ABSTRACT
A new method is proposed which combines the high spatial resolution of transmission electron microscopy with information on the dynamics of transcription. Incorporation of two different RNA precursors was used to define a time transcription window on cultured cells treated with hypometabolizing peptides which are known to modulate transcription. This procedure allows detecting a single fibril of newly synthesized RNA in the time range in which it is transcribed.
Subject(s)
Enkephalin, Leucine-2-Alanine/pharmacology , RNA Precursors/chemistry , RNA, Ribosomal/genetics , Transcription, Genetic/genetics , Cell Line, Tumor , Cell Nucleus , Cell Proliferation , Dactinomycin/pharmacology , Dichlororibofuranosylbenzimidazole/pharmacology , Enkephalin, Leucine-2-Alanine/analogs & derivatives , HeLa Cells , Humans , Immunohistochemistry/methods , Microscopy, Electron, Transmission/methods , RNA Precursors/genetics , RNA, Ribosomal/biosynthesis , Staining and Labeling/methods , Transcription, Genetic/drug effects , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.
Subject(s)
Autophagy/drug effects , DNA/metabolism , Gene Silencing/drug effects , Glioma/pathology , Oligonucleotides, Antisense/pharmacology , PrPC Proteins/genetics , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 7 , Beclin-1 , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Cytoprotection/drug effects , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glioma/genetics , Glioma/ultrastructure , Humans , Membrane Proteins/metabolism , Mice , PrPC Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Ubiquitin-Activating Enzymes/metabolismABSTRACT
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2(R33Q/R33Q) mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2(R33Q/R33Q) mice develop bidirectional VT on exposure to environmental stress whereas CASQ2(R33Q/R33Q) myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2(R33Q/R33Q) hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2(R33Q/R33Q) CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.
Subject(s)
Arrhythmias, Cardiac/genetics , Calsequestrin/genetics , Mutation, Missense/physiology , Animals , Arrhythmias, Cardiac/etiology , Calcium/analysis , Calsequestrin/physiology , Disease Models, Animal , Electrophysiology , Homozygote , Mice , Mice, Transgenic , Phenotype , Sarcoplasmic Reticulum/chemistryABSTRACT
Perichromatin fibrils represent the morphological expression of transcription and co-transcriptional processing of pre-mRNA. They can be considered, hence, an example of work in progress. High resolution techniques such as electron microscopy demonstrate that perichromatin fibrils play a role as early markers of transcriptional alterations. In this paper, we review some experimental and physiological conditions impairing or modulating transcription as well as their effects on perichromatin fibrils. In all the situations reported, perichromatin fibrils show modifications in their amount and/or their associated proteins. Their movements are also affected, as well as their export or their intra-nuclear storage forms. Perichromatin fibrils therefore represent highly sensitive markers not only for monitoring transcriptional and processing rate but also for identifying the maturation level of pre-mRNA/mRNA occurring in the cell nucleus and the functional correlation with the cellular metabolic state.
