Induction of Cyclin D1 Gene in Myeloma Cells Down-Regulates the Expression of Cyclin D2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3411-3411
Author(s):  
Yoshiaki Kuroda ◽  
Akira Sakai ◽  
Naohiro Tsuyama ◽  
Mami Mizuno ◽  
Nanae Nakaju ◽  
...  
Keyword(s):  
Western Blot ◽  
Cyclin D1 ◽  
D1 Protein ◽  
Myeloma Cell ◽  
Cyclin D3 ◽  
Proliferation Index ◽  
Cyclin D2 ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Cyclin D1 Protein

Abstract Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.

scholarly journals Bortezomib-Induced Apoptosis in Myeloma Cell Via Oxidation of Peroxiredoxin III and Thioredoxin 2

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2081-2081
Author(s):  
Yeung-Chul Mun ◽  
Jee-Young Ahn ◽  
Seung Hye Kang ◽  
Eun-Sun Yoo ◽  
Kyoung Eun Lee ◽  
...  
Keyword(s):  
Western Blot ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Reduced Form ◽  
Ros Generation ◽  
Down Regulation ◽  
Western Blot Assay ◽  
Myeloma Cells ◽  
Mitochondrial Ros ◽  
Induced Apoptosis

Abstract Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment. Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis. Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise. Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells. Disclosures No relevant conflicts of interest to declare.

Myeloma Cell Lines Are Not Sensitive to Modulation of Cyclin D1 Level.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 790-790 ◽  
Author(s):  
Helen D. Nickerson ◽  
Marta Chesi ◽  
Peter Leif Bergsagel
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Transfection Efficiency ◽  
D1 Protein ◽  
Myeloma Cell ◽  
Transfected Cells ◽  
Cyclin D1 Protein ◽  
Human Myeloma Cell

Abstract Dysregulation of D type cyclins is an almost universal event in multiple myeloma. Overexpression of cyclin D1 may occur by chromosomal translocation of t(11:14)(q13:32), and also in association with hyperdiploidy. To investigate the role of cyclin D1 in the growth of human myeloma cell lines, we used both shRNA plasmid constructs, and an siRNA Smartpool against cyclin D1 and compared to control cells transfected with anti-GFP or GAPDH constructs. siRNA was electroporated into multiple myeloma cell lines overexpressing cyclin D1 (KMS12PE, U266, H929) which gave a 50–60% transfection efficiency, assessed using a GFP plasmid. Successful reduction in levels of cyclin D1 was confirmed by western blotting and normalization to a beta actin standard. Cyclin D1 protein in sh- or siRNA transfected cells was reduced on average to 32% of that in cells transfected with an anti-GAPDH or anti-GFP siRNA. In individual experiments reduction of cyclin D1 protein to as little as 10% of control was observed. However, we found no sign of increased apoptosis by Annexin V/Propidium iodide staining. Furthermore, cell cycle analysis by ethidium bromide staining and flow cytometry revealed no significant change in the cell cycle of sh- or siRNA transfected cells when compared to control cells. qPCR analysis revealed no acute compensatory increase in the RNA levels of other D-type cyclins (D2, D3) following reduction of cyclin D1 levels. These results may be explained by the residual presence of sufficient cyclin D1 protein for cell cycle progression. An alternative explanation is that changes that frequently occur in myeloma to other cell cycle regulators, for example p16(INK4a) and Rb are able to circumvent cell cycle effects of reducing cyclin D1protein. These results suggest that the human myeloma cell lines tested are not acutely sensitive to cyclin D1 level.

scholarly journals Arrest of G1-S Progression by the p53-Inducible Gene PC3 Is Rb Dependent and Relies on the Inhibition of Cyclin D1 Transcription

2000 ◽  
Vol 20 (5) ◽  
pp. 1797-1815 ◽  
Author(s):  
Daniele Guardavaccaro ◽  
Giuseppina Corrente ◽  
Francesca Covone ◽  
Laura Micheli ◽  
Igea D'Agnano ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
D1 Protein ◽  
Down Regulation ◽  
Inhibition Of Proliferation ◽  
Protein Levels ◽  
Growth Inhibitory ◽  
Cyclin D1 Protein ◽  
Cdk4 Activity ◽  
Inducible Gene

ABSTRACT The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression ofPC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G1 arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that thePC3-mediated G1 arrest was Rb dependent. Furthermore, (i) the arrest of G1-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G1-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.

Kinetin Riboside Is a Novel Targeted Inhibitor of CCND1 and CCND2 Transactivation with Substantial Preclinical Anti-Myeloma Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 367-367
Author(s):  
Rodger E. Tiedemann ◽  
Xinliang Mao ◽  
Ronald J. Marler ◽  
Craig B. Reeder ◽  
Aaron Schimmer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cyclin D1 ◽  
Myeloma Cell ◽  
S Phase ◽  
Luciferase Reporter ◽  
Clinical Activity ◽  
G1 Arrest ◽  
Cyclin D ◽  
Cyclin D2 ◽  
Myeloma Cells

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood;106:296). To identify novel pharmaceutical inhibitors of cyclin D2 (CCND2) transactivation we therefore screened the Lopac, Prestwick and Spectrum libraries of drugs and natural compounds (n>4000) using NIH 3T3 cells stably expressing the CCND2 promoter driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Kinetin riboside, a nucleoside analogue and plant cytokinin hormone, as a novel inhibitor of CCND1+CCND2 transactivation. By immunoblotting, Kinetin riboside induced rapid suppression of cyclin D1 and D2 proteins (<6 hours) in H929, JJN3, Kms11 and U266 human myeloma cell lines (HMCL) that all over-express either cyclin D1 or D2 due to a range of transforming events relevant to myeloma (including deregulation of FGFR3, MMSET or c-Maf oncogenes, or translocation of CCND1 to the IgH locus). Similar results were obtained in primary CD138+ purified myeloma cells from 5/6 patients. To verify that cyclin D1 and D2 suppression induced by Kinetin riboside is a direct effect and does not occur secondary to cellular arrest in S-phase (when cyclin D protein levels decline) we examined the effects of Kinetin riboside on the cell cycle profile of HMCL. Within 20 hours Kinetin riboside caused the proportion of cells entering S-phase to fall by 50–70% in all HMCL tested, consistent with primary cyclin D suppression and secondary G0/G1 arrest. By MTT assay, Kinetin riboside is cytotoxic to HMCL with an IC50 of <1.7 mg/L (5uM) in 8/12 lines and <5mg/L (15uM) in 11/12 HMCL. By comparison, toxicity studies in Balb/c mice confirm that Kinetin riboside is tolerated in vivo at a dose of 80–100mg/kg i.p. or 25mg/kg i.v. Importantly, Kinetin riboside shows potent synergy with dexamethasone in HMCL that are poorly responsive to one or other agent and Kinetin riboside activity persists during co-culture with myeloma growth factors IL-6, IGF-1 and Baff. Moreover, when tested against unsorted patient bone marrow samples, Kinetin riboside preferentially killed CD138+ myeloma cells at up to 5–8 fold greater rate than normal marrow progenitors. Kinetin riboside induced myeloma cell death is mediated by apoptosis and is associated with caspase 9 cleavage and annexin V binding. Mechanistically, we show that kinetin riboside blocks CCND2 promoter transactivation induced by cAMP or by regulatory phosphoproteins (activated by Forskolin or the PP2A inhibitor, Cantharadin, respectively) and also blocks cis-activation of CCND1 induced by translocation to the IgH enhancer and trans-activation of CCND2 induced by cMaf or FGFR3 over-expression, indicating that kinetin riboside acts at a distal level to block transactivation of CCND1+CCND2 induced by multiple factors. Gene expression profiling reveals that KinR causes rapid induction of the transcriptional repressor, cAMP Response Element Modifier (CREM), which has been reported to bind the cyclin D2 promoter to regulate cyclin D2 expression (Muniz, 2006, Biol Reprod.), providing a putative mechanism for targeted suppression of CCND1+CCND2. Together these studies demonstrate a novel targeted mechanism and substantial pre-clinical activity for kinetin riboside and provide a rationale for clinical evaluation of this drug to improve the outcome of multiple myeloma.

Cyclin D1 Overexpression Increases Chemosensitivity Via the Induction of Bim in Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3418-3418
Author(s):  
Yoshiaki Kuroda ◽  
Akira Sakai ◽  
Naohiro Tsuyama ◽  
Shoso Munemasa ◽  
Nanae Nakaju ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Cdna Microarray ◽  
Risk Group ◽  
Low Risk ◽  
High Dose ◽  
Cyclin D2 ◽  
Down Regulation ◽  
Myeloma Cells

Abstract Recent study analyzing the gene expression in myeloma cells using cDNA microarray showed that myeloma cells are divided into 7 groups. Two showed a high expression of cyclin D1 together with a low expression of cyclin D2, and they belong to the low-risk group when analyzed based on event-free and overall survival. Since cyclin D1 promotes the cell cycle progression, we sought to explain why cyclin D1 overexpression appears to be a favorable prognostic variable for multiple myeloma (MM) patients treated with high-dose chemotherapy and single or double autologous transplantation. It is not clear whether the down-regulation of cyclin D2 in these myeloma cells might offset cyclin D1 overexpression in cell biology. We have established a myeloma cell line (RPMI8226) with cyclin D1 overexpression by transfection of the cyclin D1 gene using a retrovirus vector to analyze biological changes in myeloma cells with cyclin D1 overexpression. In comparison with myeloma cells transfected with the vector only (mock), the analysis of gene expression by cDNA microarray showed the down-regulation of cyclin D2 and MCL-1 and the up-regulation of CED6 in cyclin D1-transfected myeloma cells. However, there were no significant changes in Bcl-related genes between them. And as we expected, cyclin D1-transfected myeloma cells showed high proliferative activity, increased number of cells in S-phase, and increased pRb protein. Next we analyzed their sensitivity for bortezomib (Millennium Pharmaceuticals, Inc., USA), immunomodulatory thalidomide analogs (IMiD1, D2, D3) (Celgene Corporation, USA) and dexamethasone. Bortezomib and dexamethasone induced apoptosis at an earlier time point (12hr) in cyclin D1 transfectant compared to mock transfectant, concomitant with decreased expression of MCL-1, but with increased expression of Bim. Furthermore, we confirmed in cell culture condition with a low concentration of FCS that these results were not due to just promotion of the cell cycle caused by cyclin D1 overexpression. Given that myeloma cells with cyclin D1 overexpression easily undergo apoptosis upon bortezomib or dexamethasone treatment, this may explain why patients with those myeloma cells are in the low-risk group. With this finding, we then analyzed the expression of cyclin D1 by RQ-PCR and immunocytostaining before and after 2 or 3 cycles of VAD (vincristine, doxorubicin, dexamethasone). There was no significant difference between the response to VAD and the reduction rate of cyclin D1 positive myeloma cells or the decrease of cyclin D1 expression in RQ-PCR. Interestingly, cyclin D2 expression increases relatively in progressive disease (PD) after chemotherapy containing high-dose melphalan followed by autologous stem cell transplantation compared with those of cyclin D1 and D3 by RQ-PCR. Therefore, cyclin D1-induced chemosensitivity may be due to the induction of Bim, which consequently inhibits the function of MCL-1. New strategies to down-regulate of MCL-1 might be useful in the treatment of MM.

Induction of Cyclin D1 by HGF, IGF-1 and IL-6 in a Human Myeloma Cell Line with a t(11:14) Translocation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5055-5055
Author(s):  
Håkon Hov ◽  
Thea K. Våtsveen ◽  
Anders Waage ◽  
Anders Sundan ◽  
Magne Borset
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cyclin D1 ◽  
Cell Line ◽  
D1 Protein ◽  
Myeloma Cell ◽  
Chromosome 11 ◽  
Myeloma Cell Line ◽  
Ccnd1 Gene ◽  
Cyclin D1 Protein

Abstract Almost all patients with multiple myeloma have dysregulation of cyclin D1 -2 or -3. The t(11:14) translocation juxtapose the CCND1 locus coding for the cyclin D1 protein to the IgH locus. This leads to upregulation of the cyclin D1 gene, and thus to increased proliferative capacity. Despite the presence of IgH translocations, the myeloma cells are highly dependent of the bone marrow microenvironment for malignant growth. Whether the expression of cyclin Ds is additionally regulated by the microenvironment needs to be further examined. Here we report a myeloma cell line, INA-6, with a t(11;14) translocation in which expression of cyclin D1 is regulated by several cytokines. INA-6 cells were analysed by interphase FISH with probes matching the CCND1 gene (coding for the cyclin D1 protein) and the Ig heavy chain locus. We found co-localisation of the two probes indicating a translocation between chromosome 14 and chromosome 11. We detected a total of five signals for CCND1, indicating the occurrence of three extra copies of chromosome 11. This was confirmed by an analysis with a single probe detecting the CCND1 locus. We further analysed the content of cyclin D1 protein by western blotting after the cells had been washed and grown in serum free media with or without cytokines for 24 hours. INA-6 constitutively expressed cyclin D1. HGF, IL-6 and IGF-1 further enhanced the expression of cyclin D1. In combination with IL-6, HGF had a synergistic effect on cyclin D1 expression. The constitutive cyclin D1 expression is probably caused by the translocation between chromosome 11 and 14. However the increase in number of cyclin D1 genes not co-localized to the IgH chain can also contribute to the basal level of cyclin D1 expression in this cell line as well as post-transcriptional regulation. The increase in cyclin D1 expression after cytokine stimulation can be explained by an increase in transcription from the normal CCND1 copy or the other extra copies. One intriguing possibility is that the translocation itself makes the cell susceptible to cytokine regulation of cyclin D1. At least IGF-1 and IL-6 are known for their ability to enhance immunoglobulin production and may therefore regulate cyclin D1 through the IgH promotor in the t(11:14) translocation. The different ways to induce cyclin D1 protein in this cell line is a functional example of the collaboration between the dysregulated myeloma cell and the components of its microenvironment in the bone marrow in regulating a key component of cell cycling and a possible oncogene in multiple myeloma.

Halofuginone Induces Post-Transcriptional Down-Regulation of Cyclin D1, Cell Cycle Arrest and Apoptosis In Mantle Cell Lymphoma Cells through Activation of Integrated Stress Response Pathways

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 773-773
Author(s):  
Rodrigo Proto-Siqueira ◽  
Melina G. Santos ◽  
Valdemir M. Carvalho ◽  
Yumi H. Maekawa ◽  
Leonardo A. Testagrossa ◽  
...  
Keyword(s):  
Stress Response ◽  
Cyclin D1 ◽  
Cell Lines ◽  
D1 Protein ◽  
Integrated Stress Response ◽  
Down Regulation ◽  
Protein Levels ◽  
Cyclin D1 Protein ◽  
Mantle Cell

Abstract Abstract 773 Mantle cell lymphoma (MCL) remains an incurable disease and has the worst outcome among B-cell lymphomas. Patients generally have a good response to first line treatment but most relapse and tend to have shorter responses or resistant disease. Thus, novel treatment strategies capable of providing and sustaining durable responses are clearly needed. The translocation t(11;14), a hallmark of MCL, leads to cyclin D1 overexpression and is invariably accompanied by different secondary genetic lesions that collaborate for lymphomagenesis. In a previous study, we found that several genes related to the AKT, WNT and TGFβ signaling pathways were aberrantly expressed in MCL. The role of the AKT and WNT pathways in MCL pathogenesis has been well established by other groups, but little is known about the role of the TGFβ pathway. To address this issue, we tested whether halofuginone, a small molecule with recognized anti-TGFβ and antifibrotic activity, would have cytotoxic effect against a panel of MCL cell lines. We found that halofuginone at nanomolar levels had significant cytotoxic activity against MCL cell lines as measured by the MTT assay. The IC50's for Mino and HBL-2 cell lines were 30 and 61 ng/mL at 48h, respectively, with IC50's for Jeko-1, JVM-2 and Granta-519 falling in between. Halofuginone induced apoptosis in Mino and HBL-2 cells in a time- and concentration-dependent fashion, as evidenced by annexin V/7-AAD staining by flow cytometry and electron microscopy studies. However, halofuginone failed to inhibit SMAD2 phosphorylation induced by recombinant TGFβ1 in Mino and HBL-2 cells, as shown by Western blot analysis, and co-treatment experiments with TGFβ1 failed to show antagonism, suggesting that the effect of halofuginone in MCL is not mediated by TGFβ inhibition. Cell cycle analysis of Mino and HBL-2 cells exposed to halofuginone revealed time- and concentration-dependent accumulation in G1 (83% of Mino cells at G1 upon exposure to 50 ng/mL for 24h vs. 48% in untreated Mino cells), and immunocytochemical analysis showed that this effect was accompanied by striking down-regulation of cyclin D1 protein levels starting as early as 3h after exposure to halofuginone, a finding that was reproduced in primary MCL cells. Real-time RT-PCR experiments, however, revealed up-regulation of cyclin D1 mRNA levels by halofuginone over time, suggesting a post-transcriptional mechanism for the observed down-regulation of cyclin D1 protein levels. Western blot analysis of Mino and HBL-2 cells exposed to halofuginone for 24h showed a concentration-dependent phosphorylation of GCN2, PERK and EIF2α, and up-regulation of ATF4. These findings point to an activation of integrated stress response pathways (amino acid starvation response and endoplasmic reticulum stress response) that causes a general shutdown in protein synthesis and explain, at least partially, the down-regulation in cyclin D1 levels. To further characterize the proteins targeted by halofuginone in MCL we employed a proteomic profiling approach in which differentially expressed proteins were revealed by label-free liquid chromatography tandem mass spectrometry (LC-MSE) analysis on a nanoAcquity system coupled to a Synapt MS Q-Tof mass spectrometer. A comprehensive catalogue representing 147 proteins was generated from this analysis and we found that several members of the heat shock protein family are up-regulated in Mino cells exposed to 100 ng/mL of halofuginone for 14h, the relevance of which is currently under investigation. Together, our data demonstrate that halofuginone at nanomolar levels has significant antiproliferative and cytotoxic effects in MCL cells that are induced by the activation of integrated stress response pathways. More importantly, our study provides a rationale for exploring the clinical activity of this oral agent in patients with MCL. Disclosures: No relevant conflicts of interest to declare.

Cyclin D1 Overexpression Increases Chemosensitivity Via Prolonged S-Phase and TRAIL Signal in Myeloma Cell.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3530-3530
Author(s):  
Yoshiaki Kuroda ◽  
Akira Sakai ◽  
Naohiro Tsuyama ◽  
Yuta Katayama ◽  
Shoso Munemasa ◽  
...  
Keyword(s):  
Western Blot ◽  
Cyclin D1 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Cell Number ◽  
S Phase ◽  
Early Event ◽  
Phase Duration ◽  
Myeloma Cells ◽  
Trail Receptors

Abstract The mechanism of oncogenesis of plasma cells remains unclear. Since tumor cells are post-germinal center B cells, reciprocal chromosomal translocations between the IgH gene located on chromosome 14q32 and other chromosomal partners such as cyclin D1 located on chromosome 11q23, which are supposed to be candidate oncogene, arise in myeloma cells during the procedure of isotype switching. However, a recent study demonstrated that multiple myeloma (MM) with cyclin D1 overexpression belongs to the low-risk group. Previously, we reported that cyclin D1 overexpression downregulated cyclin D2 expression in myeloma cells and increased the sensitivity to treatment with anti-myeloma agents such as bortezomib (PS-341), dexamethasone, melphalan, and immunomodulatory thalidomide analogs in a comparison between RPMI8226 transfected cyclin D1 and mock. Here we have analyzed characteristics of RPMI8226 transfected cyclin D1. This cyclin D1 transfectant did not induce cell growth advantage, and cell cycle analysis by bromodeoxyuridine (BrdU) stimulation showed significant increase cell number in S-phase without an increase of that in G2/M-phase and decrease of cell number in G0/G1-phase. Therefore, Cyclin D1 overeexpression prolonged the S-phase. Western blot analysis demonstrated an increase in the hyperphosphorylated form of retinoblastoma protein (ppRb), and this ppRb was also found in KMS12BM, KMS21BM, in which myeloma cell line cyclin D1 overexpresion was detected due to t(11;14). Considering that ppRb releases free E2F, the increase in E2F could be expected to upregulate apoptosis-related genes. However, expressions of p53, Bcl-2, Bax, Bad, Bim, Mcl-1, p16, and CDK4 were not changed in cyclin D1 transfectant besides the decrease in p27 expression compared with those of the mock and parent cells. Furthermore, cyclin D1 overexpression that alone did not induce apoptosis because there were no such cells detected in sub-G0/G1 by BrdU stimulation without treatment by anti-myeloma agents. On the other hand, treatment with anti-myeloma agents induced both the intrinsic and extrinsic pathways earlier in the cyclin D1 transfectant. And this cyclin D1 transfectant cells easily lost viability in confluent samples. Interestingly, expression of the TRAIL receptors (DR4 & DR5) were significantly higher in cyclin D1 transfectant cells and treatment with recombinant TRAIL induced apoptosis earlier compared with those of mock and parent cells. There was no difference of TRAIL expression in these cells by western blot. These findings suggest that high sensitivity to anti-myeloma agents in myeloma cell with cyclin D1 overexpressin might be due to the prolonged S-phase duration and high expression of TRAIL receptor. We speculate that high ppRb induces gene instability via high E2F and leads to progressive disease in MM. This might be a reason why cyclin D1 overexpression caused by t(11;14) or hyperdiploidy is an early event in the progression of MM.

Cyclin D1 Protein Tissue Detection in Laryngeal Cancer

ORL ◽  
2005 ◽  
Vol 67 (6) ◽  
pp. 319-325 ◽  
Author(s):  
John V. Segas ◽  
Andreas C. Lazaris ◽  
Thomas P. Nikolopoulos ◽  
Nikolaos G. Kavantzas ◽  
Irene E. Lendari ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Laryngeal Cancer ◽  
D1 Protein ◽  
Cyclin D1 Protein

mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators

2005 ◽  
Vol 289 (6) ◽  
pp. C1457-C1465 ◽  
Author(s):  
Gustavo A. Nader ◽  
Thomas J. McLoughlin ◽  
Karyn A. Esser
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Ribosomal Rna ◽  
Molecular Mechanisms ◽  
D1 Protein ◽  
Rna Content ◽  
Cyclin D1 Protein ◽  
Rb Phosphorylation ◽  
Myotube Hypertrophy

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.

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