The purpose of this study was to investigate development and age‐specific novel alternative splicing patterns in the longevity genes age‐1 and clk‐1. The nematode Caenorhabditis elegans was used as the model organism, from which mRNA was harvested at different points in their developmental cycle (eggs, larval 3 (L3), dauer, adults, geriatrics). Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis was performed to detect the presence of mRNA of different sizes at different genetic locations throughout the gene, which would suggest alternative mRNA isoforms. The fragments were sequenced (eggs L3, geriatrics) to verify actual splicing reactions. The results indicate novel instances of intron inclusion and exon exclusion with the latter being associated specifically with developmental stage L3.
120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.
134 Alteration in expression and function of transcription factors has been frequently associated with neoplastic transformation. We here provide both experimental and clinical evidence that Sp1, a transcription factor that controls number of cellular processes, plays an important regulatory role in MM cell growth and survival. Although Sp1 is ubiquitously expressed, its nuclear localization observed in MM is functionally both relevant and important. We have confirmed high Sp1 activity in MM cells both by demonstrating increased DNA binding as well as increased Sp1-responsive promoter activity measured by luciferase reporter assay. MM cell-BMSC interaction led to Sp1 activation which was completely abrogated by the ERK pathway inhibitor U0126 but not by the AKT inhibitor LY29004. Furthermore, using both SiRNA and ShRNA mediated Sp1 knock-down, we have confirmed the growth and survival effects of Sp1 on MM cell growth. Using gene expression profile of MM cells from 172 uniformly treated patients, we have further confirmed these in vitro results by observing that overexpression of Sp1-related genes, including HIF-1a, HDAC1 and MAPK genes, correlate with poor prognosis in MM. This clinical correlation further suggests the role of Sp1 in MM biology, providing the rationale to preclinically target Sp1 in MM. We have investigated TMP, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA) which disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity. We have previously confirmed specific inhibition of both Sp1 binding and transcriptional activity in MM cells by TMP, including in the context of MM-BMSC interaction. Along with inhibition of Sp1 activity, we observed both in vitro and in vivo in murine models of human myeloma, anti-myeloma effect of TMP. Importantly, there was no significant synergistic effect when MM cells transfected with Sp1 siRNA were treated with TMP, confirming specificity of TMP's mechanism of action. We have further observed activation of the mitochondrial apoptotic pathway by TMP via activation of caspase-3, -9 and -7 and PARP cleavage while caspase-8 was not activated suggesting possible synergism with activators of alternate apoptotic pathways. We have also observed that lenalidomide and dexamethasone upregulate Sp1 activity, suggesting that Sp1 may be a common resistance mechanism. We have confirmed that the increased Sp1 activity by lenalidomide or dexamethasone is abrogated by TMP and the combination provides synergistic cytotoxicity, in MM cell lines as well as primary MM cells. In conclusion, we report significant role of Sp1 in myeloma cell growth, survival and drug resistance with its influence on clinical outcome in MM. Our results suggest that specific inhibition of Sp1 activity may be an important therapeutic target in MM. Disclosures: Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.
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