ABSTRACT
Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.
Subject(s)
Cell Membrane/physiology , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Membrane Fluidity/genetics , Membrane Proteins/genetics , Neoplasms/pathology , Sphingosine N-Acyltransferase/genetics , Cells, Cultured , Down-Regulation , Gene Expression Regulation, Neoplastic , HL-60 Cells , Humans , Jurkat Cells , K562 CellsABSTRACT
The transcription factor E2F1 has a crucial role in the control of cell growth and has been shown to regulate neoangiogenesis in a p53-dependent manner through inhibition of activity of the VEGF-A (vascular endothelial growth factor) promoter. Besides being regulated by transcription, VEGF-A is also highly regulated by pre-mRNA alternative splicing, resulting in the expression of several VEGF isoforms with either pro-(VEGF(xxx)) or anti-(VEGF(xxx)b) angiogenic properties. Recently, we identified the SR (Ser-Rich/Arg) protein SC35, a splicing factor, as a new transcriptional target of E2F1. Here, we show that E2F1 downregulates the activity of the VEGF-A promoter in tumour cells independently of p53, leading to a strong decrease in VEGF(xxx) mRNA levels. We further show that, strikingly, E2F1 alters the ratio of pro-VEGF(xxx) versus anti-VEGF(xxx)b angiogenic isoforms, favouring the antiangiogenic isoforms, by a mechanism involving the induction of SC35 expression. Finally, using lung tumour xenografts in nude mice, we provide evidence that E2F1 and SC35 proteins increase the VEGF(165)b/VEGF ratio and decrease tumour neovascularization in vivo. Overall, these findings highlight E2F1 and SC35 as two regulators of the VEGF(xxx)/VEGF(xxx)b angiogenic switch in human cancer cells, a role that could be crucial during tumour progression, as well as in tumour response to antiangiogenic therapies.
Subject(s)
Angiogenesis Inhibitors/biosynthesis , E2F1 Transcription Factor/metabolism , Nuclear Proteins/metabolism , Ribonucleoproteins/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Angiogenesis Inhibitors/genetics , Angiogenesis Inhibitors/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Down-Regulation , E2F1 Transcription Factor/genetics , Female , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Nuclear Proteins/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteins/genetics , Proteins/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoproteins/genetics , Serine-Arginine Splicing Factors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The transcription factor E2F1 has a key function during S phase progression and apoptosis. It has been well-demonstrated that the apoptotic function of E2F1 involves its ability to transactivate pro-apoptotic target genes. Alternative splicing of pre-mRNAs also has an important function in the regulation of apoptosis. In this study, we identify the splicing factor SC35, a member of the Ser-Rich Arg (SR) proteins family, as a new transcriptional target of E2F1. We demonstrate that E2F1 requires SC35 to switch the alternative splicing profile of various apoptotic genes such as c-flip, caspases-8 and -9 and Bcl-x, towards the expression of pro-apoptotic splice variants. Finally, we provide evidence that E2F1 upregulates SC35 in response to DNA-damaging agents and show that SC35 is required for apoptosis in response to these drugs. Taken together, these results demonstrate that E2F1 controls pre-mRNA processing events to induce apoptosis and identify the SC35 SR protein as a key direct E2F1-target in this setting.
Subject(s)
Alternative Splicing/genetics , Apoptosis/genetics , E2F1 Transcription Factor/metabolism , Nuclear Proteins/genetics , Ribonucleoproteins/genetics , Up-Regulation/genetics , Alternative Splicing/drug effects , Animals , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cyclophosphamide/pharmacology , DNA Damage , Gene Expression Regulation/drug effects , Humans , Mice , Nuclear Proteins/metabolism , Protein Binding/drug effects , RNA Precursors/metabolism , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors , Transcription, Genetic/drug effects , Up-Regulation/drug effects , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
Research into the biomechanical properties of tendons had led to a better understanding of the functional and pathophysiologic processes that occur in vivo, particularly in the setting of tendon injury and failure. Increasingly, biomechanical information is being utilized in the clinical setting, guiding patient management in certain circumstances. Differentiating minor and self-limiting abnormalities from those in which specific treatment may prevent further disability from altered biomechanics may become an important role for imaging. This article examines normal tendon anatomy and its relationship to tendon biomechanics under both physiological and pathologic conditions. Imaging characteristics of tendons in the physiologic and pathologic states are discussed. We have focused primarily on MRI, as this modality has been studied most intensively and has been shown to offer additional information regarding unsuspected pathology.
Subject(s)
Tendon Injuries/physiopathology , Tendons/physiology , Biomechanical Phenomena , Diagnostic Imaging , Humans , Tendon Injuries/diagnosis , Tendons/anatomy & histologyABSTRACT
Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.
