TRAF6 Dominant Negative Peptides Block Intracellular Signaling Resulting in Anti-Myeloma and Anti-Bone Resorptive Effects in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2919-2919
Author(s):  
Mingjie Li ◽  
Marissa P Dreyer ◽  
Cameryn P Ahles ◽  
David Ramirez ◽  
Cydney M Nichols ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Cell Formation ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Rt Pcr

Abstract Abstract 2919 Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. We have previously reported inhibition of cell proliferation and increase of apoptosis in multiple myeloma (MM) cells through regulation of these intracellular pathways through silencing of TRAF6 C-domain mRNA. To determine TRAF6 protein expression in fresh MM tumor cells, we performed an immunofluorescence assay (IFA). The results showed that expression of this factor in tumor cells from bone marrow (BM) from MM patients with progressive disease is higher than in cells from patients with monoclonal gammopathies without disease progression or normal controls. We further examined the effects of TRAF6 negative dominant peptides on intracellular signaling pathways. Briefly, cells from the RPMI8226 or MM1s MM cell lines or primary MM BM samples were treated with or without TRAF6 inhibition peptide for 24 hours and then stimulated with either IGF1 (30ng/ml) or IL1 β (20ng/ml) for 30 minutes. The cells were lysed and Western blot analysis performed to determine protein phosphorylation and RT-PCR for gene expression. TRAF6 has been found to be an E3 ligase for Akt ubiquitination. We found that IGF1 increased the phosphorylation of AKT and treatment with TRAF6 inhibition peptide markedly decreased its phosphorylation compared to treatment with a control peptide in RPMI8226 and primary MM tumor cells. Downstream of AKT, C-Raf phosphorylation was also significantly reduced with treatment with TRAF6 inhibition peptide. Notably, cyclin D gene expression in MM tumor cells treated with TRAF6 inhibition peptide was reduced as determined with an RT-PCR. In contrast, the gene expression of mTOR was increased in RPMI8226 cells treated with TRAF6 inhibition peptide whereas there was no change in its expression in MM1s and primary MM tumor cells. It is quite possible that the increase in mTOR expression in RPMI8226 cells may act as a negative feedback which results from blockage of the ubiquitination of TRAF6. We further examined the effect of the TRAF6 inhibition peptide on NF-kB and JNK signaling as determined through evaluation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-kB protein was reduced and phosphorylation of JNKK was clearly decreased with exposure to the TRAF6 inhibition peptide. We examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. Total endogenous c-Jun is reduced following exposure of RPMI8226 cells to the TRAF6 inhibition peptide. Consistent with our past findings, TRAF6 inhibition peptide significantly inhibited osteoclast formation from CD14+ induced by RANKL and M-CSF with in a concentration dependent fashion whereas control peptides showed no effects on osteoclast formation. In addition, inhibition of the TRAF6 signaling blocked not only myeloma cell proliferation induced by AKT and NF-kB activation but also osteoclast cell formation mediated through transcription at TRE/AP-1 elements. The study has been extended to our SCID-hu murine model of human myeloma. Disclosures: No relevant conflicts of interest to declare.

TRAF6-Dominant Negative Peptides Show Potent Inhibitory Effects On Multiple Myeloma, Osteoclast Formation and Bone Resorption.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 611-611
Author(s):  
Mingjie Li ◽  
Eric Sanchez ◽  
Jennifer Li ◽  
Cathy S Wang ◽  
Jing Shen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Bone Resorption ◽  
Tumor Cell ◽  
Cell Apoptosis ◽  
Cell Formation ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Control Peptide

Abstract Abstract 611 Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating NF-κB and JNK signal transduction pathway resulting in inhibition of tumor cell proliferation and osteoclast formation. The unique biological function of TRAF6 is largely determined within its TRAF-C domain which does not interact with peptide motifs that are recognized by other TRAFs including 1, 2, 3 or 5. We have recently reported inhibition of cell proliferation and increased apoptosis of multiple myeloma (MM) cells through regulation of the NF-κB and JNK pathways through silencing the TRAF6 C-domain mRNA. In this study, we determined the effects of TRAF6 dominant negative peptides on MM cells, osteoclast formation and bone resorption. We cloned a 167 amino acid (in residues 333 to 508) fragment to produce a TRAF6 negative dominant (TRAF6dn) construct and synthesized an inhibitory decoy peptide of the TRAF6 interaction domain with CD40 and another peptide interacting with the TRAF6-RANK binding domain as well as a control peptide. All peptides were synthesized with a 16 amino acid permeable peptide. Using the MM1s, RPMI8226, and U266, we evaluated the effects of these peptides on MM tumor cell growth using an MTS assay and apoptosis with an Annexin V assay. We found that TRAF6dn peptides significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on induction of apoptosis in MM cells were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration-dependent fashion. We found that TRAF6dn also markedly inhibited osteoclast cell formation from freshly derived human monocytes induced by RANKL and M-CSF in a concentration-dependent fashion comparing with cells exposed to control peptide. We further examined the effects on MM cell apoptosis of the TRAF6 decoy or CD40 decoy peptides alone and in cells exposed to the combination of both peptides. The results showed either decoy peptide alone slightly induced apoptosis of MM tumor cells whereas the combination of both peptides demonstrated marked apoptosis of MM cells. We also showed that although melphalan alone induced apoptotic cell death, this effect was markedly enhanced when this alkylating agent was combined with the TRAF6 decoy peptide. Although the CD40 peptide alone did not inhibit osteoclast formation, TRAF6 decoy peptide alone and the combination of both decoy peptides markedly inhibited formation of these bone resorbing cells. We also examined the effects of TRAF6dn on NF-κB and JNK by measuring JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels and phosphorylation of JNKK are both markedly reduced when MM cells are exposed to TRAF6dn fragment or TRAF6 decoy peptide. These studies suggest that TRAF6dn or the combination of TRAF6 decoy and CD40 decoy peptides may be excellent targets to block both myeloma cell and osteoclast cell formation. The study has been extended to assess the effects of these peptides in vivo using our SCID-hu murine model of human myeloma. Disclosures: No relevant conflicts of interest to declare.

Blockage of TRAF6 by Dominant Negative Peptides to Inhibit Multiple Myeloma (MM) Cell Proliferation and Osteoclast Formation through NF-κB, JNK and AKT Signal Transduction Pathways

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4068-4068 ◽  
Author(s):  
Mingjie Li ◽  
Eric Sanchez ◽  
Cathy Wang ◽  
Megan Schultz ◽  
Jessica Wang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Tumor Cells ◽  
Cell Apoptosis ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Control Group ◽  
Affinity Column

Abstract Abstract 4068 Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5. We have reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-κB and JNK pathways through silencing TRAF6 C-domain mRNA and the dominant negative peptide expression vector (Chen H. et al, Oncogene, 2006; Li M. et al, Blood 2009). TRAF6 have been recently found as a ligase for Akt ubiquitination (Yang WL et al, Science, 2009). Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. In this study, we first investigated whether TRAF6 is over-expressed in MM tumor cells. Twelve MM fresh bone marrow (BM) aspirates derived from MM patients were assessed using Western blot analysis and immunohistochemical staining with anti-TRAF6 antibody. We found that TRAF6 protein was highly expressed in tumor cells from MM patients compared to normal human BM samples. Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain binding residues. We found that TRAF6 dominant negative binding peptide (TRAF6dn) significantly inhibited MM cell proliferation maximally at 72 hours using the MTS cell proliferation assay whereas effects on inducing MM cell apoptosis were most prominent at 48 hours as assessed with Annexin V staining with flow cytometric analysis. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration peptide-dependent fashion. Furthermore, phosphorylation of both AKT and NF-κB were also reduced using our human TRAF6dn or decoy peptides. We also examined the effect of the TRAF6dn peptide on the JNK pathway since this signaling pathway is also associated with cell cycle effects in MM. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is markedly reduced after treatment with the TRAF6dn peptide. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. We evaluated the effect of TRAF6dn peptide on osteoclast formation using cells from human monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' BM or peripheral blood mononuclear cells. The monocytes were cultured on slide-culture dishes (2 × 105 cells/well).We found TRAF6dn markedly inhibited osteoclast cell formation from monocytes induced with RANKL and mCSF in a concentration- dependent fashion compared with a control group using tartrate resistant acid phosphatase staining. We further assessed whether TRAF6dn can reduce bone resorption using a dentin bone resorption assay. BM-derived monocytes were isolated as above and were cultured on dentin bone slides (4 × 105 cells/slide). The cells treated with a TRAF6dn peptide or the control peptide, were incubated with 50ng/ml RANKL and 10ng/ml MCSF. All cells were cultured for 21 days. It was found that TRAF6dn significantly inhibited lacunar resorption in a concentration-dependent fashion. These studies suggest that TRAF6 is over-expressed in MM and our TRAF6dn peptide inhibits many signaling pathways critical to the growth of MM and formation of osteoclasts resulting in marked anti-MM effects and reduction in osteoclast formation resulting in marked inhibition of bone resorption. Thus, this novel approach may offer a new therapeutic approach to both treat multiple myeloma and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures: No relevant conflicts of interest to declare.

Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5362-5362
Author(s):  
Mingjie Li ◽  
Suzie Vardanyan ◽  
Jillian Gottlieb ◽  
Cathy Wang ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Healthy Subjects ◽  
Progressive Disease ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Dependent Manner

Abstract Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling. First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects.  The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM. We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission. Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures: No relevant conflicts of interest to declare.

Blockage of TRAF6 Signaling Inhibits Osteoclast Formation and Tumor Cell through Blocking the NF-kB and JNK Pathways in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2511-2511
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Jennifer Li ◽  
Richard A. Campbell ◽  
Cathy S. Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Control Group ◽  
Affinity Column ◽  
Tartrate Resistant Acid Phosphatase ◽  
Control Vector

Abstract We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients’ peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

Triggering of Toll-Like Receptor 4 Expression in Human Multiple Myeloma Promotes Proliferation and Protects the Cells From Immune and Chemotherapy Drug Attack.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2843-2843
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Lijuan Wang ◽  
Yang Yang ◽  
Yi Zhao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Lines ◽  
Immune Escape ◽  
Conflicts Of Interest ◽  
Rt Pcr ◽  
Facs Analysis ◽  
Mapk Pathways ◽  
Tlr4 Ligand

Abstract Abstract 2843 Poster Board II-819 Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll like receptors (TLRs) are essential in the host defense against infections. In MM, recurrent infections could promote tumor growth and favor escape from standard therapies. TLRs initiated responses in B cells include proliferation, antiapoptosis and immune escape. In this study, we first screened four MM cell lines, MM1-r, MM1-s, RPMI8226, and U266, for the expression of major TLRs (including TLR 1–2,4-7and 9) by RT-PCR. Surprisingly, all the MM cell lines expressed multiple TLRs. We focused on TLR4, which had the most strongly expressed mRNA. Consistent with the RT-PCR result, fluorescence-activated cell sorting (FACS) analysis revealed that TLR4 protein was also present on the surface of MM cell lines. By FACS analysis, we found that MM1-s and MM1-r cells had increased TLR4 co-receptor CD14 expression when induced by lipopolysaccharide (LPS). We next asked if TLR4 was functional in MM cells. To activate TLR4 in MM cells (MM1-r, MM1-s, RPMI8226, and U266), we incubated the cells with LPS, the natural ligand for TLR4 and measured cell proliferation by [3H]thymidine incorporation. Proliferation of MM1-s and MM1-r cells increased significantly, but that of U266 and RPMI8226 cells did not. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of MM cells. We pretreated MM cells with lipopolysaccharide followed by induction by adriamycin. Our results showed that LPS could save MM1-s cells from adriamycin induced apoptosis. In addition to proliferation and apoptosis, we would like to learn whether TLR4 ligand could change cell cycle, We found that MM1-s and MM1-r MM cells showed decreased number in G0/G1 phase but increased number in S phase when induced by LPS. To explore whether MAPK pathways were implicated in MM1-s and MM1-r cells to LPS stimulation, we investigated the role of ERK1/2, p38, and JNK MAPKs in LPS-stimulated cells by using specific inhibitors of MAPK pathways, Firstly, time-dependent MAPKs phosphorylation was measured to assess the activation of these kinases upon treatment with LPS. ERK1/2, p38, and JNK phosphorylation and NF-κB were significantly up-regulated following LPS treatment, with the maximum phosphorylation occurring at 20min after stimulation. It is interesting to note that JNK inhibitors inhibited phosphorylation of JNK and inhibited phosphorylation of p-38 simultaneously. Moreover, inhibitors of ERK1/2 inhibited phosphorylation of ERK1/2, increased phosphorylation of p-38 simultaneously, suggesting that ERK, JNK and p38 pathways were associated. Our findings demonstrated that LPS-induced cell proliferation dependent on JNK, ERK and p38 signaling, suggesting that ERK and p38 pathways were involved in MM1-s and MM1-r cells proliferation. Since TLRs activate innate and adaptive immune responses, we further study immunoregulatory factor IL-6, IL-12 and IL-18 secretion in the culture supernatants induced by LPS. LPS increased the production of IL-6 in RPMI8226 and U266 cells, and increased the production of IL-18 in MM1-r and MM1-s cells. To further examine the effect of LPS on immune escape, MM1-s cells were co-cultured with T cells from donors and 3H incorporation was examined for T cells proliferation. Our results showed that T cells proliferation was decreased when the cells were treated by LPS, suggesting that TLR4 ligand LPS facilitates MM1-s cells' evasion of immune surveillance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Octreotide and Pasireotide Combination Treatment in Somatotroph Tumor Cells: Predominant Role of SST2 in Mediating Ligand Effects

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1816
Author(s):  
Jessica Amarù ◽  
Federica Barbieri ◽  
Marica Arvigo ◽  
Agnese Solari ◽  
Adriana Bajetto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cells ◽  
Intracellular Signaling ◽  
Somatostatin Receptor ◽  
Combined Treatment ◽  
Primary Cultures ◽  
Receptor Subtype ◽  
Camp Accumulation ◽  
Gh Secretion ◽  
Somatostatin Receptor Ligands

First-generation somatostatin receptor ligands (fg-SRLs), such as octreotide (OCT), represent the first-line medical therapy in acromegaly. Fg-SRLs show a preferential binding affinity for somatostatin receptor subtype-2 (SST2), while the second-generation ligand, pasireotide (PAS), has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). Whether PAS acts via SST2 in somatotroph tumors, or through other SSTs (e.g., SST5), is a matter of debate. In this light, the combined treatment OCT+PAS could result in additive/synergistic effects. We evaluated the efficacy of OCT and PAS (alone and in combination) on growth hormone (GH) secretion in primary cultures from human somatotroph tumors, as well as on cell proliferation, intracellular signaling and receptor trafficking in the rat GH4C1 cell line. The results confirmed the superimposable efficacy of OCT and PAS in reducing GH secretion (primary cultures), cell proliferation, cAMP accumulation and intracellular [Ca2+] increase (GH4C1 cells), without any additive effect observed for OCT+PAS. In GH4C1 cells, co-incubation with a SST2-selective antagonist reversed the inhibitory effect of OCT and PAS on cell proliferation and cAMP accumulation, while both compounds resulted in a robust internalization of SST2 (but not SST5). In conclusion, OCT and PAS seem to act mainly through SST2 in somatotroph tumor cells in vitro, without inducing any additive/synergistic effect when tested in combination.

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

2011 ◽  
Vol 300 (1) ◽  
pp. F177-F188 ◽  
Author(s):  
Masanori Kugita ◽  
Kazuhiro Nishii ◽  
Miwa Morita ◽  
Daisuke Yoshihara ◽  
Hiroe Kowa-Sugiyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Kidney Disease ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Early Stage ◽  
Global Gene Expression ◽  
Rt Pcr ◽  
Polycystic Kidney ◽  
Global Gene Expression Profiling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated “Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation,” “LPS/IL-1-Mediated Inhibition of RXR Function,” and “Liver X Receptor (LXR)/RXR Activation.” These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

Endothelial Cells Induce Multiple Myeloma Cell Proliferation Protect Against Conventional and Novel Therapies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2354-2354
Author(s):  
Shaji Kumar ◽  
Noopur Raje ◽  
Teru Hideshima ◽  
Klaus Podar ◽  
Kenji Ishitsuka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Tumor Cell ◽  
Blood Vessels ◽  
Intracellular Signaling ◽  
De Novo ◽  
Cell Contact ◽  
Protective Effects ◽  
Tumor Cell Proliferation

Abstract Angiogenesis or formation of new blood vessels from existing blood vessels, in contrast to vasculogenesis or de novo formation of new vessels, plays an important role in the progression and spread of most cancers. Multiple myeloma (MM) is characterized by increased microvessel density (MVD), a quantitative estimate of angiogenesis, which correlates with stage of disease. MVD increases with progression from MGUS to smoldering MM to newly diagnosed MM and relapsed MM. It is a powerful prognostic factor, predicting for overall survival. To further elucidate the biological basis for the prognostic value of increased angiogenesis in MM, we studied the interactions of MM cells with endothelial cells using HUVECS as a model system. Co-culture of MM cells (MM1.S, OPM2, U266) with HUVECS induced tumor cell proliferation. Enhanced tumor cell proliferation correlated with the number of HUVECs and was greater than that triggered by co-culture with patient bone marrow stromal cells. When HUVECs were fixed prior to co-culture there was a significant decrease in the tumor cell proliferation. Addition of HUVEC conditioned media to the MM cell lines also induced proliferation. Importantly, HUVECS protected against anti-MM agents including conventional agents (Dexamethasone, Doxorubicin, Melphalan) and novel drugs (Revlimid™). The protective effect afforded by co-culture was lost on HUVEC fixation. Intracellular signaling events following MM cell-endothelial cell contact were studied to understand the mechanisms of the proliferative and protective effects. Western blotting demonstrated activation of the JAK/STAT, PI3K/Akt and the MAPK pathways, mediating proliferation and survival. Ongoing studies focused on understanding cytokine as well as adhesion-mediated interactions between the endothelial cells and the MM cells will identify targets for new therapeutic approaches in MM.

Peripheral Blood Cells from Multiple Myeloma Patients Show Increased Osteoclast and Decreased Osteoblast Gene Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5098-5098
Author(s):  
Melinda S. Gordon ◽  
Ariana M. Berenson ◽  
Charles B. Drucker ◽  
Matthew Katz ◽  
Hee Jin Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Alkaline Phosphatase ◽  
Bone Disease ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Bisphosphonate Treatment ◽  
Rt Pcr ◽  
Circulating Cells ◽  
Osteolytic Bone Disease

Abstract Bone resorption leading to osteolytic bone disease is characteristic of multiple myeloma (MM). Recent studies show the presence of bone-resorbing osteoclasts and bone-forming osteoclasts in the circulation, and these cells may correlate with bone disease and change with anti-bone resorptive therapies. We have investigated whether there is an imbalance in the expression of osteoblast and osteoclast genes in the peripheral blood mononuclear cells (PBMCs) from MM patients relative to normal age-matched controls and the effect of bisphosphonate treatment on the expression of these genes. We analyzed the expression of a panel of osteoblast-related (bone alkaline phosphatase [bone AP], bone morphogenic protein 2 [BMP2], collagen I and osteocalcin) and osteoclast-related (b3 integrin, calcitonin, receptor for activation of nuclear factor kappa B [RANK] and tartrate-resistant alkaline phosphatase [TRAP]) genes by semi-quantitative RT-PCR on total RNA isolated from PBMCs obtained following density gradient separation. We demonstrated that the expression of the osteoblast-related gene BMP2 was reduced in eight of nine MM patients when compared with normal donors. In marked contrast, three osteoclast-related genes, b3 integrin, RANK and TRAP, were more highly expressed in all nine MM patients compared to the normal donors; only calcitonin expression was similar to the control subjects. Interestingly, patients receiving bisphosphonate treatment appeared to show increased osteoblast gene expression with higher amounts of bone AP, BMP2 and osteocalcin RNA compared to the patients not receiving anti-bone resorptive therapy. However, there was no alteration in the level of the RNA in any of the four osteoclast genes compared to patients not receiving anti-bone resorptive therapy. We are extending our analysis to a larger panel of MM patients in order to determine the relationship between these circulating cells and bone disease, overall clinical status and change in their levels with anti-bone resorptive therapy. In addition, we are also investigating whether there exist larger and smaller numbers of circulating osteoclasts and osteoblasts, respectively, in MM patients, or whether these circulating cells show alteration of their expression of these genes. Our semi-quantitative RT-PCR results are being correlated with immunohistochemical staining results from osteoblast and osteoclast markers obtained on PBMCs from MM and normal subjects. These studies provide evidence that the number of circulating osteoblasts and osteoclasts is altered in patients with MM, and also may suggest that bisphosphonate therapy may also be associated with changes in these cell populations.

Clonotypic Bone Marrow-Derived Endothelial Progenitor Cells in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3497-3497
Author(s):  
Marc J. Braunstein ◽  
Daniel R. Carrasco ◽  
David Kahn ◽  
Kumar Sukhdeo ◽  
Alexei Protopopov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Resolution ◽  
Gene Expression Profiling ◽  
Progenitor Cells ◽  
Tumor Cells ◽  
Endothelial Progenitor Cells ◽  
Expression Profiling ◽  
Gains And Losses

Abstract In multiple myeloma (MM), bone marrow-derived endothelial progenitor cells (EPCs) contribute to tumor neoangiogenesis and their levels covary with tumor mass and prognosis. Recent X-chromosome inactivation studies in female patients showed that, similar to tumor cells, EPCs are clonally restricted in MM. Genomic profiling of MM using high-resolution array comparative genomic hybridization (aCGH) has been previously utilized to mine the genome and find clinical correlates in MM patients. In this study, clonotypic aspects of bone marrow-derived EPCs and MM cells were investigated using aCGH and expression profiling analysis. Confluent EPCs were outgrown from bone marrow aspirates by adherence to laminin. EPCs were >98% vWF/CD133/KDR+ and <1% CD38+. The laminin-nonadherent bone marrow fraction enriched for tumor cells was >50% CD38+. For aCGH and for gene expression profiling, genomic DNA and total RNA from EPCs and MM cells were hybridized to human oligonucleotide arrays (Agilent Technologies) and human cDNA microarrays (Affymetrix), respectively. High resolution aCGH with segmentation analysis showed that EPCs and MM cells in one of ten cases share identical patterns of chromosomal gains and losses, while another 5 cases shared multiple focal copy number alterations (CNAs) including gains and losses. The genomes of EPCs and MM cells additionally displayed exclusive CNAs, but these were far fewer in EPCs than in MM cells. In 3 patients, EPCs harbored a common 0.6Mb deletion at 1q21 not shared by MM cells. Pertinent genes in this region that could affect proliferation and tumor suppression include N2N, NBPF10, and TXNIP. Validation studies of aCGH findings by other methods are ongoing. Gene expression profiling showed decreased expression of 1q21 region genes (e.g., calgranulin C and lamin A/C). A genome-wide comparison of patients’ MM cells and EPCs, which is focused on their shared genetic characteristics, will be presented.

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