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Link to original content: https://pubmed.ncbi.nlm.nih.gov/26059433
Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry - PubMed Skip to main page content
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Review
. 2015 May 20;6(14):11735-50.
doi: 10.18632/oncotarget.4149.

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry

Zbigniew Darzynkiewicz  1 , Hong Zhao  1 , Sufang Zhang  2 , Marietta Y W T Lee  2 , Ernest Y C Lee  2 , Zhongtao Zhang  2
Affiliations
Review

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry

Zbigniew Darzynkiewicz et al. Oncotarget. .

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.

Keywords: DNA replication; EdU labeling; S-phase; cell cycle; cell proliferation; laser scanning cytometry; polymerase δ.

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Figures

Figure 1
Figure 1. Expression of cyclin D1 (A), the CDK inhibitor p21 (B), the chromatin licensing and DNA replication factor Cdt1 (C), and the smallest subunit of DNA polymerase δ p12 (D), in relation to EdU incorporation
A549 cells were exposed to EdU for 60 min, the EdU incorporation was detected by the Click-ItTM protocol, cellular expression of cyclin D1, p21, Cdt1 and p12 was detected immunocytochemically, DNA was counterstained with DAPI and cellular fluorescence was measured by LSC, as described [11-15,20]. During the “paint-a-gate” analysis the cells incorporating EdU were colored red. The dashed contours in A (left panel) outline the cells that during duration of the 60 min exposure to EdU were entering S (eS), were constantly present during pulse duration (mid-S), or were entering G2 (eG2) [15]. The EdU incorporation is correlated on the bivariate scatterplots with expression of these proteins presented as integrated values of immunofluorescence intensity over cell nucleus (mid-panels). The dashed skewed lines in mid-panels show the upper level of fluorescence intensity of the cells stained with the secondary fluorochrome-tagged Ab only, namely +3SD of the mean fluorescence intensity of such negative control [20]. The cells below this line are thus considered negative with respect to expression of these proteins. Below the thick arrows in mid-panels presented is the percentage of cells at the G1 to S transition (with DNA content equivalent of G1) and at the S to G2 transition (G2M DNA content) that are negative with respect to expression of the measured proteins and did not incorporate EdU. A direct relationship between expression of these proteins and EdU incorporation is presented in the right panels in which the dashed line separates the EdU labeled from unlabeled cells. The regression analysis reveals the degree of correlation (Pearson; r = x) between incorporation of EdU and expression of each of these proteins in all EdU positive cells, assessed as described before [20].
Figure 2
Figure 2. Expression of cyclin E, cyclin A, PCNA and Ki-67 in relation to incorporation of EdU
Similar as described in the legend to Fig. 1 A549 cells were exposed to EdU for 60 min and incorporation of EdU was correlated with expression cyclin E, cyclin A, PCNA or Ki-67 and also related to cellular DNA content [11-15, 20]. The cells incorporating EdU were gated (left panels; red dots) and on the bivariate scatterplots correlated with expression of the respective protein, detected immunocytochemically (mid- and right- panels). Additional gating was done to select the mid-S phase cells as shown on the left panels by the parallel vertical lines. The DNA content frequency histogram representing cells from the studied culture is shown in the A right panel. As described in legend to Fig. 1 the dashed skewed lines in mid-panels show the upper level of fluorescence intensity of the cells not incubated with the primary Ab (mid-panels). The correlation coefficients between DNA replication of the mid-S gated cell population (r1 = x), or of the all EdU-positive cells (r2 = x) and expression of the respective proteins are shown in the right panels. {Figs. 1 and 2 are adapted from ref. [20] with permission of authors and publisher}.
Figure 3
Figure 3. Expression of cyclin A, and Ki-67 in relation the expression of p21WAF1 and Cdt1, measured in the same individual cells
The individual proteins were detected in A549 cells immunocytochemically using the primary Abs of either mice or goat and the secondary Ab anti-mouse or anti-goat, respectively, labeled with fluorochromes of different emission wavelength; cellular fluorescence was measured by LSC [12-15,20,21]. The gating analysis was based on the selection of cells that were positive in the expression of p21 A. or Cdt1 B. (left panels; colored red) and the expression of these proteins was juxtaposed with the expression of cyclin A A. or Ki-67 B.. The insets in the right panels show DNA content frequency histograms from the respective cultures. As described in legend to Fig. 1 the dashed skewed lines in mid-panels show the upper level of fluorescence intensity of the negative control. See the text for further details.
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