ABSTRACT
Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Oocytes/metabolism , Oogenesis/genetics , Real-Time Polymerase Chain Reaction/methods , Animals , Female , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Mice , Oocytes/cytology , Peptide Elongation Factors/toxicity , Reference Standards , Ribosomal Proteins/genetics , Tumor Suppressor Proteins/toxicityABSTRACT
The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Membrane/metabolism , Cell-Penetrating Peptides/pharmacology , Peptides, Cyclic/pharmacology , Tumor Suppressor Proteins/pharmacology , Amino Acid Sequence , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/toxicity , Helix-Loop-Helix Motifs , Humans , Lipid Bilayers/metabolism , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/toxicity , Protein Binding/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/chemical synthesis , Tumor Suppressor Proteins/toxicityABSTRACT
The presence of a complete (BH1-3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells. It is unclear, however, what distinct roles the members of the large family of BH3-only proapoptotic molecules play in apoptosis. Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict. We have, therefore, established three functional categories of BH3-only human proapoptotic proteins based on their toxicity after expression in budding yeast: directly killing (tBid), sensitizing in Bax/Bcl-2 expressing cells (Bad or Puma), and non-toxic (BNip3, BNip3L, and Noxa). The mechanism of killing by the proapoptotic molecules in yeast, however, is not due to activation of the recently described yeast metacaspase MCA1.
