New distinct compartments in the G 2 phase of the cell cycle defined by the levels of γh2ax. - PDF

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Cell Cycle ISSN: (Print) (Online) Journal homepage: New distinct compartments in the G 2 phase of the cell cycle defined by the levels of γh2ax. Idun Dale Rein, Caroline Stokke, Marwa Jalal, June H Myklebust, Sebastian Patzke & Trond Stokke To cite this article: Idun Dale Rein, Caroline Stokke, Marwa Jalal, June H Myklebust, Sebastian Patzke & Trond Stokke (2015) New distinct compartments in the G 2 phase of the cell cycle defined by the levels of γh2ax., Cell Cycle, 14:20, , DOI: / To link to this article: The Author(s). Published with license by Taylor & Francis Group, LLC Idun Dale Rein, Caroline Stokke, Marwa Jalal, June H Myklebust, Sebastian Patzke, and Trond Stokke View supplementary material Accepted author version posted online: 28 Aug Submit your article to this journal Article views: 332 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [Oslo and Akershus University College of Applied Sciences] Date: 16 March 2016, At: 04:39 Cell Cycle 14:20, ; October 15, 2015; Published with license by Taylor & Francis Group, LLC REPORT New distinct compartments in the G 2 phase of the cell cycle defined by the levels of gh2ax. Idun Dale Rein 1, Caroline Stokke 2,3, Marwa Jalal 4, June H Myklebust 5,6, Sebastian Patzke 1, and Trond Stokke 1, * 1 Group for Molecular Radiation Biology; Department of Radiation Biology; The Norwegian Radium Hospital; Oslo, Norway; 2 The Intervention Centre; Oslo University Hospital; Oslo, Norway; 3 Faculty of Health Sciences; Oslo and Akershus University College of Applied Sciences; Oslo, Norway; 4 The Francis Crick Institute; London, UK; 5 Group for Lymphoma and Lymphocyte Biology; Department of Cancer Immunology; The Norwegian Radium Hospital; Oslo, Norway; 6 Centre for Cancer Biomedicine; University of Oslo; Oslo, Norway Downloaded by [Oslo and Akershus University College of Applied Sciences] at 04:39 16 March 2016 Keywords: DNA repair, G 2 cell cycle phase, ionizing radiation, PARP inhibition, gh2ax Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (gh2ax) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of gh2ax in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable gh2ax and no gh2ax-foci in G 1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of gh2ax increased immediately upon S phase entry, and about 10 and 30 gh2ax foci were found in mid-s phase control and PARPi-treated cells, respectively. The gh2ax-labeled damage caused by DNA replication was not fully repaired before entry into G 2. Intriguingly, G 2 cells populated a continuous distribution of gh2ax levels, from cells with a high content of gh2ax and the same number of foci as S phase cells (termed G 2 H compartment), to cells that there were almost negative and had about 2 foci (termed G 2 L compartment). EdU-labeling of S phase cells revealed that G 2 H was directly populated from S phase, while G 2 L was populated from G 2 H, but in control cells also directly from S phase. The length of G 2 H in particular increased after PARPi treatment, compatible with longer DNArepair times. Our results show that cells repair replication-induced damage in G 2 H, and enter mitosis after a 2 3 h delay in G 2 L. Introduction The cell cycle of mammalian somatic cells contains 4 welldefined, non-overlapping compartments; G 1, S (DNA replication), G 2 and mitosis (M), in addition to the actual process of cell division (cytokinesis). The G 1 phase varies greatly in length, from hours to years, and the important decision of whether the cell shall proceed through a new round in the cell cycle or not is taken in G 1, specifically at the so-called restriction point, 1 reviewed by Blagosklonny and Pardee. 2 Passage through the restriction point requires both the presence of appropriate growth factors, and lack of growth inhibitory factors. However, S phase entry may also be blocked after DNA damage, e.g. after ionizing radiation or treatment with genotoxic drugs. 3,4 The processes taking place during G 2 are less well known, but e.g., disentangling of sister chromosomes may require time before the chromosomes can condense, 5 marking the onset of mitosis. 6 There may also be other time-consuming biochemical processes and restructuring required for entry into M. Although somatic mammalian cells do not regulate cell proliferation in G 2, there is a DNA damage checkpoint in this phase, serving a similar purpose as the one in G 1. 7,8 Although the length of G 2 is typically much shorter and more homogeneous than that of G 1 in cells not treated with genotoxic agents, it still varies from 2 7 hours within the same culture for the cell lines employed in this work (see also 9,10 ). This somewhat argues against the idea that untreated cells go through a strict set of processes in G 2, which would be expected to take about the same time in each cell. Lesions in DNA, particularly double-strand brakes (DSBs), initiate an array of events, including DNA repair, cell cycle arrest in G 1 and G 2, and apoptosis if repair is not successful. However, DNA damage may be the result of non-faithful DNA replication, which may also happen in the absence of external injury. Indeed, ATR is activated during DNA replication in untreated, exponentially growing cells, indicating that DNA damage may be induced during an unperturbed S phase. 11,12 Serine 139 on histone H2AX is phosphorylated in the vicinity of DSBs, these sites can be visualized as nuclear foci after staining with antibodies against the phosphoprotein (gh2ax). Several research groups have reported that S phase cells in control, untreated, cultures of both Idun Dale Rein, Caroline Stokke, Marwa Jalal, June H Myklebust, Sebastian Patzke, and Trond Stokke *Correspondence to: Trond Stokke; Submitted: 05/20/2015; Revised: 08/18/2015; Accepted: 08/22/2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. Cell Cycle 3261 transformed and immortalized cells have such foci 13,14 (reviewed in 15,16 ), showing that damage is induced during DNA replication, also in the absence of DNA damaging agents. In contrast, G 1 control cells have undetectable levels of gh2ax. Mitotic cells show varying levels of gh2ax, but the distribution is diffuse rather than focal, and has been suggested not to be associated with DNA damage. 17,18 It is possible that the levels of DNA damage carried over from S phase to G 2 varies from cell to cell, thus requiring different times in G 2 for DNA repair. To our knowledge, there have been no studies addressing the levels of gh2ax in G 2. In this work we have studied gh2ax in the G 2 phase by flow cytometry and microscopy. We show that individual cells are entering G 2 with various levels of gh2ax, and that they spend variable amounts of time in a compartment in G 2 defined by high gh2ax levels and many foci. This compartment was termed G 2 H; the H refers to high gh2ax levels. After having left this compartment, the cells enter a compartment termed G 2 L (low gh2ax), more homogeneous in length than G 2 H, before they enter mitosis. We thus define 2 entirely new compartments in G 2, and explain how different shapes of the cell cycleresolved content of gh2ax may reflect differences in fidelity of DNA replication/repair between different cell types and lines. Results Heterogeneity of gh2ax levels in G2 in control and PARP inhibitor treated cells Four exponentially growing malignant B-lymphocyte cell lines were studied for cell cycle phase-dependent phosphorylation of H2AX in interphase cells (Fig. 1). Comparison with samples stained without the primary gh2ax antibody (staining control) showed that the G 1 cells had little, if any gh2ax (Fig. S1). gh2ax levels increased immediately upon S phase entry and remained high throughout S. gh2ax levels in control S cells were lowest in Reh, and increasingly higher in U698, Granta-519 and JVM-2. Some G 2 cells had high levels of gh2ax (termed G 2 H, see arrows in Fig. 1 and Fig. S1), while others had lower levels down to almost negative (termed G 2 L ), resulting in a broader gh2ax distribution in this phase. The cell cycle-resolved gh2ax expression pattern was similar in primary (normal) B lymphocytes stimulated to enter the cell cycle (Fig. S2). The heterogeneity in gh2ax levels in G 2 was assessed by the robust coefficient of variation (rcv), which was significantly higher than the rcv for mid-s phase cells for all cell lines (data not shown). After treatment with 3 mm of the PARP inhibitor Olaparib (PARPi) for 24 h to create damage and inhibit DNA repair, 19 gh2ax in S phase cells was increased relative to the corresponding control, while G 1 cells still had no gh2ax (Fig. 1). gh2ax also increased in G 2 cells after PARPi treatment. (See accompanying article in this issue for gh2ax levels in S and G 2 cells with different concentrations of PARPi). The rcv values for G 2 compared to S were significantly higher also after PARPi treatment. Control and PARPi-treated mitotic cells had a high content of gh2ax in the cells studied here (Fig. 2A). In contrast to PARPi treatment, irradiation with 4 Gy X-rays 1 h before harvest resulted in an increase in gh2ax in all cell cycle interphases (Fig. 2A). To see how the variable levels of gh2ax in G 2 phase related to DNA damage, the cell cycle-resolved numbers of gh2ax foci were determined in sorted cells from distinct cell cycle phases (sort gates shown in Fig. S3), followed by microscopic evaluation. Most G 1 cells had no foci, with some cells displaying 1 focus (Fig. 2B), which was also the case after PARPi treatment for 24 h. Mid-S phase cells in control cultures had 10 5 (Reh; mean SD) and 12 6 (U698) foci, in agreement with the high gh2ax content measured by flow cytometry. PARPi treatment increased focus numbers in mid-s phase cells to foci in Reh and foci in U698 cells, respectively. gh2ax focus numbers thus increased 2.9 and 2.6 fold upon PARPi treatment in Reh and U698 cells, while the corresponding increase in gh2ax-associated fluorescence by flow cytometry was 3.3 and 2.3 fold (background corrected). Together, these results indicated that replication damage-associated (focal) gh2ax fluorescence was reliably measured by the total intensity in interphase cells. In contrast, control mitotic Reh and U698 cells, with high gh2ax intensities, had only 1 focus on average. PARPi treatment for 24 h increased the number of foci to 2 in mitotic Reh cells, but mitotic U698 cells still had 1 focus (Fig. 2B). Thus, the diffuse staining in mitotic cells accounted for most of the total gh2axassociated fluorescence (not shown). The broader distributions observed for the gh2ax-associated fluorescence of G 2 cells by flow cytometry showed that the content of gh2ax in G 2 was more heterogeneous than in S (Figs. 1, 2A). We therefore sorted G 2 cells with high (G 2 H) and low (G 2 L) gh2ax-associated fluorescence to reveal possible differences in focus counts between these 2 compartments (see Fig. S3 for sort gates). The G 2 cells with high gh2ax content (G 2 H) had many foci (10 5 and 16 7 for control Reh and U698 cells, respectively), similar to the S phase cells (Fig. 2B), indicating that these cells had similar DNA damage levels. In contrast, the G 2 L cells had few foci (3 2 and 2 2 for Reh and U698, respectively). Focus numbers in the 2G 2 compartments increased after PARPi treatment, most significantly for the G 2 H cells. Hence, G 2 H and G 2 L are welldefined compartments in G 2, although the lack of a clear bimodal distribution indicated a continuous distribution of gh2ax. The lack of consistent bimodal distributions precluded a straight-forward assessment of the fractions of cells in the G 2 H and G 2 L compartments. In contrast to the heterogeneity seen for PARPi treated G 2 cells, focus counts were homogeneously increased in G 1, S and G 2 cells 1 h after irradiation with 4 Gy (Fig. 2B), in agreement with the homogeneously enhanced gh2ax-associated fluorescence in all cell cycle interphases measured by flow cytometry (Fig. 2A). The order of G 2 H and G 2 L Non-irradiated cells entered mitosis continuously, also in the presence of PARPi, although at a lower rate than in the control samples (see later). Entry into mitosis is known to be prevented by the presence of DSBs, which can be signaled by the presence of gh2ax foci. As mitotic cells had only 1 2 foci, it seems likely 3262 Cell Cycle Volume 14 Issue 20 Figure 1. Cell cycle-resolved phosphorylation of H2AX in interphase control and PARPi-treated cells. Cells were grown for 24 h in the absence (left panels), or presence of 3mM the PARPi Olaparib (right panels). They were thereafter fixed and stained for gh2ax, ps10h3, apoptosis, and DNA content and measured by flow cytometry. Aggregates of cells and apoptotic cells (few at this time point, see the accompanying article in this issue), as well as mitotic cells were removed before displaying interphase cells. (See Fig. S4 in the accompanying article in this issue for details.) Fig. 2A shows the position of mitotic cells in the cytograms. that the cells are entering mitosis with a low number of gh2ax foci, possibly from G 2 L. To this end, we studied in which order cells proceeded through the 2 compartments in G 2,G 2 H and G 2 L, by pulse-labeling S phase cells with EdU and chasing them (experimental strategy shown in Fig. S3). Data corrected for the spillover of S phase cells are shown in Fig. 3 (raw data are shown in Fig. S4). The G 2 H compartment was populated directly from S phase, as the fraction of EdU C G 2 H cells was increased after 1 h. The entry of EdU-labeled cells into the G 2 L compartment always lagged entry into G 2 H, with lower initial increases than the corresponding ones for G 2 H. The fractions of EdU C G 2 L cells after 1 h were close to zero in the presence of PARPi, indicating that G 2 L was populated exclusively via another compartment (G 2 H) under those conditions. Mitosis was populated after both G 2 compartments, and eventually EdU C cells entered the next G 1. The plateau levels reached after 8 h for control G 2 H and M cells were about 80% ( i.e. lower than the theoretical 100%, which may have been caused by a less than ideal separation between the EdU - and EdU C populations). However, the fractions of EdU C control cells in the G 2 L compartment consistently flattened out at lower levels compared to G 2 H and M, particularly pronounced for JVM-2 cells (Fig. 3, experimental data). Hence, these results suggest the presence of a subpopulation of G 2 L cells that is not leaving the compartment within the time frame of the experiment, and is thus not populating M, possible due to delayed cell death. Cell cycle traverse was slower in the presence of PARPi, and plateau levels reached after 24 h were about 60%. These levels were lower than for control cells, which may be explained by lower DNA replication rates and less intense EdU-labeling. However, plateau levels were similar for G 2 H, G 2 L, and M, indicating that all cells entering G 2 L (from G 2 H) proceed to M within the time frame of the experiment. The duration of the G 2 H and G 2 L compartments We further simulated the time evolution of population of the different phases and compartments with EdU C cells to extract durations. A similar strategy has been employed to study the duration of G 2 plus M. 9,10 If the duration of G 2 H had been constant (T), the fraction of EdU C cells in this compartment should have increased linearly with time with a slope proportional to 1/T (an example is shown in Fig. S5A). It was evident from the experimental data in Fig. 3 that a model with constant duration of G 2 H could not fit the data in any case. A Cell Cycle 3263 Figure 2. Cell cycle-resolved gh2ax levels and number of gh2ax foci. (A) Reh (upper panels) and U698 cells (lower panels) were grown for 24 h in the absence (Ctrl) or presence of Olaparib (3mM PARPi 24 h), or they were irradiated with 4 Gy 1 h before harvest. Cells were fixed and stained for gh2ax, ps10h3, apoptosis, and DNA content and measured by flow cytometry. Aggregates of cells and apoptotic cells were removed before displaying interphase cells (colored dots) and mitotic cells (black dots). (B) Cells stained as under (A) were sorted according to the scheme in Fig. S3, and analyzed for the number of gh2ax foci. There were no mitotic cells and no cells in G 2 L in cultures harvested 1 h after 4 Gy IR, and results for irradiated cells are given for G 1, S and G 2 H. The plot shows mean SD. model was therefore employed where we allowed for sequential movement from S into G 2 H, allowed to vary in length from cell to cell, and further into G 2 L before entering M (see Materials and Methods). First we assumed that the flux of cells from S phase into G 2 H and further into G 2 L and M was the same for the different residence times in G 2 H ( flat distribution, i.e., f(t) D constant; an example is shown in Fig. S5B). The time dependency of EdU C cells in G 2 H of control cells could be reasonably fitted with flat distributions for all 3 cell lines, 0.5 7, and h for Reh, U698 and JVM-2, respectively (simulation curves in Fig. 3). However, the data for G 2 L and M were not fitted, irrespective of the durations employed for the latter 2 compartments (Fig. S5B). Since some cells may enter G 2 L directly, i.e. G 2 H has zero duration, f (0) was allowed to take non-zero values. Notice that the time course of EdU C cells in G 2 H will be determined by f(t 0) only. In contrast, the time course of EdU C cells in G 2 L and M will be affected if a fraction of the cells enter G 2 L directly (f(0) 0). The data for G 2 L and M at early times were better fitted with this approach, but the time evolutions of EdU C cells in G 2 L and M at the later times were not fitted employing the flat distribution model (Fig. S5C). This, and the lower plateau levels in control cells observed for G 2 L compared to G 2 H and M, indicated that the cells with the longest duration of G 2 H did not leave G 2 L to enter M within the time frame of the experiment. We therefore also allowed for variable weighting of the flux of cells out of the compartments with different residence times in G 2 H. Mathematically, this was done by allowing f(t) to vary within the time interval found to fit G 2 H (see Materials and Methods and Sup. File), thus obtaining simulations for G 2 L and M. In the case of control cells, good fit was obtained if the distribution of cells entering M through G 2 L had a skewed distribution with higher amplitudes toward the lower residence times in G 2 H (simulation curves in Fig. 3). To fit the G 2 L plateau levels of control cells at 8 h, a long lifetime component was added (G 2 L: 8 h, not entering M), representing cells that, by implication, were recruited from a skewed distribution with higher amplitudes toward longer residence times in G 2 H(Table 1). We considered possible sources of error in these calculations. The gating, and sorting, of mitotic cells was based on ps10h3 staining, which results in clear bimodal peaks and good separation between the ps10h3-positive and -negative cells. Microscopy revealed that there were almost no interphase cells in the 3264 Cell Cycle Volume 14 Issue 20 Figure 3. Fraction of EdU C cells as a function of time after pulse-labeling. Control cells, or
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