Clonage et analyse de la protéine suppresseur de tumeur P53 chez l'axolotl

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer

<p>Abstract</p> <p>Background</p> <p>Urodele amphibians like the axolotl are unique among vertebrates in their ability to regenerate and their resistance to develop cancers. It is unknown whether these traits are linked at the molecular level.</p> <p>Results</p> <p>Blocking p53 signaling in axolotls using the p53 inhibitor, pifithrin-α, inhibited limb regeneration and the expression of p53 target genes such as Mdm2 and Gadd45, suggesting a link between tumor suppression and regeneration. To understand this relationship we cloned the p53 gene from axolotl. When comparing its sequence with p53 from other organisms, and more specifically human we observed multiple amino acids changes found in human tumors. Phylogenetic analysis of p53 protein sequences from various species is in general agreement with standard vertebrate phylogeny; however, both mice-like rodents and teleost fishes are fast evolving. This leads to long branch attraction resulting in an artefactual basal emergence of these groups in the phylogenetic tree. It is tempting to assume a correlation between certain life style traits (e.g. lifespan) and the evolutionary rate of the corresponding p53 sequences. Functional assays of the axolotl p53 in human or axolotl cells using p53 promoter reporters demonstrated a temperature sensitivity (ts), which was further confirmed by performing colony assays at 37°C. In addition, axolotl p53 was capable of efficient transactivation at the Hmd2 promoter but has moderate activity at the p21 promoter. Endogenous axolotl p53 was activated following UV irradiation (100 j/m²) or treatment with an alkylating agent as measured using serine 15 phosphorylation and the expression of the endogenous p53 target Gadd45.</p> <p>Conclusion</p> <p>Urodele p53 may play a role in regeneration and has evolved to contain multiple amino acid changes predicted to render the human protein defective in tumor suppression. Some of these mutations were probably selected to maintain p53 activity at low temperature. However, other significant changes in the axolotl proteins may play more subtle roles on p53 functions, including DNA binding and promoter specificity and could represent useful adaptations to ensure p53 activity and tumor suppression in animals able to regenerate or subject to large variations in oxygen levels or temperature.</p

Activation of Smad2 but not Smad3 is required to mediate TGF-β signaling during axolotl limb regeneration

Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-β1 is highly upregulated during regeneration and that TGF-β signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-β antagonist SB- 431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-β during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-β targets (e.g. Mmp13) are unaffected. This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-β signaling supporting the regenerative process

Senescence gives insights into the morphogenetic evolution of anamniotes

Senescence represents a mechanism to avoid undesired cell proliferation that plays a role in tumor suppression, wound healing and embryonic development. In order to gain insight on the evolution of senescence, we looked at its presence in developing axolotls (urodele amphibians) and in zebrafish (teleost fish), which are both anamniotes. Our data indicate that cellular senescence is present in various developing structures in axolotls (pronephros, olfactory epithelium of nerve fascicles, lateral organs, gums) and in zebrafish (epithelium of the yolk sac and in the lower part of the gut). Senescence was particularly associated with transient structures (pronephros in axolotls and yolk sac in zebrafish) suggesting that it may play a role in the elimination of these tissues. Our data supports the notion that cellular senescence evolved early in vertebrate evolution to influence embryonic development

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer-2

<p><b>Copyright information:</b></p><p>Taken from "Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer"</p><p>http://www.biomedcentral.com/1471-2148/7/180</p><p>BMC Evolutionary Biology 2007;7():180-180.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2072957.</p><p></p>itions inferred by the program Treefinder with a WAG+Γ8 model. Numbers at internal nodes are corresponding to bootstrap support values, obtained in the analysis of 100 replicates using the same program and model of sequence evolution. Due to the dense species sampling within the mammals interesting aspects of mammalian evolution are becoming apparent. There are clear differences in the evolutionary rates among the different groups, indicated by the branch length of the rooted tree, this is especially true for the four Neoteleost fish. There is also an acceleration observed for the mouse-like rodents, with a striking exception represented by the sequence of the mole rat , which is despite the fact of being a rather small rodent even more slowly evolving than the related rabbit (, lagomorph). In fact the only sequences among the tetrapods (amphibians, reptilian and mammalian) that are more slowly evolving than the one from are from the urodeles (axolotl and newts). The primary sequence of salamander p53 is more closely related to the ancestral protein of tetrapod vertebrates than the p53 proteins of any other of the studied groups

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer-3

<p><b>Copyright information:</b></p><p>Taken from "Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer"</p><p>http://www.biomedcentral.com/1471-2148/7/180</p><p>BMC Evolutionary Biology 2007;7():180-180.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2072957.</p><p></p>ciferase activities were significantly different from non-p53 controls (at least p < 0.05, data not shown). Error bar ± s.e.m. human and axolotl p53 induced luciferase expression were significantly different with the Hdm2 promoter (p < 0.01). Each assay was performed in triplicate at least 3 separate times

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer-1

<p><b>Copyright information:</b></p><p>Taken from "Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer"</p><p>http://www.biomedcentral.com/1471-2148/7/180</p><p>BMC Evolutionary Biology 2007;7():180-180.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2072957.</p><p></p>ivation Domain; DBD, DNA Binding Domain; NLS, Nuclear Localisation Signal; TET, tetramerisation domain; REG, Regulatory domain; Regions I-V, highly conserved regions. Lysine (K), serine (S) and threonine (T) residues implicated in post-translational modifications are indicated. The protein domains depicted in the diagrams are not to scale. (B) Sequences alignment of human and axolotl p53 proteins. The conserved regions I to V are highlighted and many changes between the axolotl and human sequence are identified (arrows). See table-2 for a complete list of changes associated with mutations in the human protein