Tài liệu Báo cáo khoa học: Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells pptx

Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells Maria van Betteraey-Nikoleit, Karl-Heinz Eisele, Dirk Stabenow and Hans Probst Physiologisch-Chemisches Institut der Universita ¨ tTu ¨ bingen, Germany It was shown previously [Riedinger, H. J., van Betteraey- Nikoleit, M & Probst, H. (2002) Eur. J. Biochem. 269, 2383–2393] that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of repli- cation proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G 1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the ‘hypoxic preini- tiation state’, ready to initiate replication as soon as normal pO 2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The use- fulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the ‘classi- cal’ prereplication complex is already formed during hypoxia. Keywords: chromatin; DNA replication; hypoxia; nuclei; synchronization. Apart from control by cell cycle signals, DNA replication in mammalian cells is subject to a regulation which depends on the O 2 tension in the cellular environment. Presumably, this regulatory phenomenon, adapting the intensity of DNA replication of growing cells to the supply of O 2 , is important during embryonic growth and wound healing, and influen- ces the propagation of malignant tumors. The O 2 -depend- ent regulation concerns cells which are in S-phase or are definitively committed to enter S-phase. When the concen- tration of O 2 drops to about 0.2–0.02%, scheduled replicon initiations are suppressed and already-active replication forks are slowed down. Of the cell lines examined so far, only Ehrlich ascites cells exhibit suppression of replicon initiation without a significant slowing down of fork progression [1–3]. During hypoxia cells accumulate repli- cons in a state ready to initiate (almost instantaneously) within a few minutes after oxygen recovery. Thus, sudden reoxygenation after several hours of hypoxia triggers a synchronous burst of initiations of the accumulated repli- cons followed by normal replication. So far, this regulatory phenomenon has been published for Ehrlich ascites, HeLa and CCRF cells [2,4,5]. Further cell lines examined so far, e.g.T24,A549,PC3,TC7,BHK,SW2,HL60andHUVEC arealsosubjecttothefastO 2 -dependent control of replication (G. Probst, H. Probst & M. van Betteraey- Nikoleit, unpublished results). We therefore suggest that it represents a general phenomenon in mammalian cells, although the molecular mechanisms involved are still largely obscure. The remarkably fast resumption of initiations after reoxygenation suggests that the O 2 -dependent replication control acts very directly on the replication apparatus itself. As published earlier, replication of the SV40 genome in virus infected cells also obeys the oxygen-dependent regu- lation [6,7]. Reoxygenation of virus infected cells after several hours of hypoxia triggers a burst of hypoxically accumulated viral replicon initiations followed by a syn- chronous round of completely regular replication of viral genomes. Studying several replication proteins bound to SV40 minichromosomes before and after reoxygenation, i.e. before and after triggering initiation, we found that a large number of polypeptides taking part in viral replication were bound to the SV40 minichromosome already under hypo- xia. However, the multiprotein complexes necessary for unwinding, primer synthesis and elongation lacked essential components and remained incomplete as long as hypoxia lasted [8]. Reoxygenation triggered fast completion to a Correspondence to M. van Betteraey-Nikoleit, Physiologisch-Chem- isches Institut der Universita ¨ tTu ¨ bingen, Hoppe-Seyler-Straße 4, D-72076 Tu ¨ bingen, Germany. Fax: + 49 7071293339, Tel.: + 49 70712973329, E-mail: maria.van-betteraey@uni-tuebingen.de Abbreviations: PCNA, proliferating cell nuclear antigen; BrdU, 5¢-bromodeoxyuridine; FITC, fluorescein isothiocyanate. (Received 30 April 2003, revised 24 July 2003, accepted 28 July 2003) Eur. J. Biochem. 270, 3880–3890 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03769.x functional complex, indicating a specific influence of O 2 -dependent cellular changes on critical steps of the assembly of a functional viral replication machinery. Consequently, we wanted to extend these studies from the SV40 model to the far more complex replication of the cellular genome of mammalian (human) systems. For this purpose we had to meet two demands. Firstly to find a cell line that can be induced to bear a maximal number of replicons arrested in the ‘hypoxic preinitiation state’ and as few as possible in other states of replication, and secondly the elaboration of a protocol for preparing a cell fraction that contains the cellular chromatin and specifically retains the functionally bound (replication) proteins. In this communication we demonstrate that by a simple starvation procedure followed by stimulation with fresh medium and concomitant establishment of hypoxia, the human bladder carcinoma cell line T24 can be induced to accumulate replicons scheduled to initiate in the early S-phase in most cells, while other stages of replicon activity are virtually absent. Reoxygenation triggers these replicons to initiate replication at a high degree of synchrony, followed by subsequent normal elongation. The immediate answer to sudden reoxygenation resembles in principle that of SV40 replicons in virus infected cells. However, replicons started in the noninfected T24 cells are much longer and not identical. Extraction of T24 nuclei with a Triton X-100 buffer yields a fast sedimenting nuclear fraction containing the cellular DNA and proteins associated with replicating chromatin. We take this material as a functional equivalent to SV40 minichromo- somes elutable from the nuclei of virus infected cells and operationally define replication proteins remaining after the Triton X-100 extraction (in accordance with [9]) as functionally chromatin-bound. To justify this view we examined whether reoxygenation triggers the association of PCNA, the processivity clamp of polymerase delta in mammalian replication, with the fast sedimenting Triton- extracted fraction. In a first experiment we employed this protocol thereby demonstrating that important components of the ‘classical’ prereplication complex [10] become bound to chromatin already during the hypoxic incubation. This suggests that the ‘hypoxic preinitiation state’ is similar to the known ‘classical’ prereplication complex, and that hypoxia directly influences mechanisms activating this complex. Materials and methods Cell culture, transient hypoxia, reoxygenation and radioactive labeling T24 cells (gift from Altana Pharma, Konstanz, Germany) were grown in plastic flasks in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ 100 lgÆmL )1 ). The cells were subcultured when they reached confluence. Under these conditions the cells exhib- ited a partially tetraploid caryotype. For synchronization, the desired number of glass Petri dishes was seeded from an almost confluent large culture with 150 000 cellsÆmL )1 (35 mm, 1.5 mL; 145 mm, 25 mL) 44 h before the start of an experiment. Thereby, most cells became arrested in G 1 due to starvation. When a 14 C prelabel was desired, the seeding medium was supplemented with 2.5 nCiÆmL )1 [ 14 C]Thd. Experiments started with stimulation of the cells by a complete exchange of the culture medium with prewarmed fresh medium supplemented with 10% (v/v) fetal bovine serum. Subsequent gassing of the cell cultures was performed with a continuous flow of humidified artificial air containing 5% (v/v) CO 2 for normoxic incubations, and with 0.02% O 2 ,5%CO 2 , and Ar to 100% for hypoxic gassing. For gassing, the equipment and the procedures described by [7] were used. For reoxygenation 0.25 volumes of medium equilibrated with 95% O 2 /5% CO 2 (v/v) were added to hypoxic cell cultures, and gassing was continued with artificial air. [Methyl- 3 H]deoxythymidine was added either directly to the cells, or under hypoxic culture conditions by plunging a spatula carrying the appropriate quantity in dried form into the culture medium. To stop incubations medium was removed by aspiration and the cells were washed once with ice-cold phosphate-buffered saline (NaCl/P i : 150 m M NaCl, 10 m M NaHPO 4 , pH 7) and either processed for determin- ation of acid insoluble radioactivity as described [11] or otherwise for analyses as described below. Alkaline sedimentation analyses of cellular DNA For analyzing the length distribution of growing daughter strands of T24 DNA, cultures on 35 mm glass Petri dishes were pulse-labeled for 8 min with 7 lCi [methyl- 3 H]deoxy- thymidineÆmL )1 . Labeling was stopped by washing the cells with ice cold phosphate-buffered saline (NaCl/P i : 150 m M NaCl, 10 m M NaHPO 4 , pH 7). The cells were trypsinized for 5 min at 4 °C and layered onto the top of 10–30% alkaline sucrose gradients [12]. After denaturation of the DNA for 6 h, centrifugation was performed at 20 000 r.p.m., 23 °Cfor10hinaBeckmanSW28rotor. 1.2 mL fractions were collected from the top of the gradient and processed to analyze acid insoluble radioactivity. DNA cytofluorometry For cytofluorometry of cellular DNA cells were trypsinized, washed with NaCl/P i and fixed with 90% methanol. Histograms of DNA contents were recorded with a FACSCalibur (Becton-Dickinson) after staining the cells with propidium iodide (0.05 mgÆmL )1 in 0.1% sodium citrate) and simultaneous RNase digestion (1 mgÆmL )1 )for 30 min at 37 °C. Cell fractionation Cells were washed once with NaCl/P i and twice with hypotonic buffer (20 m M Hepes, pH 7.5, 20 m M NaCl, 5m M MgCl 2 ) and suspended in 10 mL of hypotonic buffer. After 10 min on ice, cells were disrupted to free nuclei by 25 strokes with the tight fitting pestle of a dounce homogenisator and were then centrifuged for 5 min and 1500 g at 4 °C to separate the cytosolic supernatant from the nuclear pellet. Nuclei were resuspended in extraction buffer (50 m M Hepes, pH 7.5, 100 m M KCl, 0.25% Triton X-100, 2.5 m M MgCl 2 ,1m M dithiothreitol) containing Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3881 aprotinine (1 l M ), leupeptine (50 l M ), 4-(2-aminoethyl)- bezenesulfonylfluoride/HCl (1 m M )andNaF(10m M )and centrifuged for 3 min and 600 g at 4 °C. Nuclei were resuspended in extraction buffer three more times to fully lyse the nuclear envelope and complete extraction. Super- natants were combined and yielded nucleosolic proteins. The remaining pellet contains all DNA and structure bound proteins and is further referred to as chromatin-fraction. Electrophoresis of proteins and Western blotting Cytosolic and nucleosolic proteins were precipitated from the respective supernatants by adding five volumes of ice- cold acetone. Proteins remaining in Triton-extracted nuclei were recovered after nuclease digestion with DNase and RNase. Nuclei prepared as above were suspended in extraction buffer containing DNase (0.1 mgÆmL )1 ), RNase (0.025 mgÆmL )1 )andMgCl 2 (5 m M ). Digestion was for 1 h on ice. Proteins for Western blot analyses were then either isolated by phenol extraction and subsequent acetone precipitation as described [13] or directly denatured and solubilized with SDS electrophoresis sample buffer, as it turned out that remaining DNA fragments did not interfere with the running properties of the proteins in SDS-gels. Proteins were separated on an 8% SDS/polyacrylamide gel [14], blotted to Nylon-P membrane (Amersham) and subsequently immunodetected using the ECL Western blotting procedure (Amersham) according to the manufac- turer’s instructions. Dilution of antibodies used were as follows: PCNA (mouse monoclonal antibody, Santa Cruz) 1 : 3000, Cdc6 (mouse monoclonal antibody, Santa Cruz Biotechnologies) 1 : 500, MCM2 (rabbit polyclonal anti- body, Transduction Laboratories, Heidelberg, Germany) 1 : 10,000, MCM3 (rabbit polyclonal antibody, Transduc- tion Laboratories, Heidelberg, Germany) 1: 3000, Cdk2 (rabbit polyclonal antibody, Santa Cruz Biotechnologies) 1 : 500. Immunofluorescence staining of total PCNA and chromatin-bound PCNA Cells grown on coverslips were washed once with ice-cold NaCl/P i . For subsequent staining of total PCNA, cells were directly fixed with ice-cold acetone/methanol (1 : 1, v/v) for 10 min at 4 °C. When only chromatin-bound PCNA had to be stained, soluble proteins were extracted by washing the cells three times withextraction buffer (see Cell fractionation) and afterwards fixed with acetone/methanol (1 : 1, v/v) for 10 min at 4 °C. Subsequently all coverslips were processed for detection of PCNA after air drying. Cells were blocked with 1% (w/v) BSA in NaCl/P i for 20 min and incubated with anti-PCNA Ig (Boehringer Mannheim, dilution 1 : 100) in NaCl/P i /BSA for 1 h at room tempera- ture. After washing three times with NaCl/P i for 5 min they were further incubated for 30 min with anti-mouse IgG labeled with Alexa FluorÒ 586 (Molecular Probes, dilution 1 : 200) in NaCl/P i /BSA. Cells were again washed three times for 5 min with NaCl/P i . During the last wash total DNA was stained with bisbenzimide (2 lgÆmL )1 in NaCl/ P i ). Finally PCNA (Alexa FluorÒ 568 stain) and total DNA (bisbenzimide stain) were visualized with a Zeiss fluorescence microscope (Axioskop) using the appropriate filter combinations. Immunofluorescence staining of chromatin-bound PCNA and of replicating DNA Cells grown on coverslips were labeled by adding 15 l M 5¢-bromodeoxyuridine (BrdU) 15 min before the end of the respective incubation conditions, in case of hypoxic labeling by plunging a spatula carrying the appropriate quantity in dried form into the cell culture medium. To stop incubations cells were washed once with NaCl/P i . For extraction of soluble proteins cells were washed three times with extraction buffer (see Cell fractionation) and subsequently fixed with methanol for 10 min at 4 °C. Cells were then sequentially stained and fixed as reported previously in [15]. Briefly, cells were blocked with 1% (w/v) BSA in NaCl/P i for 20 min, incubated with anti- PCNA Ig (Boehringer Mannheim, dilution 1 : 100) in NaCl/P i /BSA for 1 h at room temperature and for 30 min with Alexa FluorÒ 568 antibody (red fluorescence, dilu- tion 1 : 200) in NaCl/P i /BSA. The primary and secondary antibodies were fixed in place with 4% (v/v) formaldehyde for 20 min at room temperature. Subsequently cells were washed twice with NaCl/P i . For DNA denaturation cells were treated with 2 M HCl at 37 °Cfor1h.After neutralization with NaCl/P i they were finally incubated for one h with a fluorescein isothiocyanate (FITC)-labeled anti-BrdU Ig (green fluorescence, Boehringer Mannheim, dilution 1 : 50). Between the antibody incubation steps cells were washed three times for 5 min with NaCl/P i . During the last wash total DNA was stained with bisbenzimide (2 lgÆmL )1 in NaCl/P i ). Finally PCNA (Alexa FluorÒ 568 stain), replicating DNA (FITC stain) and total DNA (bisbenzimide stain) were visualized with a Zeiss fluorescence microscope (Axioskop, Zeiss, Go ¨ ttin- gen, Germany) using the appropriate filter combinations. Results Inducing the ‘hypoxic preinitiation state’ in cellular replicons About 35 h after infection with SV40 virus, CV1 cells replicate almost exclusively SV40 minichromosomes at high intensity. These represent a highly homogenous population of conveniently small subcellular entities. During a hypoxic period of 6–7 h, a large amount of them is arrested in the ‘hypoxic preinitiation state’ [6,7] and can be released within 2–3 min into effective initiation and a succeeding synchron- ous replication round by reoxygenation. Thereby, among total viral genomes exhibiting replicative activity (‘viral replicons’), the fraction of hypoxically synchronized repli- cons reaches > 90%. Consequently, the equipment of the minichromosomes with replication proteins reflects with sufficient reliability the state of the replication machinery before and after oxygen recovery, respectively. Cellular replicons on the other hand, are highly hetero- geneous in their sizes and replication states within an asynchronous cell cycle. Therefore in addition to ‘hypoxic pre-initiation states’, 7 h of hypoxia accumulated significant amounts of replicons hit by hypoxia in other states of 3882 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003 activity [4]. Thus, accumulation of cellular ‘hypoxic prein- itiation states’ cannot be achieved as easily as that of SV40. Therefore we tried to subject cell populations enriched with G 1 cells to hypoxia, as successfully performed previously with Ehrlich ascites cells, by selecting G 1 cells by zonal zentrifugation [3,16]. In the course of investigating several cell lines (see Discussion), we came across the human bladder carcinoma cell line T24, which is easily arrested in G 1 by starvation [17]. Using this cell line, we developed an appropriate protocol. Briefly, cells were grown for 44 h after seeding which caused shortage of nutrients and growth factors in the medium. Starved cells were stimulated by exchanging the medium with prewarmed fresh medium, followed by hypoxic or normoxic gassing of the cells. The experiments described below demonstrate that replicative activity released immediately after O 2 admission to pre- treated hypoxic T24 cells represents almost exclusively synchronous replicon initiation followed by normal elon- gation. DNA synthesis rate The course of the [methyl- 3 H]deoxythymidine incorpor- ation rate into DNA of starved T24 cells was monitored after stimulation by medium renewal under normoxic, hypoxic and reoxygenated incubation conditions. Figure 1 shows that, in normoxically incubated cells, the incorpor- ation rate remained relatively low up to 4 h after medium exchange and then gradually increased up to 10 h, when maximal incorporation was attained. This was followed by a decrease. Under hypoxia, in contrast, incorporation decreased to a background level during the first 2 h and remained at this level until reoxygenation. Immediately after reoxygenation, incorporation of radioactivity increased strongly within a very short interval and decreased 6–8 h later. The profile of [ 3 H]Thd incorporation after reoxygen- ation appears double-peaked. The first peak is possibly caused by cells that proceded to the end of G 1 phase during the 7 h hypoxic gassing, accumulating replicons ready to initiate immediately after reoxygenation. Cells causing the second peak possibly had not yet reached this border during the 7 h hypoxic period. Alkaline sedimentation analyses of growing daughter strands A fast increase of the DNA synthesis rate either reflects release of replicon initiations or stimulation of elongation, or both. To determine the cause of the increase in Fig. 1, we analyzed the chain length distribution of pulse-labeled nascent daughter strands by means of alkaline sedimenta- tion. Synchronous replicon initiations first produce homo- geneously sized small daughter strands, which subsequently grow homogeneously to longer sizes, thus causing a synchronous shift of growing DNA chains to higher S-values. Figure 2A shows a survey of alkaline sedimentation profiles of acid-insoluble radioactivity from pulse labels applied to normoxic, hypoxic and reoxygenated T24 cells. The cells were prelabeled with [ 14 C]Thd when seeded, 44 h before the start of the experiment. The resulting [ 14 C]Thd profile (Fig. 2B, crosses) typically exhibits a peak in the last third of the gradient representing matured bulk DNA. The [ 14 C]Thd gradients were omitted from Fig. 2A for clarity. After medium exchange the normoxically incubated cultures exhibited a sedimentation profile (Fig. 2A, first profile) attributable to asynchronously acting replicons, because of a typical label distribution across the gradient, resulting from the normal steady-state of asyn- chronous initiation, elongation and termination. The gradient of hypoxically treated T24 cells contains almost no [ 3 H]Thd, as expected according to the incorporation curve (Fig. 1). As soon as 15 min after reoxygenation, a strong incorporation of [ 3 H]Thd into growing daughter strands occurs, preferentially sedimenting in the first third of the gradient and attributable to short chains originating from newly initiated replicons. In the course of further 25 min of reoxygenated growth, the incorporation of [ 3 H]Thd still increased, while the peak shifted to higher S-values. To visualize the chain growth between 15 and 40 min better, the last two profiles of Fig. 2A are depicted as the percentage of total c.p.m. in Fig. 2B. From 15 to 40 min after reoxygenation the peak distinctly shifted to higher S-values. The extent of the shift reflects the chain elongation during 25 min and can be calculated as about 0.5 lmÆmin )1 at either end of growing daughter strands. This is a very common elongation rate for mammalian cells. Note that up to 15 min after reoxygenation there is hardly any incorporation into fast sedimenting ‘old’ daughter strands. Thus, almost no active replicons occur that have been initiated before reoxygenation. The shapes of the two gradient profiles are narrow and very similar, suggesting that the cellular replicons grow synchronously at relatively homogenous elongation rates. Thus, alkaline Fig. 1. Rate of [ 3 H]Thd incorporation into DNA of starved T24 cells under normoxic (s) and hypoxic/reoxygenated (d) incubation condi- tions after medium renewal. T24 cells were prelabeled with [ 14 C]Thd and grown for 44 h. Subsequently the medium was renewed and cells were either incubated normoxically for 7 h, or hypoxically for 7 h and then reoxygenated. At the times indicated cells were pulse-labeled for 8min with 7lCiÆmL )1 [ 3 H]Thd while maintaining the respective incubation conditions during labeling and processed for measuring the ratio between acid-insoluble 3 Hand 14 C radioactivity. Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3883 gradient centrifugation confirmed that replicon initiation is inhibited under hypoxia. Upon reoxygenation, suppressed initiations are released very fast in a highly synchronous fashion. DNA cytofluorometry A large portion of the partially tetraploid T24 cells exhibited G 1 DNA content at 44 h growth after seeding (Fig. 3A). Subjecting such cells after medium renewal to a 7-h hypoxic period markedly increased the cell fraction with G 1 DNA content (Fig. 3B). Three hours after reoxygenation, the majority had entered the S-phase while a minor part still exhibited G 1 DNA content (Fig. 3C). This result also supports the assumption that after medium renewal most of the cells proceed during hypoxia through G 1 up to the point at which the first replicons of a (scheduled) S-phase would normally be activated. However, for a minor part of the cells a 7-h hypoxic incubation following medium exchange does not seem to be sufficient to accumulate replicons ready to initiate immediately after O 2 recovery. Perhaps these cells already were in G 0 at the time of medium exchange. Yan et al. [18] presented flow cytometric analyses of T24 cells 4 days after seeding in high density and following release from contact inhibition. In the ATCC catalogue T24 cells are described as hypertriploid with 8% polyploidy. In contrast to the diploid T24 cells used by Yan et al. [18], the T24 cells we used were tetraploid for unknown reasons. Nevertheless their flow cytometric analyses also show that the cells are arrested with a G 1 DNA content. As they enter S-phase about 20 h after replating, the cells must have been in a G 0 state before this. We intended to arrest the cells in G 1 , from where they can proceed to DNA synthesis within about 6 h. As shown in Fig. 3C, the majority of the cells exhibiting S-phase DNA content 3 h after reoxygenation probably only experienced a G 1 arrest. These cells are obviously identical to those initiating immediately upon reoxygenation, and may be the cause of the first peak in Fig. 1 and the sedimentation profiles shown in Fig. 2B. Mitotic index To demonstrate that after release of the hypoxic block T24 cells further proceed through the cell cycle normally and at high synchrony, we determined the percentage of mitotic cells. Figure 4 shows that after medium exchange and further normoxic gassing first mitotic cells appear after about 13 h, their number increases within the next 5 h and decreases again at longer incubation. A similar increase of DNA synthesis occurs in the same cells 8–10 h before (Fig. 1), compatible with an elapse of a S- and G2-phase. Cells exposed to hypoxia directly after medium renewal and reoxygenated 7 h later exhibited sharp rise of mitotic cells 10 h after reoxygenation, which resembles the sharp rise in the DNA synthesis rate directly after reoxygenation. The Fig. 3. Histograms of cellular DNA content recorded by flow cyto- fluorometry. T24 cells were grown for 44 h. Subsequently the medium was renewed and the cells were incubated hypoxically for 7 h or reoxygenated for 3 h thereafter. After stopping the respective incu- bation conditions, cells were trypsinized, fixed and stained as described in the Materials and methods. (A) Cells after medium renewal; (B) cells after medium renewal and 7 h of hypoxia (200 p.p.m.); (C) the same cells after 7 h of hypoxia (200 p.p.m) and 3 h aerated incubation. Fig. 2. Alkaline sedimentation patterns of pulse-labeled T24 DNA after lysis on top of the gradients. T24 cells were grown for 44 h, after which the medium was renewed and cells were either incubated normoxically or hypoxically for 7 h, or reoxygenated after 7 h of hypoxia. Nascent daughter DNA chains were pulse-labeled with 10 lCi [ 3 H]ThdÆmL )1 8 min before the end of the respective incubation conditions. (A) Comparison of the gradient profiles of normoxic, hypoxic, 15 min and 40 min reoxygenated T24 cells. Profiles are depicted consecutively in total c.p.m. Normoxia, 97215 c.p.m.; hypoxia, 365 c.p.m.; reoxygen- ated 15 min, 57735 c.p.m.; reoxygenated 40 min, 176754 c.p.m. Each profile consists of 31 fractions. (B) Comparison of the profiles of 15 min (m)or40min(d) reoxygenated cells (same as in Fig. 2A) in percentage of total c.p.m. ·,Matured 14 C-labeled bulk DNA of T24 cells. Sedimentation was from left to right. 3884 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003 mitotic index also exhibits a double-peaked profile similar to the profile of the [ 3 H]Thd incorporation. The double peak of the 3 H incorporation curve is therefore possibly caused by cells entering S-phase in succession. Separating a cell fraction containing DNA bound proteins Entire replicative SV40 minichromosomes bearing func- tionally bound replication proteins can be eluted from nuclei of virus infected cells by hypotonic buffer [19]. The DNA of mammalian chromatin, however, is organized into loops of about 5–150 kb firmly attached to the nuclear matrix [20]. Thus, intact cellular chromatin cannot be eluted from isolated nuclei. Interrupting the continuity of the DNA (e.g. by suitable endonucleases) yields elutable chromatin fragments preferably originating from regions far from matrix attachment points. As DNA replication foci are probably located near the nuclear matrix, preferably nonreplicative chromatin fragments might be eluted while replicative chromatin regions remain attached. Therefore, preserving the natural chromatin/matrix relations and extracting unbound replication proteins from the nuclei seemed to be more appropriate for studying the influence of oxygen recovery after a hypoxic period on DNA-bound proteins. For this purpose, we adopted a protocol described in [9] with some modifications. The modified protocol yields three fractions which are denoted according to the proteins they contain. Fraction 1 includes all ‘non-nuclear proteins’, i.e. cytosolic proteins separated during hypotonic prepar- ation of nuclei, fraction 2 contains ‘soluble nuclear proteins’ which are extractable from nuclei by Triton X-100 contain- ing buffer, and in fraction 3, the chromatin fraction, all proteins remain that resist Triton extraction. We supposed that the latter fraction included, besides the common chromatin proteins, functionally DNA-bound replication proteins. Because the PCNA protein is loaded by a well- defined actively controlled process onto replicative DNA structures [21], it can be taken as an example of replication proteins recruited to DNA according to the demands of replication. Western blot analyses T24 cells synchronized by starvation/hypoxia were fract- ionated as described. Equal amounts of protein from each fraction were separated by SDS gel electrophoresis, blotted onto a Nylon-P membrane and PCNA was immunodetec- ted. Figure 5 shows the results obtained from cells incuba- ted hypoxically for 7 h and then stopped or reoxygenated for 5 min, 30 min or 1 h. In cytosolic and soluble nuclear proteins, the amounts of PCNA did not vary under any incubation conditions. By contrast, in hypoxic chromatin only very little PCNA was detected. However PCNA increased strongly as soon as 5 min after reoxygenation and continued to increase after 30 min and 1 h. The pattern of chromatin-bound PCNA suggests that the protein is recruited to DNA as soon as its function in replication is required, after replicon initiation had taken place. Immunofluorescence staining of total cellular and chromatin-bound PCNA T24 were grown and incubated on coverslips. Two sets of hypoxic cells and of cells reoxygenated for 10 min and 30 min were prepared. One set of cells was directly fixed after the incubation. From the second set, soluble proteins were extracted by washing with buffer containing Triton X-100 prior to fixation. As shown in Fig. 6A, directly fixed cells show a very similar PCNA content after any incubation condition. No visible differences exist between hypoxically incubated and reoxygenated cells. The mainly nuclear localization of PCNA is due to the fixation procedure. Acetone fixation leads to cell shrinkage and loss of membranes. Therefore cytosolic PCNA is not as prominent as in the Western blot (Fig. 5). In contrast, when the cells were extracted prior to fixation by Triton Fig. 4. Mitotic index of starved T24 cells under normoxic (s)and hypoxic/reoxygenated (d) incubation conditions after medium renewal, respectively. T24 cells were grown on coverslips for 44 h. The medium was then renewed and cells were either incubated normoxically for 7 h, or hypoxically for 7 h and then reoxygenated. At the times indicated incubations were stopped, cells were fixed with acetone/methanol and total DNA was stained with bisbenzimide. Subsequently cells were photographed and counted. The percentage of mitotic cells was calculated as indicated. Fig. 5. Western blot analyses of cytosolic, soluble nucleosolic and chromatin-bound PCNA from hypoxic and reoxygenated T24 cells. Cytosolic, soluble nucleosolic and chromatin-bound proteins were prepared after the indicated incubation conditions (for details see Materials and methods) and equal amounts were separated on an 8% SDS/polyacrylamide gel. After blotting onto Hybond-P membrane (Amersham) PCNA was visualized with an anti-PCNA Ig (Santa Cruz Biotechnologies) using the ECL detection procedure. H, hypoxic; 5¢, 5minreoxygenated;30¢, 30 min reoxygenated; 1 h, 1 h reoxygenated. Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3885 Fig. 6. Immunofluorescence staining of total cellular PCNA and chromatin-bound PCNA. Cells were grown on coverslips for 44 h, after which the medium was renewed and cells were incubated hypoxically for 7 h or subsequently reoxygenated for the indicated periods. Cells were then either fixed directly or washed three times with extraction buffer to remove soluble proteins prior to fixation. PCNA was visualized using anti-PCNA Ig (Boehringer Mannheim, dilution 1 : 100) as the primary and Alexa FluorÒ 568 (dilution 1 : 200) as the secondary antibody. Total DNA was stained with bisbenzimide. (A) PCNA immunfluorescence staining of directly fixed T24 cells. (B) PCNA immunfluorescence staining of T24 cells extracted prior to fixation. The respective incubation conditions of cell cultures are indicated below the images. B 0 corresponds to B 1 and is shown in this special case to visualize all cell nuclei present. The other bisbenzimide images are not shown as the red PCNA fluorescence is almost identical to the blue DNA fluorescence. 3886 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003 buffer, PCNA was barely detectable in nuclei from hypoxic cells but became visible in nuclei as soon as 10 min after reoxygenation. The proportion of unextractable PCNA increased significantly from hypoxic to reoxygenated incubations. These results again confirm that PCNA becomes chromatin-bound only when required for DNA synthesis. Simultaneous staining of replicating DNA and DNA-bound PCNA To demonstrate the connection between active DNA replication and the appearance of bound PCNA in nuclei, simultaneous immunodetection of replicating DNA after BrdU incorporation and PCNA was performed. T24 cells grown on coverslips were incubated hypoxically and then stopped or reoxygenated for 30 min. Labeling with 15 l M BrdU was started 15 min before the end of either incuba- tion. The cells were extracted prior to fixation and processed for BrdU and PCNA immunodetection. As shown in Fig. 7, hypoxic cells exhibit neither visible BrdU incorporation nor bound PCNA. However, 30 min after reoxygenation, BrdU incorporation into replicating DNA was detectable and the amount of PCNA not extractable by Triton buffer was high in the same cells. These results clearly show that the PCNA staining is colocated with the BrdU staining and this again signifies that PCNA is only bound to chromatin portions where actively replicating DNA is present. Recruitment of proteins involved in replication to chromatin during the hypoxic period In contrast to starved T24 cells, which begin to initiate replication after about 4 h following medium stimulation, T24 cells that were exposed to hypoxia after medium exchange start replicon initiation immediately upon reoxy- genation. This suggests that the ‘classical’ prereplication complex was already formed under hypoxia. We applied the elaborated protocol to investigate the binding of MCM2, MCM3 and Cdc6, which are known to be important components of the prereplication complex as well as Cdk2, which is considered to be (one of) the activating kinase(s) of the complex, after medium renewal before and at the end of hypoxic gassing as well as under normoxic conditions. As shown in Fig. 8 MCM3 and Cdc6 are not, and MCM2 and Cdk2 are barely, detectable on chromatin of starved T24 cells (lane 1). This may be caused partly by different sensitivities of the antibodies used. However, Fig. 7. Immunofluorescence staining of replicative T24 DNA and chromatin-bound PCNA under hypoxic and reoxygenated incubation conditions. T24 cells were grown on coverslips for 44 h. The medium was renewed prior to hypoxic gassing. Replicative DNA was labeled by incubating the cells for 15minwith15 l M BrdU at the end of the respective incubation. Cytosolic and soluble nuclear proteins were extracted prior to fixation by washing the cells three times with extraction buffer (see Materials and methods). BrdU incorporated in replicating DNA was visualized after denaturation with anti-BrdU–FITC conjugated Ig . PCNA was visualized by using anti-PCNA Ig, followed by anti-mouse IgG labeled with Alexa FluorÒ 568. Total DNA was stained with bisbenzimide. The respective incubation conditions are indicated below the images. Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3887 after medium renewal these proteins become obviously bound to chromatin under hypoxic and normoxic condi- tions. The signal intensities are slightly stronger under hypoxic than under normoxic conditions. This seems reasonable, as hypoxic suppression of replicon initiation accumulates prereplication complexes which disappear after initiation is completed. The latter gradually occur in cultures not subjected to hypoxia after medium renewal. The lack of Cdc6 may explain the 4 h lag phase in replication after medium exchange under normoxic conditions, since prior to replicon initiation prereplication complexes have to be formed. This requires certain proteins that have to be translated before (especially proteins with a short half life, such as Cdc6) or whose mRNA has to be transcribed first. Nevertheless, the known ‘classical’ prereplication com- plex seems to be formed under hypoxia, rendering the cells ready to activate scheduled replicon initiations immedi- ately upon reoxygenation. In this context it is noteworthy that the prereplication complex activating kinase Cdk2 becomes bound to chromatin during hypoxia. The band- ing pattern shows differences compared to Cdk2 of normoxic cells. Under hypoxia the form of the protein that migrates faster seems to predominate. Under norm- oxic conditions both forms seem to be present at roughly equal proportions. Discussion Although common interest focuses on the replication of cells’ own genome, replication of SV40 DNA frequently serves as a convenient model of mammalian (human) DNA replication. However, when the cellular replication equip- ment is abused for viral multiplication, cellular mechanisms are often falsified or put out of function, in particular the regulatory mechanisms involved. Decisive experiments concerning regulatory phenomena have to be performed in a cellular system in the long term. The aim of the present study was to establish means for extending a recent study [8] on changes of replication proteins bound to SV40 minichromosomes, occurring in the context of the fast O 2 -dependent regulation of replica- tion [6–8], from the viral system to a (preferably human) cellular system. Thus we were confronted with two main problems. Firstly, inducing in as many as possible cellular replicons the ‘hypoxic preinitiation state’ and excluding as completely as possible active replicons in other states. Secondly, preparing a cell fraction containing only those replication proteins which are functionally associated with cellular chromatin and not those located in cytosolic or nucleosolic fractions. With respect to the first problem, we initially tried to use Ehrlich ascites cells. With these cells we first demonstrated the existence of the fast O 2 -dependent regulation of replication [12,22,23]. We had already developed means to select vital G 1 cells from cell cultures by a zonal centrifu- gation procedure [16] and succeeded to bring them homo- geneously to hypoxic arrest in which they bore exclusively early S-phase replicons in the desired ‘hypoxic preinitiation state’ [3]. Although resuming the old experiments principally confirmed the suitability of the Ehrlich ascites cell system for the present purpose, we searched for alternatives because the selection procedure is complicated, time consuming and works only with a mouse cell line (i.e. Ehrlich ascites), while most available antibodies are directed against human replication proteins. Consequently we next examined a set of human cell lines, e.g. CCRF, HeLa [4], PC3, A549, BHK, TC7, SW2, HL60 and HUVEC with respect to their response to hypoxia and reoxygenation. The alkaline sedimentation profiles of HeLa and CCRF cells after hypoxia and reoxygenation already revealed [4] that hypoxic incubation caused significant accumulation of initiation competent replicons, which could be released into a more or less synchronous round of replication upon reoxygenation. However the extent of replicon synchrony attained by the hypoxic incubation alone, i.e. absence of active replicons in the state of elongation, turned out to be insufficient for examining the transition reaction between the hypoxic and the reoxygen- ated state in a satisfying specific manner. The same problem occurred with the other cell lines examined. Inhibitors such as thymidine or aphidicolin were not used, as they inhibit elongation and not replicon initiation. Furthermore, we had shown previously that initiation is not blocked in SV40- infected CV1 cells treated with aphidicolin prior to reoxy- genation [6]. Fortunately, we observed that in the human bladder cancer cell line T24 the effect of hypoxia/reoxygenation could be intensified five- to 10-fold when the medium was renewed prior to hypoxia. We suspected that these cells had been (at least partly) arrested in G 1 simply by preceding starvation as formerly described by Prescott [17]. Our experiments confirmed this suspicion. After the optimal starvation conditions were found, starved T24 cells were incubated hypoxically directly after stimulation by medium renewal. This treatment accumulated cellular replicons Fig. 8. Western blot analyses of chromatin-bound MCM2, MCM3, Cdc6 and Cdk2 from normoxic and hypoxic T24 cells. Chromatin- bound proteins were prepared after the indicated incubation condi- tions (for details see Materials and methods) and equal amounts were separated on an 8% SDS/polyacrylamide gel. After blotting onto Hybond-P membrane (Amersham) the respective proteins were immunodetected using the ECL detection procedure. Lane 1, norm- oxia without medium renewal (i.e. beginning of the experiment); lane 2, normoxia 7 h after medium renewal; lane 3, 7 h hypoxia after medium renewal. 3888 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003 almost exclusively in the ‘hypoxic preinitiation state’. It should be mentioned that T24 cells proceed normally through the cell cycle after hypoxia/reoxygenation for several days. No signs of apototic cell death could be detected by the CaspaTag TM Caspase (VAD) Activity Kit (Intergen, Oxford, UK) during and after the hypoxic treatment (data not shown). With respect to the second problem, we demonstrated by means of the PCNA example that T24 nuclei extracted by Triton X-100 buffer contain functionally chromatin- bound replication proteins, switching from another cellular compartment into the chromatin fraction (Figs 5–7) or undergoing changes of modifications (e.g. phosphoryla- tion) in response to O 2 recovery of hypoxic cells. PCNA seemed most suitable because elongation is not affected [2] or just slowed down under hypoxia [4] and ongoing elongation is dependent on functional PCNA. We suggest that the absence of chromatin-bound PCNA under hypoxia is rather a direct consequence of missing initi- ation, i.e. lost activation of the ‘hypoxic preinitiation complex’, than an impairment of ‘clamp loading’ by replication factor C. Since T24 cells start to replicate immediately upon reoxygenation, transcriptional or translational processes can be excluded as cause of the hypoxic arrest. It was already shown for Ehrlich ascites cells that the expression of growth related mRNA is not influenced during transient hypoxia [1]. DNA replication in eukaryotes is initiated by the stepwise assembly of proteins to the replication origin [10,24,25]. First the hexameric origin recognition complex binds [26], which then recruits Cdc6 [27,28], cdt1 [29,30] and the minichromosome maintenance proteins [31]. This prerepli- cation complex is built up during G 1 of the cell cycle. The complex is presumably activated by cyclin-dependent kinase Cdk2 [32,33] and the Dbf4/cdc7 [34] kinase, which is required to load the initiation factor Cdc45 on the prereplication complex [35–37]. To investigate whether this prereplication complex is built under hypoxia we performed a first experiment using the above described protocol. We show that MCM2/MCM3 and Cdc6, as well as the activating kinase Cdk2, present in two modifications with different electrophoretic mobilities, become bound to chro- matin already under hypoxia, thus enabeling hypoxic cells to initiate as soon as the hypoxic suppression of replicon initiation is released. The relative intensities of the two Cdk2 bands differ under hypoxia and normoxia. Possibly, this represents a modification of the kinase influencing its activity/inactivity. Post-translational processes such as modifications (e.g. phosphorylations or dephosphoryla- tions) of proteins have already been found to be important regulators in SV40 replication [38,39]. Our ongoing work now focuses on changes arising in the pattern of further chromatin-bound proteins of hypoxic and reoxygenated cell T24 cells. We use classical Western blot analyses with immunodetection of replication proteins or regulators as well as high resolution 2D-gels. With the aid of the T24 system presented here, we hope to characterize the special state of the protein equipment of the replication of human cells under the hypoxic block and to elucidate the fast events occurring as an effect of oxygen recovery. Acknowledgements We thank G. Probst for critical reading of the manuscript. References 1. Riedinger, H.J., Gekeler, V. & Probst, H. (1992) Reversible shutdown of replicon initiation by transient hypoxia in Ehrlich ascites cells. Dependence of initiation on short-lived protein. Eur. 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Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24. cytometric analyses of T24 cells 4 days after seeding in high density and following release from contact inhibition. In the ATCC catalogue T24 cells are