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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.

Darzynkiewicz Z, Zhao H, Zhang S, Lee MY, Lee EY, Zhang Z - Oncotarget (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Brander Cancer Research Institute, Department of Pathology, New York Medical College, Valhalla, NY.

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.

No MeSH data available.


Related in: MedlinePlus

Expression of cyclin E, cyclin A, PCNA and Ki-67 in relation to incorporation of EdUSimilar 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}.
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Figure 2: Expression of cyclin E, cyclin A, PCNA and Ki-67 in relation to incorporation of EdUSimilar 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}.

Mentions: The relationship between expression of cyclin E and incorporation of EdU is shown in Figure 2A. The gating analysis clearly demonstrates that most of the cyclin E negative cells did not initiate DNA replication. This is evident in the middle panel which shows that the cells incorporating EdU, including essentially the cells which were initiating incorporation during the pulse of EdU, were cyclin E positive. The initiation of EdU incorporation thus starts with accumulation of cyclin E to the level that can be immunocytochemically detected. However, a notable number of cells with a G1 DNA content and high expression of cyclin E that did not initiated EdU incorporation is evident (Figure 2A, mid-panel). These cells, thus, despite an increase in cyclin E content, are still being held in G1 apparently by other factors than by the deficit in amount of this cyclin. The level of cyclin E expression drops during progression of cells through S and even more at the transition to G2. Since the cells with G2M DNA content are essentially cyclin E negative the stepwise degradation of cyclin E during progression through S appears to be nearly completed at the time of S to G2 transition or early in G2.


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.

Darzynkiewicz Z, Zhao H, Zhang S, Lee MY, Lee EY, Zhang Z - Oncotarget (2015)

Expression of cyclin E, cyclin A, PCNA and Ki-67 in relation to incorporation of EdUSimilar 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}.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494901&req=5

Figure 2: Expression of cyclin E, cyclin A, PCNA and Ki-67 in relation to incorporation of EdUSimilar 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}.
Mentions: The relationship between expression of cyclin E and incorporation of EdU is shown in Figure 2A. The gating analysis clearly demonstrates that most of the cyclin E negative cells did not initiate DNA replication. This is evident in the middle panel which shows that the cells incorporating EdU, including essentially the cells which were initiating incorporation during the pulse of EdU, were cyclin E positive. The initiation of EdU incorporation thus starts with accumulation of cyclin E to the level that can be immunocytochemically detected. However, a notable number of cells with a G1 DNA content and high expression of cyclin E that did not initiated EdU incorporation is evident (Figure 2A, mid-panel). These cells, thus, despite an increase in cyclin E content, are still being held in G1 apparently by other factors than by the deficit in amount of this cyclin. The level of cyclin E expression drops during progression of cells through S and even more at the transition to G2. Since the cells with G2M DNA content are essentially cyclin E negative the stepwise degradation of cyclin E during progression through S appears to be nearly completed at the time of S to G2 transition or early in G2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Brander Cancer Research Institute, Department of Pathology, New York Medical College, Valhalla, NY.

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.

No MeSH data available.


Related in: MedlinePlus