Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry

Abstract

Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G₂ and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

1. Introduction

1.1. Virtues and vices of different synchronization methods

Different approaches are being used to obtain populations of cells synchronized in the cell cycle (reviews: ^1–4). Each of these approaches offers some advantages but also suffers limitations. One of the widely used methods is based on isolation of mitotic cells by their detachment from flasks during culturing (5). Its virtue stems from the fact that the cell progression through the cycle is not being perturbed. However, the technique is applicable to few cell lines, such as Chinese hamster ovary (CHO) or HeLa cells, that grow attached in cultures and detach during mitosis. Another technique selecting mitotic cells was designed for murine leukemic L1210 cells that grow in suspension but are being enforced to attach to polylysine-coated membranes. During mitosis the daughter cells detach from the mother cells, still attached to membranes, and float in culture medium (6,7). It is unknown how widely this approach can be used because cells of other than L1210 lines cannot be synchronized this way (8). Cells of certain lines can be synchronized in G₁ by inhibitors of protein farnesylation and geranyl-geranylation such as statins (9), or CDK2 protein kinase inhibitors (10–12). These techniques also are not universally applicable since many lines do not respond to statins or protein kinase inhibitors by reversible arrest in G₁.

Normal, non-tumor cells can be synchronized in G₀/₁ by removal of growth factors e.g. by “serum starvation” (13), or depletion of a particular amino-acid (14), or by contact inhibition (15). These approaches generally fail to synchronize tumor cell lines. Moreover, the metabolism of so synchronized cells is often perturbed which affects their rate of progression through the cycle upon release from the arrest (16). It should also be noted that the cells synchronized in early G₁ or at mitosis start to lose synchrony while progressing through G₁ and thereby become less synchronous in S or G₂.

The approach based on separation of cells based on their size, centrifugal elutriation, is also being used to obtain relatively synchronous cell populations (17). However, while this procedure does not perturb cell cycle progression the synchrony of uniformly sized elutriated cells is not always sufficiently narrow. Furthermore, elutriation requires rather complex and expensive instrumentation and an experienced operator. Density gradient centrifugation yields cell populations even less synchronized than the elutriation (18). Fluorescence-activated cell sorting (FACS) is also used to obtain synchronous cell populations e.g. based on DNA content analysis upon supravital staining with fluorochromes such as Hoechst 33342 (19). However, Hoechst 33342 elicits DNA damage response (20), long-term toxicity (21), and undergoes redistribution from labeled to unlabeled cells in mixed cell populations (22).

Cell synchronization at mitosis by mitotic spindle poisons such as colchicines, vinca alkaloids or nocodazole is another common approach (23,24). The advantage of this approach is simplicity, low cost and high degree of synchrony when one opts to obtain mitotic or immediately postmitotic cell populations (25).As mentioned, so synchronized cell populations become less synchronous after progression through G₁. Furthermore, undesirable effects such as cytotoxicity and growth imbalance are seen during arrest in mitosis (26–28).

1.2. Inhibitors of DNA replication

Among the most common approaches to obtain populations of synchronized cells, particularly in S-phase, is cell synchronization with the use of DNA replication inhibitors such as hydroxyurea, methotrexate, aphidicolin or high concentration of thymidine (29–32). A combination of replication inhibitors with other synchronizing agents is also widely used (24,33). The advantage of cell synchronization by DNA replication inhibitors is the simplicity and low cost. However, the major drawback is the induction of growth imbalance (26,34). While cells become arrested in their progression through S, their growth, in terms of RNA and protein synthesis, and cell enlargement continues which leads to perturbation of metabolic functions. Furthermore the cell cycle progression machinery of cells synchronized by DNA replication inhibitors is severely perturbed as reflected by unscheduled expression of cyclin proteins (35).

Inhibitors of DNA replication such as hydroxyurea, aphidicolin, thymidine and aphidicolin were shown to induce phosphorylation of histone H2AX on Ser-139 (36,37). Phosphorylated H2AX is defined as γH2AX (38). Phosphorylation of H2AX and activation of ataxia telangiectasia mutated protein kinase (ATM) through its phosphorylation on Ser 1981 (ATM-S1981^P) are the key markers signalizing DNA damage response (DDR) reflecting DNA damage that involves formation of DNA double-strand breaks (DSBs) (38,39; reviews ^40,41). Both, activation of ATM and H2AX phosphorylation can be detected immunocytochemically using phospho-specific Abs, and the extent of their phosphorylation reporting severity of DNA damage can be measured by flow or laser scanning cytometry (42–46). Since during replication stress induced by inhibitors of DNA replication H2AX is phosphorylated by ATM Rad3-related protein kinase (ATR) and not by ATM, the latter kinase remains not activated (36; see Fig 1). While no phospho-specific Ab are available at present to directly detect activated ATR its activation can be detected indirectly using Ab to ATM/ATR substrate that is phosphorylated on Ser/Thr at SQ/TQ cluster domains (sATM/ATR^P) (47). The response induced by DNA replication inhibitors thus can be assessed my detecting activation of ATR and phosphorylation of H2AX (expression of γH2AX) concurrent with the lack of activation of ATM (36). It should be noted, however that in certain instanced the DNA replication stress may also induce activation of ATM and DNA-dependent protein kinase (DNA-PKcs) (48).

Fig. 1. Detection of histone H2AX phosphorylation and ATM activation in HL-60 cells treated with DNA polymerase alpha inhibitor aphidicolin (Aph).

The cells were untreated (Ctrl) or treated in culture with 1 or 4 µM Aph and expression of γH2AX and ATM-S1981^P was measured as described in the protocol (indirect immunofluorescence). The bivariate distributions (scatterplots) of γH2AX or ATM-S1981^P immunofluorescence (IF) vs DNA content allow one to identify subpopulations of cells in G₁, S and G₂M phases of the cell cycle as shown in left panels. Increased expression of γH2AX is primarily seen in cells at the interphase of G₁ and S (marked by rectangular dashed outlines) and also in S and G₂M in cells after treatment with Aph. No increase in expression of ATM-S1981^P is apparent. Apoptotic cells (Ap) are detected as having markedly elevated expression of both γH2AX and ATM-S1981^P after treatment with 4 µM Aph for 4 h. Note that the γH2AX and ATM-S1981^P coordinates are exponential while DNA content scale is linear.

2.3. Analysis of DDR induced by DNA replication inhibitors

The protocol presented in this chapter is designed to assess intensity of γH2AX and/or ATM-S1981^P and/or phosphorylated ATM/ATR substrate (sATM/ATR^P) immunofluorescence (IF) for evaluation of the degree of DNA damage response. The detection of γH2AX, sATM/ATR^P or ATM-S1981^P is combined with differential staining of DNA to assess cellular DNA contents which reveals the cell cycle phase (See Note 1). The procedure is based either on indirect IF detection of these phosphorylated proteins using the primary Ab unlabeled and the secondary Ab conjugated with FITC or Alexa Fluor 488, or using direct IF with the fluorochrome-tagged primary Abs (See Note 2). DNA is counterstained with propidium iodide (PI) whose emission spectrum (red) is separated from the green color emission of FITC or Alexa Fluor 488. The cells are briefly fixed in methanol-free formaldehyde and then transferred into 70% ethanol in which they can be kept briefly (≥2h) or stored at −20 °C for weeks or longer. Ethanol treatment makes the plasma membrane permeable to the γH2AX Ab; further permeabilization is achieved by including the detergent Triton X-100 into a solution used to incubate cells with the Ab. After incubation with the primary γH2AX Ab the cells are incubated with FITC or Alexa Fluor 488-labeled secondary Ab and their DNA is counterstained PI in the presence of RNase A to remove RNA, which otherwise, similar as DNA, is also being stained with PI. Intensity of cellular green (FITC or Alexa Fluor 488) and red (PI) fluorescence is measured by flow cytometry.

It should be noted that H2AX undergoes constitutive phosphorylation in healthy cells, untreated by radiation or genotoxic agents. This constitutive expression of γH2AX, which is more pronounced in S and G₂M than in G₁ cells, is considered to be in large part a reflection of oxidative DNA damage caused by the metabolically generated oxidants (49,50). It should also be noted that DNA fragmentation during apoptosis (51) leads to formation of large quantity of DSBs inducing high level of H2AX phosphorylation (52; see Figure 1). Strategies are presented to distinguish the DNA damage induced by topo inhibitors from the constitutive damage occurring in untreated cells (See note 3), or from the apoptosis-associated (AA) DSBs (See note 4).

2. Materials

2.1 Required reagents

• 1Cells to be analyzed: 10⁶ – 5×10⁶ cells, untreated (control) and treated in culture with inhibitors of DNA replication (e.g. hydroxyurea, aphidicolin or thymidine at concentration generally used to inhibit DNA synthesis and synchronize cells at the entrance to S phase) suspended in 1 ml of tissue culture medium.
• 2Methanol-free formaldehyde (Polysciences, Warrington, PA)
• 370 % ethanol
• 3Phosphate-buffered saline (PBS)
• 4Triton X-100 (Sigma Chemical Co., St. Louis, MO)
• 5Bovine serum albumin (BSA; Sigma)
• 6Abs: There are several commercially available phospho-specific Abs mono- as well as poly- clonal, unconjugated and FITC- or Alexa Fluor 488- conjugated, applicable to cytometry, that can be used to detect γH2AX, ATM-S1981^P or ATM/ATR substrate [e.g. from BioLegend (San Diego, CA), Cell Signaling/Santa Cruz Biotechnology (Danvers, MA), Molecular Probes/Invitrogen (Eugene, OR), Millipore/Upstate (Lake Placid, NY). The same companies also offer FITC- or Alexa 488- tagged secondary Abs to be used in conjunction with the non-conjugated primary Abs to these phosphorylated proteins.
• 8Propidium iodide (PI; Molecular Probes/Invitrogen)
• 9DNase-free RNase A (e.g. from Sigma)
• 1012 × 75 mm polypropylene tubes
• 11Centrifuge and rotor capable of 300g

2.2. Reagents to be prepared

1. Methanol-free formaldehyde fixative: Prepare 1% (v/v) solution of methanol-free formaldehyde in PBS. This solution may be stored at 4°C for up to two weeks

2. BSA-T-PBS: Dissolve BSA in PBS to obtain 1 % (w/v) BSA solution. Add Triton X-100 to obtain 0.2% (v/v) of its concentration. This solution may be stored at 4 °C for up to two weeks.

3. PI stock solution: Dissolve PI in distilled water to obtain 1 mg/ml solution. This solution can be stored at 4°C in the dark (e.g. in the tube wrapped in aluminum foil) for several months.

4. PI staining solution: Dissolve RNase A (DNase-free) in PBS to obtain 0.1% (w/v; 100 mg/ml) solution. Add an appropriate aliquot of PI stock solution (e.g. 5 µl per 1 ml) to obtain its 5 µg/ml final concentration. Store the PI staining solution in the dark. This solution may be stored at 4°C for up to two weeks.

2.2. Instrumentation

Flow cytometers of different types, offered by several manufacturers, can be used to measure cell fluorescence following staining according to the protocol given below. The most common flow cytometers are from Coulter Corporation (Miami, FL), Becton Dickinson Immunocytometry Systems (San Jose, CA), PARTEC (Zurich, Switzerland), Accuri Cytometers Inc (Ann Arbor, MI), Millipore-Guava (Billerica, MA) or Applied Biosystems (Foster City, CA).

3. Methods

• 1Centrifuge cells collected from tissue culture (suspended in culture medium) at 300g for 4 min at room temperature. Suspend cell pellet (1–2 × 10⁶ cells) in 0.5 ml of PBS.
• 2With a Pasteur pipette transfer this cell suspension into polypropylene tube (see Note 5) containing 4.5 ml of ice-cold 1% methanol-free formaldehyde solution in PBS. Keep on ice for 15 min.
• 3Centrifuge at 300g for 4 min at room temperature and suspend cell pellet in 4.5 of PBS. Centrifuge again as above and suspend cell pellet in 0.5 ml of PBS. With a Pasteur pipette, transfer the suspension to a tube containing 4.5 ml of ice-cold 70% ethanol. The cells should be maintained in 70% ethanol for 1 h but may be stored in 70 % ethanol at −20 °C for several weeks.
• 3Centrifuge at 200g for 4 min at room temperature, remove the ethanol and suspend cell pellet in 2 ml of BSA-T-PBS solution.
• 4Centrifuge at 300g for 4 min at room temperature and suspend the cells again in 2 ml of BSA-T-PBS. Keep at room temperature for 5 min.
• 5Centrifuge at 300g for 4 min at room temperature and suspend the cells in 100 µl of BSA-T-PBS containing 1 µg of the primary γH2AX Ab (See Notes 2 and 6).
• 6Cap the tubes to prevent drying and incubate them overnight at 4 °C (See Note 7).
• 7Add 2 ml of BSA-T-PBS and centrifuge at 300g for 4 min at room temperature.
• 8Centrifuge at 300g for 4 min at room temperature and suspend the cells in 2 ml of BSA-T-PBS.
• 9Centrifuge at 300g for 4 min at room temperature and suspend the cells pellet in 100 µl of BSA-T-PBS containing the appropriate (anti-mouse or anti-rabbit, depending on the source of the primary Ab) FITC- or Alexa Fluor 488- tagged secondary Ab (See Note 6).
• 10Incubate for 1 h at room temperature, occasionally gently shaking. Add 5 ml of BSA-T-PBS and after 2 min centrifuge at 300g for 4 min at room temperature.
• 11Suspend the cells in 1 ml of the PI staining solution. Incubate at room temperature for 30 min in the dark.
• 12Set up and adjust the flow cytometer for excitation with light at blue wavelength (488-nm laser line or BG-12 excitation filter).
• 13Measure intensity of green (530 ± 20 nm) and red (> 600 nm) fluorescence of the cells by flow cytometry. Record the data.