ABSTRACT
BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-X(L), involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-X(L), we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Membrane Proteins/metabolism , Peptides/metabolism , Proto-Oncogene Proteins/metabolism , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolism , Amino Acid Sequence , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Cells, Cultured , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Lymphocytes/cytology , Lymphocytes/physiology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , Neoplasm Transplantation , Peptides/chemistry , Peptides/genetics , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Transplantation, Heterologous , Tumor Cells, Cultured , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/geneticsABSTRACT
The p53 family comprises three genes that encode for p53, p63 and p73. These genes have a significant degree of sequence homology, especially in the central sequence-specific DNA-binding domain. The high homology among the three DNA-binding domains indicates that these transcription factors have identical residues interacting with DNA, and thus potentially can recognize the same transcriptional targets. In this study, we demonstrate that PKCdelta is induced by p63 and p73 in Saos2 cells. The putative human PKCdelta promoter harbours three p53-like binding sites (RE I, RE II, RE III). In order to confirm the transactivation of PKCdelta by p53 family members, we performed transcription assays using the entire or selected regions of the promoter upstream of a luciferase reporter gene. The results obtained demonstrated that, at least in vitro, the p53 family members tested (TAp63alpha, TAp73alpha, DeltaNp63alpha, but not DeltaNp73alpha) were able to drive transcription from the PKCdelta promoter.
Subject(s)
DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Protein Kinase C-delta/genetics , Transcriptional Activation/physiology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Humans , Keratinocytes , Mutagenesis, Site-Directed , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Protein Kinase C-delta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
The role of p53 in modifying sensitivity to cytotoxic drugs has been commonly studied by creating transfection pairs of wt p53 parental cells and altered p53 daughter cells, or vice versa. Authors inevitably tended to extrapolate and generalize their experimental observations, and conflicting reports have been more the rule than the exception. We have performed a meta-analysis of 356 independent studies. Average changes of drug sensitivity after a change of p53 status were observed. E6 transfection predominantly induces sensitization to cytotoxic drugs, whereas p53-/- knockout cells are more drug-resistant than their normal p53+/+ counterpart. Unexpectedly, transfection with a mutated p53 does not change much the drug sensitivity of most wt p53 cancer lines, with the notable exception of A2780, a predominant cell line in the studies analyzed (A2780 cells show increased resistance after transfection with a mutated p53). Rather interestingly, mitotic spindle poisons acted differently from other classes of cytotoxic drugs. A crucial indication of our findings is that the role of p53 alone in determining sensitivity/resistance to cytotoxic drugs is limited: the individual molecular pathology and differentiation of a given cancer line prevail over any average trend, and are causal to a broad spreading of the data. We also identify major "confounding factors", alias independent categorical variables, capable of affecting the average outcome.