Abstract
During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.
Highlights
Multiparameter cytometry combined with gating analysis offers the means to correlate expression of different cellular entities and events with each other in individual cells
Numerous attempts have been made to use this methodology to assess a relationship between DNA replication and expression of particular nuclear proteins in order to explore their role in this critical event of the cell cycle
Using the prior described methodology [11,12,13,14,15, 20] we report new data, these related to expression of cyclin D1 and cyclin E with respect to EdU incorporation
Summary
Multiparameter cytometry combined with gating analysis offers the means to correlate expression of different cellular entities and events with each other in individual cells. The protein p21WAF1 is a cyclin-dependent kinase inhibitor (CKI) which binds and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and functions as a checkpoint regulator of cell cycle progression at G1 and S phase [34,35,36,37] The expression of this gene is induced by the tumor suppressor p53 in response to a variety of stimuli, from DNA damage [36]. The use of EdU labeling and LSC approaches do reveal the temporal sequence with which these substrates are degraded, at least in relation to the initiation of DNA synthesis This analysis encompasses www.impactjournals.com/oncotarget the earliest stages of DNA synthesis and captures those cells just beginning to initiate DNA synthesis that formally by their DNA content are still identified as in G1.these approaches may be of some utility as a means of studying temporal events in the complex process of the initiation of DNA replication. The assessments of initiation and the rate of DNA replication are unlikely to be affected by limitations of the accessibility and conversion of EdU to its triphosphate precursor
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