Biomedical subjects

C P Velloso

Publications and source records attributed to C P Velloso.

6 recordsLinked to original sources

PubMed2001

Mammalian postmitotic nuclei reenter the cell cycle after serum stimulation in newt/mouse hybrid myotubes.

Cell cycle reentry and dedifferentiation of postmitotic cells are important aspects of the ability of an adult newt and other urodele amphibians to regenerate various tissues and appendages [1]. In contrast to their mammalian counterparts, newt A1 myotubes are able to reenter S phase after serum stimulation of a pathway leading to phosphorylation of the retinoblastoma protein, pRb [2]. The activity in serum is not due to mitogenic growth factors but is generated indirectly by the activation of thrombin and subsequent proteolysis [3]. In this paper we describe the formation of interspecies hybrid (heterokaryon) myotubes by the fusion of mouse C2C12 [4] and newt A1 [5, 6] myogenic cells. The C2C12 nuclei reenter the cell cycle upon serum stimulation of the hybrids, while C2C12 homokaryon myotubes remain arrested under these conditions. These findings indicate that the postmitotic arrest of the mouse nuclei is undermined by the pathway activated in the newt cytoplasm. The hybrid myotubes provide a new model for the manipulation of the postmitotic arrest in both mammalian and newt differentiated cells.

Animals↗

PubMed2000

Plasticity of retrovirus-labelled myotubes in the newt limb regeneration blastema.

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.

Animals↗

PubMed2000

Generation of mononucleate cells from post-mitotic myotubes proceeds in the absence of cell cycle progression.

The remarkable regenerative ability of adult urodele amphibians depends in part of the plasticity of differentiated cells at the site of injury. Limb regeneration proceeds by formation of a mesenchymal growth zone or blastema under the wound epidermis at the end of the stump. Previous work has shown that when cultured post-mitotic newt myotubes are introduced into the blastema, they re-enter the cell cycle and undergo conversion to mononucleate cells which divide and contribute to the regenerate [11, 13]. In order to investigate the interdependence of these two aspects of plasticity, we have blocked cell cycle progression of the myotubes either by X-irradiation or by transfection of the CDK4/6 inhibitor p16. In each case, the efficacy of the block was evaluated in culture after activation of S phase re-entry by serum stimulation. The experimental myotubes were implanted into limb blastemas along with a differentially labelled control population of myotubes containing an equivalent number of nuclei. X-irradiated myotubes gave rise to mononucleate cells in the limb blastema, and the progeny were blocked in respect of S phase entry. Comparable results were obtained with the p16-expressing myotubes. We conclude that progression through S or M phase is not required for generation of mononucleate cells and suggest that such cells may arise by budding from the muscle syncytium.

Animals↗

PubMed1998

Immortalization of rat embryo fibroblasts by a 3'-untranslated region.

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3'-UTR of an alpha-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.

Animals↗

PubMed1995

Acquisition of nerve dependence for the formation of a regeneration blastema in amputated hindlimbs of larval Xenopus laevis: the role of limb innervation and that of limb differentiation.

In larval and adult urodeles and late-stage larval anurans, blastema formation after limb amputation requires an adequate nerve supply. Experimental evidence obtained from aneurogenic limbs indicates that, in urodeles, the acquisition of nerve dependence during embryonic development is due to the "addiction" of limb tissues to factors released by the ingrowing nerves rather than to limb differentiation. The aim of this work was to establish whether, in the toad Xenopus laevis, nerve-dependence for blastema formation after hindlimb amputation, which is acquired gradually during larval development and becomes complete at stage 57 is due to limb innervation or to limb differentiation. Two series of experiments were carried out. In the first series, limb differentiation was inhibited by treating the larvae with an anti-thyroid drug, and innervation was maintained for an interval much longer than that normally required for development from nerve-independent stages to stage 57. In the second series, the limb was caused to differentiate in the absence of nerves by maintaining the limbs denervated. Limb differentiation was often accelerated by treating early-stage larvae with thyroxine or by grafting early-stage limbs onto denervated limbs of late larvae, which, being near metamorphic climax, possessed high levels of circulating thyroid hormones. Results showed that in the first series of experiments the denervated limbs formed regeneration blastemas after amputation, but in the second series they did not. It was therefore concluded that the acquisition of nerve dependence for blastema formation in larval Xenopus laevis is not directly imposed by factors released by the nerve fibers, but is strongly related to differentiation of limb tissues.

Amputation, Surgical↗

PubMedDate not supplied

Regeneration as an evolutionary variable.

Regeneration poses a distinctive set of problems for evolutionary biologists, but there has been little substantive progress since these issues were clearly outlined in the monograph of T. H. Morgan (1901). The champions at regeneration among vertebrates are the urodele amphibians such as the newt, and we tend to regard urodele regeneration as an exceptional attribute. The ability to regenerate large sections of the body plan is widespread in metazoan phylogeny, although it is not universal. It is striking that in phylogenetic contexts where regeneration occurs, closely related species are observed which do not possess this ability. It is a challenge to reconcile such variation between species with a conventional selective interpretation of regeneration. The critical hypothesis from phylogenetic analysis is that regeneration is a basic, primordial attribute of metazoans rather than a mechanism which has evolved independently in a variety of contexts. In order to explain its absence in closely related species, it is postulated to be lost secondarily for reasons which are not understood. Our approach to this question is to compare a differentiated newt cell with its mammalian counterpart in respect of the plasticity of differentiation.

Adaptation, Physiological↗