Survey of studies examining mammalian cardiomyocyte DNA synthesis

MH Soonpaa, LJ Field - Circulation research, 1998 - Am Heart Assoc
MH Soonpaa, LJ Field
Circulation research, 1998Am Heart Assoc
The generation of monoclonal antibodies against the nucleotide analogue BrdU has
facilitated the development of immune-based assays to monitor DNA synthesis.
Experimental animals receive one or more injections of BrdU, which is incorporated into the
nucleus of cells synthesizing DNA. After a brief chase period, the desired tissues are
harvested and sectioned, and the cells synthesizing DNA during the labeling period are
visualized by anti-BrdU immune histology. This approach has the advantage of being much …
The generation of monoclonal antibodies against the nucleotide analogue BrdU has facilitated the development of immune-based assays to monitor DNA synthesis. Experimental animals receive one or more injections of BrdU, which is incorporated into the nucleus of cells synthesizing DNA. After a brief chase period, the desired tissues are harvested and sectioned, and the cells synthesizing DNA during the labeling period are visualized by anti-BrdU immune histology. This approach has the advantage of being much more sensitive, rapid, and technically easier than assays based on thymidine incorporation. On the negative side, loss of tissue integrity resulting from the requisite permeabilization steps during sample processing may decrease the fidelity of cell identification. In addition, the great sensitivity of this immune-based assay increases the possibility of scoring false positives. For example, marked variation in nuclear immune reactivity is frequently observed between individual cells. In such instances, it is difficult to determine whether nuclei with comparatively weak signals should be scored as positive or negative for DNA synthesis. The use of BrdU for long-term (ie, pulse-chase) studies may also be problematic, because this analogue is known to affect proliferation and differentiation of cells in culture.
Direct quantification of nuclear DNA content has also been used to monitor DNA synthesis. Such analyses typically rely on microfluorometric analysis of Feulgen-stained nuclei and have the distinct advantage that ploidy can be accurately determined. As such, it is relatively easy to distinguish between de novo genomic replication versus repair DNA synthesis (a distinction that is potentially subjective with the metabolic assays and, in particular, with BrdU incorporation). Flow cytometric analysis of either intact cells or isolated nuclei prepared from the tissue of interest has also been used to monitor cell cycle progression in vivo. Both approaches suffer from the inherent disadvantage of being static assays. As such, it is difficult to determine whether elevated DNA levels reflect active DNA synthesis or, alternatively, cumulative changes in nuclear content. In light of this limitation, these approaches cannot accurately score samples with low levels of proliferation.
Am Heart Assoc