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.

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1695-1695
Author(s):  
Norina Tanaka ◽  
Yan-Hua Wang ◽  
Masayuki Shiseki ◽  
Minoko Takanashi ◽  
Toshiko Motoji
Keyword(s):  
Cell Cycle ◽  
Acute Leukemia ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
S Phase ◽  
Leukemic Cells ◽  
Rt Pcr

Abstract Abstract 1695 Introduction: The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods: The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results: A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions: Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures: No relevant conflicts of interest to declare.

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.

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

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.

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.

scholarly journals Effects of Electroacupuncture on Ovarian Expression of the Androgen Receptor and Connexin 43 in Rats with Letrozole-Induced Polycystic Ovaries

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Ge Xu ◽  
Andong Zhang ◽  
Jiandang Liu ◽  
Xi Wang ◽  
Jiwei Feng ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Connexin 43 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Endocrine Dysfunction ◽  
Rt Pcr

Background. Polycystic ovarian syndrome (PCOS) occurs in women of reproductive age and is often characterized by reproductive and endocrine dysfunction. Androgens play a major role in PCOS, and previous studies reported abnormal expression of Connexin 43 (Cx43) in animal models of PCOS, suggesting an association of Cx43 with PCOS pathogenesis. Experimental and clinical evidence indicated that acupuncture may be a safe and effective approach for treating reproductive and endocrine disorders in women with PCOS. This study aimed to determine the effects of electroacupuncture (EA) on PCOS and its relationship with the expression of the androgen receptor (AR) and Cx43. Methods. In total, 30 female Sprague Dawley rats (6 weeks old) were randomly divided into three groups: control group, letrozole (LE) group, and LE + EA group. Rats were administered LE solution (1.0 mg/kg) for 21 consecutive days to induce PCOS. For the LE + EA group, additional EA treatment was conducted (2 Hz, 20 min/d) with “Guanyuan” (CV3) for 14 consecutive days. After hematoxylin-eosin staining, the ovarian structure was observed with an optical microscope, and serum levels of the following hormones were examined via enzyme-linked immunosorbent assay (ELISA): testosterone (T), estradiol (E2), sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH); luteinizing hormone (LH), insulin (INS), anti-Müllerian hormone (AMH), and inhibin B (INHB). Fasting blood glucose (FBG) levels were evaluated using glucose oxidase-peroxidase. Ovarian mRNA and protein expressions of AR and Cx43 were determined by real-time RT-PCR and Western blot analysis. Results. EA was found to restore the cyclicity and ovarian morphology in the PCOS rat model. Serum derived from the LE + EA group showed significant decreases in the levels of T, free androgen index (FAI), LH, LH/FSH ratio, AMH, INHB, and fasting serum insulin (FINS), and significant increases in the levels of E2, FSH, and SHBG. Western blot analysis showed a decreased protein expression of ovarian AR and Cx43; real-time RT-PCR showed reduced expression of ovarian mRNA levels of AR and Cx43. Conclusions. In conclusion, our results showed that EA can ease hyperandrogenism and polycystic ovary morphology in PCOS rats. Furthermore, EA counteracted the letrozole-induced upregulation of AR and Cx43. These results suggested that acupuncture can break the vicious cycle initiated by excessive androgen secretion and may be an effective treatment method for improving the reproductive and endocrine dysfunction caused by PCOS.

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.

Cyclin B1, D1 overexpression and its correlation with clinical prognostic factors in ovarian carcinoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 15067-15067
Author(s):  
J. W. Kim ◽  
Y. T. Kim ◽  
H. Y. Kim ◽  
M. H. Kang ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Prognostic Factors ◽  
Western Blot ◽  
Ovarian Carcinoma ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cyclin B1 ◽  
Rt Pcr ◽  
The Mean ◽  
The Relationship

15067 Background: The change of cell cycle is one of the characteristics of cancer. The various proteins related to the cell cycle have been revealed and their expression in ovarian carcinoma has been demonstrated. Therefore, this study was conducted to determine the expression of cyclin B1, D1 and evaluate the relationship between cyclin B1, D1 and clinical prognostic factors in patients with ovarian carcinoma. Methods: 41 fresh ovarian tissue samples including 36 ovarian carcinomas and 5 normal ovarian tissues were surgically obtained at YUMC from May 2002 to February 2005. Cyclin B1, D1 expression were detected using the quantitative real-time RT-PCR and Western blot analysis. For clinical prognostic factors, age, stage, grade, histopathology, LN metastasis, residual tumor size, CA 125 and DNA flow cytometry were evaluated. Results: By quantitative real time RT-PCR, the mean 2−ΔΔCT value of cyclin B1 and D1 mRNA in ovarian carcinoma was 5.83 ± 12.03, 17.60 ± 22.20, slightly higher than that of the control. (p = 0.67, 0.07). The mean value of relative protein levels of cyclin B1 and D1 in Western blot analysis was also higher in ovarian carcinoma (1.30 ± 0.73, 1.81 ± 1.28, respectively) (p = 0.76, 0.06). No significant relationship of cyclin B1, D1 expression and clinical prognostic factors was observed. Conclusions: The expression of cyclin B1, D1 in ovarian carcinoma was higher than that of the control, although there was no statistical significance. This suggests that cyclin B1, D1 might be involved in the tumorigenesis and the progression of malignancy. Even though there was no significant correlation between cyclin expression and prognostic factors, further studies are needed assessing the relationship between cyclin expression and survival rate to elucidate the role of cyclin as a prognostic factor in ovarian carcinoma. No significant financial relationships to disclose.

scholarly journals Silencing of the WFS1 gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage

2013 ◽  
Vol 45 (5) ◽  
pp. 182-190 ◽  
Author(s):  
Sulev Kõks ◽  
Rupert W. Overall ◽  
Marilin Ivask ◽  
Ursel Soomets ◽  
Mithu Guha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Expression Profiles ◽  
Mitochondrial Damage ◽  
Rt Pcr ◽  
Hek Cells ◽  
Wfs1 Gene

The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72, and 96 h after transfection with three different WFS1 siRNAs. To verify silencing we performed quantitative RT-PCR and Western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and Western blot analysis confirmed clear silencing of the expression of WFS1 after 48 h. Significant (FDR value <10%) changes in the expression of 11 genes was identified with most of these genes being related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlations between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest a role of WFS1 gene in cell survival and its involvement in degenerative diseases.

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.

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