ABSTRACT
TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.
Subject(s)
Cell Membrane Permeability , Epidermal Cells , Keratinocytes/cytology , Membrane Proteins/physiology , Morphogenesis/physiology , Animals , Blotting, Western , Cell Differentiation , Cell Proliferation , Cells, Cultured , Epidermis/metabolism , Female , Humans , Keratinocytes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organogenesis/physiology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin Physiological PhenomenaABSTRACT
BACKGROUND: Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. METHODS: MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. RESULTS: MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. CONCLUSION: Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance.
Subject(s)
Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/genetics , Membrane Proteins/genetics , Transcriptome/genetics , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Hypoxia , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Drug Resistance, Neoplasm/drug effects , Epirubicin/pharmacology , Etoposide/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Kaplan-Meier Estimate , Keratinocytes/drug effects , Keratinocytes/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Membrane Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Paclitaxel/pharmacology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transcriptome/drug effectsABSTRACT
BACKGROUND: In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers. RESULTS: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle. CONCLUSIONS: These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Muscle, Skeletal/metabolism , Neoplasm Proteins/biosynthesis , Animals , Cell Cycle/genetics , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/genetics , Mice , Mice, Knockout , Mice, Mutant Strains , Muscle Development , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/injuries , Muscle, Skeletal/pathology , Muscular Diseases/pathology , Muscular Diseases/physiopathology , Neoplasm Proteins/genetics , Regeneration , Satellite Cells, Skeletal MuscleABSTRACT
Human tumor development is often associated with a DNA demethylation process. This results in the activation of germline-specific genes, such as MAGE-A1, which rely on DNA methylation for repression in somatic tissues. Here, we searched to identify a cell line possessing ongoing DNA demethylation activity targeted to MAGE-A1. We first assessed MAGE-A1-expressing human tumor cell lines, by evaluating their ability to induce demethylation of MAGE-A1 transgenes that were methylated in vitro before transfection. All cell lines lacked such activity, suggesting that MAGE-A1 hypomethylation in tumors results from a past demethylation event. We then turned to mouse embryonic stem (mES) cells, which are characterized by a high level of methylation plasticity. Interestingly, in vitro methylated MAGE-A1 transgenes became demethylated after transfection into mES cells. Demethylation was targeted to the 5'-region of MAGE-A1 and was strongly reduced at mutated MAGE-A1 transgenes exhibiting impaired promoter activity. Our results indicate that mES cells induce demethylation of MAGE-A1 and represent therefore a valuable system to study this tumor-related process.
