C/EBPβ Expression in ALK+ Anaplastic Large Cell Lymphomas (ALCL) Is Regulated by Stat3 Signaling Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3570-3570
Author(s):  
Natasa Anastasov ◽  
Martina Rudelius ◽  
Margit Klier ◽  
Therese Dau ◽  
Daniela Angermeier ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Facs Analysis ◽  
Akt Inhibitor ◽  
Factor C

Abstract Background: ALK+ ALCL is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein called NPM-ALK. We recently reported the abnormal expression of the transcription factor C/EBPβ in ALCL, and demonstrated that C/EBPβ expression is dependent on NPM-ALK kinase activity. However, it is unclear how this signal is transduced. The aim of this study is to investigate the different signaling pathways that have been implicated in NPM/ALK signaling to elucidate their role in the expression of C/EBPβ. Materials and methods: To analyze the different signaling pathways induced by NPM-ALK, Ba/F3 cells were transfected with an NPM-ALK kinase-inhibitable construct (NPM-ALK-ATP-Abl). Imatinib was used to block NPM-ALK activity. Highly effective shRNA sequences (>85% knockdown) were identified for AKT, mTOR, and Stat3 proteins using a specific lacZ reporter fusion assay in HEK-293T cells, and corroborated by Western blot analysis. Each of these shRNAs were cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Three ALK+ ALCL cell lines were analyzed (SUDHL-1, Karpas 299 and Ki-JK), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. These studies were supplemented with pharmacological inhibitors: rapamycin, MAPK inhibitors (U0126 and PD98059) and AKT inhibitor (Calbiochem). The effect of Stat3, AKT, mTOR and MAPK knockdown on proliferation and cell viability was analyzed by MTT assay and FACS analysis. Results: Ba/F3 cells transfected with NPM-ALK-ATP-Abl construct resulted in induction of C/EBPβ expression and phosphorylation of Stat3, AKT and MAPK with no changes observed in mTOR phosphorylation. The opposite effect was observed when the NPM-ALK-ATP-Abl activity was inactivated with Imatinib. The infection rates of the specific shRNA constructs in the three ALK+ALCL cell lines were almost 100%. Downregulation of Stat3 in ALK+ALCL cells inhibited C/EBPβ at mRNA and protein level with impairment in cell proliferation and viability. In contrast, downregulation of AKT and mTOR showed no changes in C/EBPβ expression, whereas their downstream targets (rpS6 and 4E-BP1) phosphorylations were inactivated. These results were corroborated with rapamycin and AKT pharmacological inhibitory studies. MEK inhibitors (U0126 and PD98059) blocked the ERK1/2 phosphorylation reflected in growth retardation and its downstream target TSC2 phosphorylation without changing the expression of C/EBPβ. However, the phosphoThr-235 C/EBPβ was deactivated, confirming the importance of ERK1/2 in the phosphorylation and activation of C/EBPβ. Conclusions: In this study, we demonstrated that the induction of C/EBPβ expression by NPM-ALK correlates with the phosphorylation of AKT, MAPK and Stat3. However, only the downregulation of Stat3 has influence on C/EBPβ mRNA and protein expression, whereas MAPK is important for the phosphorylation and modulation of CEBPβ function. The downregulation of C/EBPβ, as a consequence of Stat3 inhibition has an important effect on cell growth and survival.

Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1584-1584
Author(s):  
Margit Klier ◽  
Natasa Anastasov ◽  
Daniela Angermeier ◽  
Mark Raffeld ◽  
Falko Fend ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Infection Rates ◽  
Facs Analysis ◽  
Cyclin D2 ◽  
Qrt Pcr

Abstract Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system. Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2. Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.

PI3K/AKT-and JAK3-Dependent Regulation of SKP2 Expression Is Mediated by E2F1 in Anaplastic Large Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3742-3742
Author(s):  
Jean-Marc Fontaine ◽  
Kojo S.J. Elenitoba-Johnson ◽  
Megan S Lim
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Cycle Progression ◽  
Gene Promoter ◽  
Large Cell ◽  
Cycle Progression ◽  
F Box Protein

Abstract The majority of anaplastic large cell lymphomas (ALCL) are characterized by the chromosomal translocation t(2;5)(p23;q35) leading to the expression of NPM/ALK. The constitutive activation of the NPM/ALK tyrosine kinase induces downstream mediators such as phosphoinositide 3-kinase (PI3-kinase)/AKT, JAK3 and STAT3 that result in increased cell proliferation and enhanced survival. Although the molecular mechanism by which these pathways deregulate the cell cycle machinery is not fully understood, previous studies have shown that NPM/ALK-mediated PI3K/AKT activation is required for cell cycle progression and that inhibition of PI3K/AKT results in decreased p27Kip1 degradation and cell cycle arrest. The expression of S-phase kinase protein 2 (SKP2), an F-box motif-containing protein which targets cell cycle regulators including cyclin-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation, was evaluated in a panel of ALCL cell lines. Western blot analysis of five t(2;5)-positive ALCL-derived cell lines demonstrated an inverse pattern of expression between F-box protein SKP2 and p27Kip1. We hypothesized that SKP2 deregulation contributes to the oncogenic activity of NPM/ALK by regulating the degradation of p27Kip1. In this study we investigated regulation of SKP2 and p27Kip1 expression as a consequence of inhibition of two well-known pathways downstream of NPM/ALK. Inhibition of PI3K/AKT with Ly294002 (20 mM) or JAK3 with WHI-P154 (10 mM) resulted in a dose and time-dependent decrease in cell viability (50% or 20% respectively at 24h). To determine the mechanism of SKP2 transcriptional regulation by PI3K, we performed quantitative RT-PCR and western blot analysis which demonstrated a decrease in both SKP2 transcript and protein levels after PI3K/AKT and JAK2 inhibition (33% or 47% at 24h respectively), with increase in the levels of p27 transcript and protein (47% or 71% at 24h respectively). Furthermore, the levels of E2F1 (a transcription factor associated with cell cycle progression) also decreased upon PI3K/AKT and JAK3 inhibition. Chromatin immunoprecipitation (ChIP) assays revealed that E2F1 binding to the SKP2 gene promoter was reduced as early as 4 hours after inhibition of PI3K/AKT or JAK3 (80% and 59% respectively) while no binding was detected with the GAPDH gene promoter (control). In conclusion, these results indicate that the expression of the F-box protein SKP2 is regulated by NPM/ALK mediators, PI3K/AKT and JAK3, and that E2F1 mediates the transcriptional control of SKP2 expression. Our data supports the role of SKP2–mediated regulation of p27Kip1 in ALCLs and implicates SKP2 and E2F1 as a potential therapeutic target in ALCLs.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 323 ◽  
Author(s):  
Hyun Jung ◽  
Dae-Sung Lee ◽  
Seong Park ◽  
Jung Choi ◽  
Won-Kyo Jung ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Nasal Polyp ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Type I ◽  
Molecular Signaling ◽  
Tgf Β1

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4640-4640
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Low Grade ◽  
Adapter Proteins ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Sirna Knockdown

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

Monoclonal Antibody Against ROR1 in Chronic Lymphocytic Leukemia Cells Induced Apoptosis Via PI3-Kinase/AKT/CREB Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1769-1769
Author(s):  
Amir Hossein Daneshmanesh ◽  
Mohammad Hojjat-Farsangi ◽  
Asa Sandin ◽  
Abdul Salam Khan ◽  
Ali Moshfegh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pi3 Kinase ◽  
Signaling Proteins ◽  
Downstream Signaling ◽  
Induced Apoptosis ◽  
Creb Pathway

Abstract Abstract 1769 Background: Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims: To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods: Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results: ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion: Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures: No relevant conflicts of interest to declare.

Detection and Quantification of Cereblon Protein and mRNA in Multiple Myeloma Cell Lines and Primary CD138+multiple Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4043-4043
Author(s):  
Anita K Gandhi ◽  
Herve Avet-Loiseau ◽  
Michelle Waldman ◽  
Anjan Thakurta ◽  
Sharon L Aukerman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Full Length ◽  
Employment Equity ◽  
Equity Ownership ◽  
Protein Variants

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Relationship between BRCA1 promoter methylation and sensitivity of breast cancer cell lines to cisplatin and paclitaxel

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21083-21083 ◽  
Author(s):  
C. Collins ◽  
D. Huo ◽  
J. Xu ◽  
W. K. Bleibel ◽  
M. E. Dolan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cell Lines ◽  
Breast Cancer Cell ◽  
Promoter Methylation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cell Lines ◽  
Brca1 Promoter ◽  
Brca1 Promoter Methylation

21083 Background: While BRCA1 germline mutations are uncommon, and contribute to fewer than 5% of breast cancer cases, epigenetic alterations in BRCA1 occur more frequently. BRCA1 promoter methylation has been detected in 10–30% of breast tumors. Given the role of BRCA1 in DNA repair and cell cycle regulation, we hypothesize that cells with decreased expression of BRCA1 secondary to promoter methylation will be sensitive to DNA damaging agents and resistant to microtubule inhibitors, as has previously been shown for cells deficient in BRCA1 secondary to mutation. Methods: BRCA1 methylation was determined using methylation specific PCR (MSP) as previously described (Wei et al, Cancer Research 2005). The relative sensitivities of BRCA1 methylated, mutated and competent cells to cisplatin and paclitaxel were determined in five representative breast cancer cell lines using the AlamarBlue cytotoxicity assay. Exponentially growing cells were treated with increasing concentrations of cisplatin and paclitaxel for 96 hours. IC50 values and 95% confidence intervals (CI) were calculated from sigmoidal dose response curves fitted with SAS 9.1 Proc NLIN. Western blot analysis for BRCA1 was performed on each cell line. Results: Conclusions: Only one of the two BRCA1 methylated cell lines studied (UACC3199) was sensitive to cisplatin and resistant to paclitaxel, as hypothesized. While both cell lines are methylated, western blot analysis revealed that both express BRCA1, but to a lesser degree than unmethylated cells. BRCA1 methylation, as assessed by non-quantitative MSP, does not correlate with sensitivity to cisplatin and resistance to paclitaxel. Quantification of BRCA1 promoter methylation may better predict chemosensitivity. Identification of the degree of BRCA1 methylation which does correlates with sensitivity to cisplatin and resistance to paclitaxel could improve treatment selection for patients with breast cancer. This work was supported by the US Army Grant W81XWH-04–1-0545. [Table: see text] No significant financial relationships to disclose.

Loss of Beta-Catenin Expression in Metastatic Gastric Cancer

2003 ◽  
Vol 21 (9) ◽  
pp. 1708-1714 ◽  
Author(s):  
Matthias P.A. Ebert ◽  
Jun Yu ◽  
Juliane Hoffmann ◽  
Alba Rocco ◽  
Christoph Röcken ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Gene Mutations ◽  
Cancer Cell Lines ◽  
Beta Catenin ◽  
Gastric Cancers

Purpose: Beta-catenin (β-catenin) participates in intercellular adhesion and is an integral part of the Wnt signaling pathway. The role of β-catenin in the pathogenesis of gastric cancer and its metastasis is largely unknown. Patients and Methods: Immunohistochemistry and Western blot analysis were used to analyze the expression of β-catenin in 87 human gastric cancers, in metastasis and cancer cell lines. The β-catenin and the adenomatous polyposis coli (APC) genes were analyzed for gene mutations. Furthermore, methylation of the β-catenin promoter in cell lines was assessed by treatment with 5′-azadeoxycytidine and sodium bisulfite genomic sequencing. Results: β-Catenin expression was present at either the cell membrane or the cytoplasm in 34 of 75 primary gastric cancers. Expression of β-catenin was significantly more frequent in intestinal-type (P = .0049) and well-differentiated gastric cancers (P < .001). There were no quantitative differences between gastric cancers and the nonmalignant gastric tissues, as determined by Western blot analysis. One of 18 metastatic cancer lesions and four of five gastric cancer cell lines expressed β-catenin protein. N87 cells, derived from the liver metastasis of a gastric cancer, did not express β-catenin. Treatment with 5′-azadeoxycytidine restored β-catenin protein levels in this cell line, which exhibited significantly more 5-methylcytosines in the β-catenin promoter compared with the other cell lines. Conclusion: β-Catenin expression is lost in a subgroup of primary gastric cancers, is frequently absent in metastases, and exhibits nuclear localization in cancers with either β-catenin or APC gene mutations. Interestingly, the loss of β-catenin expression in metastatic gastric cancers may result from hypermethylation of the β-catenin promoter.

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