reticulum (ER) resident proteins indicating that the ER is a novel target of Bcr-Abl. Recently, this organelle has been described as an important apoptotic gateway. Indeed, by measuring calcium levels (steady state and following drug treatment) in the ER and cytoplasm we could elucidate that the calcium dependent apoptotic signaling is disturbed in Bcr-Abl transformed cells. Moreover, inhibition of Bcr-Abl activity by Imatinib reversed this phenotype indicating that Bcr-Abl is directly modulating calcium homeostasis by yet unknown mechanisms. Two Bcr-Abl expressing human cell lines, K562 and BV173, also showed reduced amounts of calcium in the ER compared to the Bcr-Abl negative cell lines HL60 and Jurkat. Etoposide was chosen for apoptosis induction as this drug was classified to target not only the mitochondria (classical mitochondrial pathway) but also the ER (calcium dependent apoptotic pathway). Cell death was induced in the parental and Bcr-Abl transformed cell lines and calcium changes in the cytosol and the mitochondria were measured using organelle specific calcium probes. In C2 and C4 cells an early mitochondrial calcium overload was absent possibly due to the decreased amount of free releasable calcium in the ER. Moreover, other ER/calcium activated pathways were greatly diminished, such as cytosolic calcium elevation and calpain activation during apoptosis. It has been shown that decreased levels of releasable calcium in the ER is associated with cell survival. It is possible therefore that Bcr-Abl may exert some of its pro-survival or anti-apoptotic effects at the ER, by directly influencing the levels of releasable calcium. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium dependent apoptotic signaling is likely to be a major pro-survival mechanism in Bcr-Abl transformed cells.

Bcr-Abl reduces endoplasmic reticulum releasable calcium levels by a Bcl-2–independent mechanism and inhibits calcium-dependent apoptotic signaling

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 4003-4010 ◽  
Author(s):  
Katarzyna Piwocka ◽  
Susanne Vejda ◽  
Thomas G. Cotter ◽  
Gerald C. O'Sullivan ◽  
Sharon L. McKenna
Keyword(s):  
Drug Resistance ◽  
Endoplasmic Reticulum ◽  
Disease Progression ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
Apoptotic Signaling ◽  
Calcium Dependent ◽  
Established Cell ◽  
Apoptotic Pathways ◽  
Mitochondrial Calcium Uptake

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis and that this plays a significant role in the achievement of drug resistance. We have established cell lines expressing low and high levels of Bcr-Abl to study the molecular mechanisms involved in disease progression and drug resistance. It is now known that the endoplasmic reticulum (ER) can play a major role in the regulation of apoptosis. We therefore investigated whether Bcr-Abl expression modulates ER homeostasis and interferes with ER-mediated apoptotic pathways to promote survival. Bcr-Abl–expressing cells exhibit a decreased amount of free releasable calcium in the ER as well as a weaker capacitative calcium entry response, relative to parental cells. This effect is independent of Bcl-2, which is a known modulator of ER calcium homeostasis. The reduction in ER releasable calcium results in inhibition of the ER/mitochondria-coupling process and mitochondrial calcium uptake. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium-dependent apoptotic signaling is likely to be a major prosurvival mechanism in Bcr-Abl–expressing cells.

scholarly journals Molecular Simulations Identify Target Receptor Kinases Bound by Astaxanthin to Induce Breast Cancer Cell Apoptosis

2020 ◽  
pp. 72-82
Author(s):  
Mossa Gardaneh ◽  
Zahra Nayeri ◽  
Parvin Akbari ◽  
Mahsa Gardaneh ◽  
Hasan Tahermansouri
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Combination Therapy ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Molecular Mechanisms ◽  
Cancer Cell Lines ◽  
Cancer Drug ◽  
Breast Cancer Cell Lines

Background: We investigated molecular mechanisms behind astaxanthinmediated induction of apoptosis in breast cancer cell lines toward combination therapy against cancer drug resistance. Methods: Breast cancer cell lines were treated with serial concentrations of astaxanthin to determine its IC50. We used drug-design software to predict interactions between astaxanthin and receptor tyrosine kinases or other key gene products involved in intracellular signaling pathways. Changes in gene expression were examined using RT-PCR. The effect of astaxanthin-nanocarbons combinations on cancer cells was also evaluated. Results: Astaxanthin induced cell death in all three breast cancer cell lines was examined so that its IC50 in two HER2-amplifying lines SKBR3 and BT-474 stood, respectively, at 36 and 37 ?M; however, this figure for MCF-7 was significantly lowered to 23 ?M (P<0.05). Astaxanthin-treated SKBR3 cells showed apoptotic death upon co-staining. Our in silico examinations showed that some growth-promoting molecules are strongly bound by astaxanthin via their specific amino acid residues with their binding energy standing below -6 KCa/Mol. Next, astaxanthin was combined with either graphene oxide or carboxylated multi-walled carbon nanotube, with the latter affecting SKBR cell survival more extensively than the former (P<0.05). Finally, astaxanthin coinduced tumor suppressors p53 and PTEN but downregulated the expression of growth-inducing genes in treated cells. Conclusion: These findings indicate astaxanthin carries' multitarget antitumorigenic capacities and introduce the compound as a suitable candidate for combination therapy regimens against cancer growth and drug resistance. Development of animal models to elucidate interactions between the compound and tumor microenvironment could be a major step forward towards the inclusion of astaxanthin in cancer therapy trials.

scholarly journals Elucidating the expression and function of Numbl during cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM)

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuejiao Huang ◽  
Xianting Huang ◽  
Chun Cheng ◽  
Xiaohong Xu ◽  
Hong Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Clinical Problem ◽  
Integrin Β1 ◽  
Cell Viability Assay ◽  
Adhesion Model

Abstract Background Cell adhesion-mediated drug resistance (CAM-DR) is a major clinical problem that prevents successful treatment of multiple myeloma (MM). In particular, the expression levels of integrin β1 and its sub-cellular distribution (internalization and trafficking) are strongly associated with CAM-DR development. Methods Development of an adhesion model of established MM cell lines and detection of Numbl and Integrinβ1 expression by Western Blot analysis. The interaction between Numbl and Integrinβ1 was assessed by a co-immunoprecipitation (CO-IP) method. Calcein AM assay was performed to investigate the levels of cell adhesion. Finally, the extent of CAM-DR in myeloma cells was measured using cell viability assay and flow cytometry analysis. Results Our preliminary date suggest that Numbl is differentially expressed in a cell adhesion model of MM cell lines. In addition to binding to the phosphotyrosine-binding (PTB) domain, the carboxyl terminal of Numbl can also interact with integrin β1 to regulate the cell cycle by activating the pro-survival PI3K/AKT signaling pathway. This study intends to verify and elucidate the interaction between Numbl and integrin β1 and its functional outcome on CAM-DR. We have designed and developed a CAM-DR model using MM cells coated with either fibronectin or bone marrow stromal cells. We assessed whether Numbl influences cell-cycle progression and whether it, in turn, contributes to activation of PI3K/AKT signal pathway through the adjustment of its carboxyl end. Finally, we showed that the interaction of Numbl with integrin β1 promotes the formation of CAM-DR in MM cells. Conclusions Our findings elucidated the specific molecular mechanisms of CAM-DR induction and confirmed that Numbl is crucial for the development of CAM-DR in MM cells.

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.

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.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1772-1772 ◽  
Author(s):  
Jahangir Abdi ◽  
Yijun Yang ◽  
Patrick Meyer-Erlach ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Molecular Mechanisms ◽  
Marrow Stromal Cells ◽  
Survivin Gene ◽  
Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

Antagonizing HDAC6 Activity: A New Strategy to Maximize the Anti-Myeloma Effects of BET Inhibition

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3022-3022
Author(s):  
Jennifer S. Carew ◽  
Claudia M. Espitia ◽  
Weiguo Zhao ◽  
Valeria Visconte ◽  
Kevin R. Kelly ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Disease Progression ◽  
Cell Lines ◽  
Anticancer Agents ◽  
Caspase 3 ◽  
Hematologic Malignancy ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
The United States

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy and represents the second most common adult hematologic malignancy in the United States. MM is relatively asymptomatic during its early stages and as a result, the majority of patients have advanced disease at diagnosis. Innovations in the treatment of MM, including the development of proteasome inhibitors such as bortezomib (Velcade) have improved clinical outcomes. However, many patients fail to respond to these agents or relapse after initial response highlighting the need for novel therapeutic strategies. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression. Aberrant MYC transcriptional activity can increase the levels of a number of factors that are associated with disease progression and drug resistance making it an appealing therapeutic target. Recent studies have demonstrated that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BET antagonist JQ1 decreased the expression of c-MYC, BCL-xL, and BCL-2, induced p21 levels, diminished cell viability, and triggered apoptosis in MM cell lines. Comprehensive gene expression profiling of the pharmacodynamic effects of JQ1 revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with this agent. Several earlier studies demonstrated that aberrant HDAC6 expression/activity contributes to malignant progression and resistance to a number of classes of anticancer agents including proteasome inhibitors. Based on the roles of HDAC6 in malignant pathogenesis, we hypothesized that its induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized both genetic and pharmacological approaches to impair HDAC6 function [shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat)] and evaluated the consequential impact on the anti-MM effects of JQ1. Notably, antagonzing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with further reductions in c-MYC, BCL-2, and BCL-xL along with significant increases in apoptosis induction as evidenced by enhanced caspase-3 cleavage and DNA fragmentation. Importantly, administration of ACY-1215 was very well tolerated (less than 5% mean transient reduction in body weight) and significantly augmented the in vivo anti-myeloma activity of JQ1 in the RPMI-8226 MM xenograft model as disease burden in combination treated animals was substantially lower than those that received either monotherapy. Immunohistochemical analyses demonstrated that the combination of JQ1 and ACY-1215 led to significantly lower MM cell proliferation (PCNA), increased apoptosis (active caspase-3), and diminished expression of c-MYC and BCL-2. These data suggest for the first time that induction of HDAC6 may represent a key mechanism that promotes drug resistance and limits the efficacy of bromodomain inhibitor therapy. Taken together, our findings demonstrate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel approach to augment the efficacy of bromodomain inhibitors in MM that warrants further investigation. Disclosures Carew: Boehringer Ingelheim: Research Funding.

scholarly journals Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells

1982 ◽  
Vol 206 (3) ◽  
pp. 499-503 ◽  
Author(s):  
K Majamaa ◽  
R Myllylä ◽  
K Alitalo ◽  
A Vaheri
Keyword(s):  
Enzyme Activity ◽  
Cell Lines ◽  
Enzyme Activities ◽  
Cell Transformation ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Hydroxylase Activity ◽  
Transformed Cell ◽  
Transformed Cell Lines

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.

Cell surface topography in adhesion and detachment

1978 ◽  
Vol 36 (2) ◽  
pp. 300-301
Author(s):  
T.D. Allen
Keyword(s):  
Cell Lines ◽  
Thin Sheet ◽  
Liver Parenchyma ◽  
Soft Agar ◽  
Transformed Cells ◽  
Normal Cells ◽  
Epithelial Cell Lines ◽  
Normal Rat ◽  
Transformed Cell Lines ◽  
Peripheral Cytoplasm

The effects of trypsin and EGTA were investigated in parallel on two epithelial cell lines: a normal rat-liver parenchyma line (Fig. 1) and a spontaneously transformed line which had been subcloned in soft agar. The basic ultrastructural differences between these two cell lines has already been reported; briefly, the transformed cells, although typically epithelial in morphology, lack the well ordered microtubular and microfilamentous organisation of the normal cells but exhibit a marked increase in the number of surface microvilli.The reaction of each cell line to trypsination was broadly similar between the normal and transformed cell lines, with the transformed cells rounding up and detaching at a slightly quicker rate than the normals. In both cases the central region of the cell became domed with the peripheral cytoplasm being withdrawn in a thin sheet, some of which remained extended in short retraction fibrils (Fig. 2). Collection of the detached cells (after fixation in suspension) by aspiration onto silver filters showed the increased density of microvilli on the transformed cells to be maintained.

E-Selectin Ligand Expression Increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which Is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1805-1805 ◽  
Author(s):  
Alessandro Natoni ◽  
Theodore A.G. Smith ◽  
Niamh Keane ◽  
Silvia C. Locatelli-Hoops ◽  
Isabela Oliva ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Disease Progression ◽  
Cell Lines ◽  
Saline Control ◽  
Selectin Ligands ◽  
Ligand Expression ◽  
Selectin Ligand ◽  
Transplant Model

Abstract There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance. The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines. The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche. Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008) These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. Disclosures Smith: GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.

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