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.

The CALM/AF10 Interactor CATS Is a Substrate of KIS, a Positive Regulator of Cell Cycle Progression in Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2549-2549
Author(s):  
Leticia Fröhlich Archangelo ◽  
Fabíola Traina ◽  
Philipp A Greif ◽  
Alexandre Maucuer ◽  
Valérie Manceau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Cycle Progression

Abstract Abstract 2549 The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that interacts with and influences the subcellular localization of CALM/AF10, a leukemic fusion protein found in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and in malignant lymphoma. CATS is highly expressed in leukemia, lymphoma and tumor cell lines but not in non-proliferating T-cells or in peripheral blood lymphocytes (PBLs). The protein levels of CATS are cell cycle-dependent, induced by mitogens (e.g. PHA) and correlate with the proliferative state of the cell. Thus, CATS is as a marker for proliferation. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) as a CATS interacting partner. KIS is a serine/threonine kinase that positively regulates cell cycle progression through phosphorylation of p27KIP in leukemia cell lines. The interaction between CATS and KIS was confirmed by GST pull-down, and co-immunopreciptation. KIS interaction region was mapped to CATS N-terminal portion. Searching through the phosphorylation site databases PhosphoSitePlus™ (http://www.phosphosite.org) and Phosida (http://www.phosida.com/) we identified 9 residues within CATS shown to be subject of post-translational modification. Phosphorylation assay with recombinant KIS demonstrated that this kinase efficiently phosphorylated full length CATS and its N-terminal part, but not the C-terminal of the protein. To map the KIS phosphorylation site of CATS, peptides comprising all known phospho-sites of CATS N-terminal (S16, S129, S131, T133 and S135) and mutations of the putative KIS target motif (S129 and S131) were tested for KIS phosphorylation. Thereby, we identified CATS S131 as the unique target site for KIS phosphorylation. Western blot analysis of U2OS cells, which had undergone cell cycle synchronization by a double thymidine block, revealed that KIS fluctuated throughout the cell cycle and counteracted CATS levels. Furthermore, we analyzed KIS protein expression on bone marrow mononuclear cells (MNCs) of MDS and AML patients. We studied 5 healthy donors, 13 MDS patients (7 low-risk [RA/RARS] and 6 high-risk [RAEB/RAEBt] according to FAB classification) and 10 AML patients (7 de novo and 3 secondary). Western blot analysis revealed elevated levels of KIS in MDS and AML compared to the control samples. We used a reporter gene assay in order to determine the influence of KIS on the CATS-mediated transcriptional repression and to elucidate the role of KIS-dependent phosphorylation of CATS at serine 131 in this context. Coexpression of GAL4-DBD-CATS and KIS enhanced the inhibitory function of CATS on transactivation of the GAL4-tk-luciferase reporter. This effect of KIS was observed for both CATS wild type and CATS phospho-defective mutant (CATS S131A) but not when the kinase dead mutant KISK54R was used. Moreover, CATS phosphomimetic clone (CATSS131D) exerted the same transcriptional activity as the CATS wild type. These results demonstrate that KIS enhances the transcriptional repressor activity of CATS, and this effect is independent of CATS phosphorylation at S131 but dependent on the kinase activity of KIS. Finally, we investigated whether CATS would affect the CALM/AF10 function as an aberrant transcription factor. Coexpression of constant amounts of GAL4-DBD-CALM/AF10 and increasing amounts of CATS lead to reduced transactivation capacity of CALM/AF10 in a dose dependent manner. Our results show that CATS not only interacts with but is also a substrate for KIS, suggesting that CATS function might be modulated through phosphorylation events. The identification of the CATS-KIS interaction further supports the hypothesis that CATS plays an important role in the control of cell proliferation. Moreover the elevated levels of KIS in hematological malignances suggest that KIS could regulate CATS activity and/or function in highly proliferating leukemic cells. Thus our results indicate that CATS function might be important to understand the malignant transformation mediated by CALM/AF10. Disclosures: No relevant conflicts of interest to declare.

NPM-ALK Promotes Cell Cycle Progression through Activation of JNK and c-Jun in Anaplastic Large Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2057-2057
Author(s):  
Vasiliki Leventaki ◽  
Elias Drakos ◽  
Megan Lim ◽  
Kojo S. Elenitoba-Johnson ◽  
Francois-Xavier Claret ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Anaplastic Large Cell Lymphoma ◽  
Blot Analysis ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) chimeric protein. NPM-ALK mediates its oncogenic effects through phosphorylation of a number of proteins involved in known signal transduction pathways including PLC, PI3K-AKT and JAK-STAT. ALK+ ALCL cells also are known to overexpress c-Jun, a member of the activator protein-1 (AP-1) transcription factor family that controls cell proliferation, differentiation, growth and apoptosis. Phosphorylation of c-Jun at serine 73 and serine 63 residues substantially increases AP-1 transcriptional activity and the levels of c-Jun protein through an autoregulatory positive feedback loop. In this study, we hypothesized that NPM-ALK activates JNK which , in turn, phosphorylates and activates c-Jun, resulting in uncontrolled cell cycle progression in ALCL. 293T and Jurkat (T-acute lymphoblastic leukemia) cells were transfected with a vector expressing NPM-ALK with active kinase domain (pDest40-NPM-ALK) or a construct lacking NPM-ALK kinase activity (pDest40-K210R) or empty vector. Cells were harvested at 48 hours and analyzed for protein expression by Western blot analysis and for AP-1 activity by luciferase reporter assay. Two ALK+ ALCL cell lines Karpas 299 and SU-DHL-1, found to express high levels of serine phosphorylated and total c-Jun in immunoblots, were treated with JNK (SP600125), ERK (U0126), or ALK (WHI-P154) inhibitors or were transiently transfected with siRNAs specific for JNK1 and c-Jun. Cell proliferation was assessed by MTS assay, and cell cycle was analyzed by BrdU assay or propidium iodide staining and flow cytometry. Forced expression of NPM-ALK in 293T and Jurkat cells resulted in increased levels of JNK and c-Jun phosphorylation in immunoblots and a dramatic increase in AP-1 activity. Conversely, pharmacologic inhibition of ALK activity in Karpas 299 and SU-DHL1 resulted in a concentration-dependent decrease of JNK and c-Jun phosphorylation levels. Co-immunoprecipitation studies revealed that NPM-ALK physically binds to JNK1 and its upstream activator MKK7 in ALK+ ALCL cells. Selective inhibition of JNK, but not ERK, in Karpas 299 and SU-DHL1 decreased the level of c-Jun phosphorylation in a dose-dependent manner as shown by Western blot analysis and in vitro kinase assays. Inhibition of JNK by SP600125 or silencing of the JNK1 gene by siRNA also resulted in decreased cell proliferation associated with decreased AP-1 activity, cell cycle arrest mostly at G2 phase, and up-regulation of the cyclin-dependent inhibitor p21, a transcriptional target of c-Jun. Similarly, silencing of c-Jun by specific siRNA led to decreased S-phase fraction of cell cycle, which was associated with up-regulation of p21 and downregulation of cyclin D3. These findings reveal a novel function of NPM-ALK oncoprotein, phosphorylation and activation of JNK, which may contribute to uncontrolled cell cycle progression through activation of c-Jun. Modulation of JNK or c-Jun activity may be a target for therapy in patients with ALCL.

Proteasome Inhibitors Can Induce Caspase-3 Activation and Apoptosis in Human CML Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4681-4681
Author(s):  
Byung-Su Kim ◽  
Chang Up Kim ◽  
Young-Ju Kim ◽  
Eun Kyung Bae ◽  
Jinhee Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Proteasome Inhibitor ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Proteasome Inhibitors ◽  
Acridine Orange Staining

Abstract The proteasome is a multi-enzyme complex that provides the ubiquitin-dependent degradation of many cytoplasmic and nuclear proteins involved in cell cycle progression and apoptosis. Inhibition of the proteasome represents a promising approach for the treatment of cancer because it can lead to cell cycle arrest and activation of caspases in tumor cells. There are several proteasome inhibitors that have been reported to induce apoptosis in various tumors. However, the effect of proteasome inhibition in human myeloid leukemia has not been reported so far. In this study, we tested two peptide-aldehyde proteasome inhibitors (MG115, MG132) on two human CML cell lines (K562, KCL22). At first, we treated both cell lines for 24, 48 and 72 hours with different doses of MG115 and MG132 and cell viability was tested by MTT assay. It showed substantial time and dose dependent cytotoxicity in both CML cell lines. Acridine orange staining also revealed DNA fragmentation. We then performed caspase-3 colorimetric assay after treating both cell lines for 6, 12 and 24 hours with 0.78μM of MG115, MG132. K562 showed the continuous rising of caspase-3 activity, while KCL22 exhibited the initial increase and subsequent mild decrease of caspase-3 activity. In addition, western blot analysis showed the reduction of procaspase-3 expression. The expression of Bcl-2 and Bcl-XL was reduced by western blot. p21 expression was slightly increased and that of cyclin D1 was decreased. Additionally, the treatment of proteasome inhibitor in CML cell lines initially induced phosphorylation of Jun kinase. We next examined the expression of heat shock proteins (Hsp70, Hsp90) after treating for 6, 12, 24 hours with the same proteasome inhibitors. Western blot analysis results indicated that expression patterns were different between MG115 and MG132. MG115 induced the slight increase of Hsp70 and Hsp90 in K562, but the reduction of both in KCL22. Meanwhile, MG132 produced the decrease of Hsp70 and Hsp90 in both K562, KCL22. In summary, our work supports that a proteasome inhibitor can induce apoptosis in human CML cell lines. We are currently focusing on the combined effect of proteasome inhibitor and Hsp90 inhibitor on CML. IC50 of Proteasome Inhibitors Cell line Proteasome Inhibitor 24hr 48hr 72hr K562 MG115 3.01 μM 1.14 μM 0.59 μM K562 MG132 μ 2.13 M 1.03 μM 0.57 μM KCL22 MG115 156.92 μM 1.36 μM 0.73 μM KCL22 MG132 1.56 μM 0.93 μM μ 0.75 M

cJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2620-2620
Author(s):  
Vassiliki Leventaki ◽  
Elias Drakos ◽  
Francois-Xavier Claret ◽  
L. Jeffrey Medeiros ◽  
George Z. Rassidakis
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
S Phase ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.

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.

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4213-4213
Author(s):  
Priya Khoral ◽  
Robert J Guo ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Blot Analysis ◽  
Drug Combination ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

The F-Box Protein Skp2 Regulates Expression of p27 in BCR-ABL Transformed Cell Lines and Is Required for Induction of Myeloproliferative Disease in a Murine Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 742-742
Author(s):  
Anupriya Agarwal ◽  
Thomas G.P. Bumm ◽  
Amie Corbin ◽  
Thomas O’Hare ◽  
Marc Loriaux ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Dependent Manner ◽  
Myeloproliferative Disease ◽  
Cycle Progression ◽  
Cell Counts ◽  
F Box Protein ◽  
Transformed Cell Lines

Abstract Background:The cyclin-dependent kinase inhibitor p27 is a central regulator of cell cycle progression, whose function is perturbed in many human cancers, either due to decreased expression or abnormal localization. p27 levels are negatively correlated with Skp2, the F-box protein of SCFSKP2, a E3 ubiquitin ligase targeting nuclear p27 for degradation. Skp2 has been shown to cooperate with mutant Ras in in vitro transformation assays, implicating Skp2 as a bona fide oncogene. In chronic myeloid leukemia cell lines, p27 is down-regulated in a Bcr-Abl dependent fashion, while cytoplasmic accumulation has been described in primary CML cells. We herefore hypothesized that Bcr-Abl may regulate p27 via Skp2. Experimental approach and results:Mo7e-p210BCR-ABL treated with 2.5 μM imatinib arrested in G0/G1 in a time-dependent manner (53.6±2, 58.3±2, 71.9±1% at 4, 8 and 16h), correlated with reduced in vitro kinase activity of Cdk2 (32% of controls at 16h). Western blot analysis showed a marginal increase of cytoplasmic p27 and 2.5-fold accumulation of nuclear p27 that preceded G0/1 arrest. Despite the reduced Cdk2 activity, most p27 was phosphorylated on T187, a target of cdk2/cyclinE, suggesting reduced degradation of phospo-p27 (T187). Degradation of nuclear p27 is mediated by SCFSKP2 and degradation of cytoplasmic p27 by the recently discovered KPC complex. We therefore assayed expression SCFSKP2 components and KPC1/2 by immunoblot analysis of imatinib-treated cells. Skp2 expression was greatly reduced compared to controls while expression of other SCFSKP2 components and KPC1/2 was unchanged, consistent with up-regulation of nuclear but not cytoplasmic p27 and suggesting a central role of Skp2 in mediating p27 degradation in Bcr-Abl positive cells. To test whether Skp2 is crucial for Bcr-Abl-driven leukemogenesis, we infected bone marrow of Skp2+/+ and Skp2−/− mice with BCR-ABL retrovirus. No consistent difference was observed in B-cell transformation assay (Whitlock-Witte cultures). However, formation of myeloid colonies in semisolid media was reduced in Skp2−/− compared to Skp2+/+ marrow [46.4±10% of controls (p=0.002) without and 76.6±9% of controls (p=0.008) with cytokines, n=6]. Skp2+/+ mice transplanted with BCR-ABL infected Skp2−/− marrow had significantly longer median survival (19days, range 12–60days, n=8) compared to recipients of Skp2+/+ marrow (13days, range 12–22days, n=10) (p=0.0034) with significant reduction of spleen weight (0.42±0.07g vs. 0.28±0.09g, p=0.004) and white blood cell counts (median 59x103/μl, range 9.6–142x103/μl, vs. 7.9x103/μl, range 0.8–87x103/μl, p=0.02). Histology and immunophenotyping of tissues (blood, marrow, spleen) revealed no signinificant differences between Skp2+/+ and Skp2−/− mice. Conclusions: Our data suggest that the primary cell cycle effect of Bcr-Abl kinase is up-regulation of Skp2. This leads to increased activity of SCFSKP2, inducing degradation of T187 phosphorylated p27 which in turn promotes cell cycle progression by relieving suppression of Cdk2. Skp2 is required for Bcr-Abl to fully realize its potential to induce myeloproliferative disease, providing the first in vivo evidence that SKP2 is an oncogene. Targeting Skp2-p27 interactions to prevent p27 degradation may be therapeutically useful in malignancies with a high Skp2/p27 ratio.

scholarly journals Cancer cells are selectively eliminated through UPR driven apoptosis during treatment with Aquilegia nivalis extracts

2021 ◽  
Author(s):  
Nazia Hilal ◽  
Ozaira Qadri ◽  
Irshad A Nawchoo ◽  
Seema Akbar ◽  
Khalid Majid Fazili
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Methanolic Extract ◽  
Dapi Staining ◽  
Dose Dependent

Abstract BackgroundAquilegia nivalis Flax Jackson, also called Aquilegia vulgaris sub sp. nivalis (Bak.) Brühl or columbine, locally known as “Zoe-neel”, is a wild edible plant traditionally used as an anti-inflammatory medicine by the local nomadic tribes inhabiting the Himalayas of Jammu and Kashmir. The plant has been used as herbal medicine since middle ages in treating ailments that include chronic rhinitis and various infectious diseases. The extracts from the plant possess antioxidant properties and have been reported to be hepatoprotective in rats. Our preliminary studies, however, pointed to hitherto unexplored anti-apoptotic potential of the plant which lead us to carry the in-depth study using breast cancer cell lines to validate its anti-cancerous properties and explore the affected pathways.MethodsMTT assay was used to draw the dose response curve and evaluate the effect of increasing concentrations of the extract on cell lines to determine the appropriate dosage to be used for further experimentation. DNA fragmentation analysis was followed through gel electrophoresis and DAPI staining was pursued by phase contrast microscopy to study apoptosis. Quantitative PCR was used to study the expression of UPR signaling and RIDD markers at the level of mRNA. Western blot analysis was used in studying the expression of the various markers of the signaling pathways. The cell cycle analysis was carried out using flow cytometry.ResultsMTT assay revealed that the methanolic extract of the plant (ANME) was selectively cytotoxic to various cancer cell lines as revealed by lower IC50 values relative to normal cell lines. The results of cell cycle analysis were similar as ANME caused Sub G1 arrest of the cell cycle. DNA fragmentation analysis, DAPI staining and western blot analysis for PARP and caspases revealed that the extract selectively induced apoptosis in cancerous cell lines. UPR markers p-Ire1α and Xbp1 splicing were consistently alleviated in a dose dependent manner, the rate of phosphorylation of eIF2a and ATF4 also decreased with increasing concentration of ANME. The RT PCR results of the RIDD marker, Blos1S1 revealed a similar dose dependent association. The methanolic extract was especially chosen for it could be easily internalized by the cells and any resultant potential bioactive compounds could gain access to the cells because of their hydrophobic nature.ConclusionOur results suggest that ANME causes deactivation of UPR signaling pathway facilitating apoptosis selectively in cancerous cells, paving the way forward for a novel approach in cancer therapeutics.

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.

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