A Differential Response to Newt Regeneration Extract by C2C12 and Primary Mammalian Muscle Cells

Abstract

Tissue regeneration in mammals does not occur via the process of dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state. McGann et al. (2001) showed that mouse C2C12 myotubes treated with newt extract derived from regenerating limbs can re-enter the cell cycle, fragment and proliferate ( a characteristic of muscle dedifferentiation). However, the validity of these studies has been called into question since others have been unable to repeat them. My research attempts to replicate the results of McGann et al, and to carry them further. I examined several strategies for tracking the extract-treated cells and I also repeated the studies in a primary muscle culture system. Furthermore, I examined the effect of the extract on myoblast differentiation. The most effective dedifferentiation assay that I developed involved the microinjection of myotubes with extract and with a GFP plasmid that allowed tracking of the injected cells. Cells were then examined for cell cycle re-entry using BrdU incorporation or Ki-67 immunostaining. In addition, immunocytochemistry and RT-PCR analysis were used to examine the expression or down-regulation of muscle-specific markers. Finally, a preliminary GeneChip analysis was conducted to examine which genes were up or down regulated following extract treatment. The results show that newt extract is able to block the differentiation of confluent myoblasts, resulting in fewer multinucleated, myosin heavy chain expressing myotubes. However, when myoblasts were differentiated into myotubes and subsequently treated with newt extract, the results suggest that cell cycle re-entry and down-regulation of differentiation markers can occur in C2C12 myotubes, but not in primary myotubes. Fragmentation though, was seen in both C2C12 and primary myotubes following treatment or injection with newt extract. Moreover, the fragmented cells appeared to be viable. Transcriptional profiling indicated that newt extract affects genes implicated in cell cycle, transcription, stress, chromatin modification, growth, cell adhesion, extracellular matrix, wound healing and microtubule binding. These findings confirm that mammalian myotubes can be induced to dedifferentiate following treatment with newt extract; however, a differential response was observed between C2C12 and primary muscle cells.