Cdk2 activity is dispensable for triggering replicon initiation after transient hypoxia in T24 cells Dirk Stabenow, Hans Probst and Maria van Betteraey-Nikoleit Interfakulta ¨ res Institut fu ¨ r Biochemie der Universita ¨ tTu ¨ bingen, Germany In eukaryotic cells, orderly cell cycle progression is believed to be regulated by the action of Cdks and their binding partners, i.e. the cyclins, their inhibitors, and the E2F family of transcription factors [1]. The initiation of the first replicons at the G 1 - to S-phase transition, marks a key step of cell cycle progression. In living cells, the stepwise assembly of proteins at the replication origins of replicons prepares for initiation [2–5]. First, the hexameric origin recognition complex binds; this then recruits Cdc6 [6,7], Cdt1 [8,9] and the minichromosome maintenance proteins [10]. This pre- replication complex (pre-RC) is built up during the G 1 -phase. The complex is suggested to be activated by Cdk2 [11] and the Dbf4 ⁄ Cdc7 kinase [12] which is required to load the initiation factor Cdc45 on the pre- RC [13–15]. Commonly, Cdk2 in association with cyclin E is thought to be essential for driving the cells through this transition. Cdk2-mediated phosphoryla- tions are reported to be important in numerous steps preceding initiation of DNA replication; for example, together with Cdk4, the phosphorylation of pRb and the subsequent release of E2F results in the transcrip- tion of essential factors of the replication initiation complex such as MCM and Cdc6 [16,17]. Protein phosphorylations by Cdk2 were suggested to be important for initiation complex assembly and activa- tion [17,18]. Cdk2 ⁄ cyclin A mediated phosphorylation of Cdc6 is thought to induce its translocation from the Keywords hypoxia; reoxygenation; Cdk2; replication; chromatin Correspondence M. van Betteraey-Nikoleit, Physiologisch- Chemisches Institut der Universita ¨ t Tu ¨ bingen, Hoppe-Seyler-Straße 4, D-72076 Tu ¨ bingen, Germany Fax: +49 7071293339 Tel: +49 70712973329 E-mail: maria.van-betteraey@ uni-tuebingen.de (Received 6 July 2005, revised 16 August 2005, accepted 5 September 2005) doi:10.1111/j.1742-4658.2005.04957.x We examined whether the fast release of replicon initiation after sudden O 2 recovery of hypoxically incubated mammalian cells depends on kinase activity of Cdk2. We used a system based on starved ⁄ refed T24 cells elab- orated previously for such investigations [van Betteraey-Nikoleit M, Eisele KH, Stabenow D & Probst H (2003) Eur J Biochem 270, 3880–3890]. Cells subjected to hypoxia concurrently with refeeding accumulate the G 1 DNA content within 5–6 h. In this state they are ready to perform, within 1– 2 min after O 2 recovery, a burst of replicon initiations that marks the start of a synchronous S-phase. We found that Cdk2 binds to the chromatin fraction within 4–6 h after refeeding with fresh medium, irrespective of whether the cells were incubated normoxically or hypoxically. However, inhibition of Cdk2 by olomoucine, roscovitine or the Cdk2 ⁄ cyclin inhibi- tory peptide II had no influence on the synchronous burst of replicon initi- ations. Cdc6 and pRb, possible targets of Cdk2 phosphorylation, behaved differentially. Inhibition did not affect phosphorylation of Cdc6 after reoxygenation, whilst chromatin bound pRb remained hypophosphorylated beyond the initiation burst. Thus, neither Cdk2 activity, though present at the end of the hypoxic period, nor pRb phosphorylation are necessary for releasing the burst of replicon initiations upon oxygen recovery. Conse- quentially, Cdk2 dependent phosphorylation(s) cannot be a critical trigger of replicon initiation in response to reoxygenation after several hours of hypoxia, at least in the T24 cells studied. Abbreviations FITC, fluorescein isothiocyanate; PCNA, proliferating cell nuclear antigen; Pre-RC, prereplication complex. FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5623 nucleus to the cytoplasm, thus preventing reinitiation. Altogether, these observations suggest that Cdk2 activ- ity is necessary for progression of cells from G 1 to act- ive DNA replication. The importance of Cdk2 has been substantiated by a number of different approa- ches. Microinjection of antibodies against Cdk2, cyclin E or cyclin A and the antisense mRNA of Cdk2 block initiation of DNA synthesis in mammalian cells [19,20]. In vitro initiation in G 1 nuclei is dependent on Cdk2 ⁄ cyclin A and Cdk2 ⁄ cyclin E complexes [21]. Fur- thermore, in general, elevated levels of the Cdk2 inhib- itor p27 and decreased activity of Cdk2 in conjunction with hypophosphorylated pRb have been shown to result in growth arrest [22,23]. However, these results have been challenged by the finding that Cdk2 knock- out mice are viable [24]. In addition, Cdk4 can sub- stitute for Cdk2 in pRb phosphorylation, and proliferation of cancer cells that do not contain pRb, may be completely independent of Cdk2 or Cdk4 activity [25]. In order to reveal whether Cdk2 activity is also involved in the very fast (requiring a few min only) release of replicon initiations from the hypoxic arrest described first by us [26], we studied the association of Cdk2 with the chromatin fraction of T24 cells and its enzymatic activity during the course of a starvation– refeeding ⁄ hypoxia–reoxygenation experiment. As out- lined in detail before and substantiated by cytofluoro- metry, analysis of DNA replication at the level of replicons and determination of the fraction of mitotic cells [27] our especially elaborated starvation protocol accumulates T24 cells in a G 1 arrest, from which they can be released by medium renewal. Normally, the cells then proceed to the S-phase within approximately 5 h, passing through a number of the above mentioned regulatory steps until the start of orderly DNA synthe- sis at the origins of replication (scheduled to be activa- ted as the very first of the S-phase). We demonstrated [27] that subjecting the cells to hypoxic conditions directly after restimulation with fresh medium reversi- bly interrupts this process at a state situated extremely close to the actual occurrence of replicon initiation. The cells accumulate in a state which we refer to as the ‘hypoxic preinitiation’ state on the basis of prior studies on DNA replication of a number of other cell lines [27] and Simian virus 40 replication in vivo [28]. From this state, characterized by a still incomplete and not yet functional set of replication proteins associated with chromatin, they can be released within 1–2 min by restoring atmospheric pO 2 , into a synchronous wave of replicon initiations. During the transition trig- gered by the reoxygenation event, chromatin bound replication proteins become completed [28]. We have demonstrated that, in the T24 system described before [27], most cells pass through a normal synchronous S-phase after reoxygenation. In the present study we found that substantial chro- matin associated Cdk2 emerged during the hypoxic period. However, inhibitors of Cdk2 activity did not affect replicon initiation after reoxygenation in vivo. Cdc6 phosphorylation in the course of initiation of DNA replication was also not affected, pRb remained hypophoshorylated for at least 30 min beyond the ini- tiation burst. Thus, at least in the T24 cells studied, Cdk2 activity as well as Rb phoshorylation is dispensable for the fast release of hypoxically suppressed initiation of DNA replication at the beginning of the S-phase. Results Hypoxia or reoxygenation do not interfere with the emergence of chromatin bound Cdk2 activity after restimulation by fresh medium In [27] we found no Cdk2 protein in the chromatin fraction of starved T24 cells by western blot analysis. However, significant amounts exist 7 h after medium renewal, irrespective of whether the cells were grown normoxically or hypoxically. Immunofluorescence staining of the cells using a Cdk2 specific antibody (Fig. 1D, final column) confirmed this result. We then examined the protein kinase activity of Cdk2 from dif- ferent fractions of hypoxic and reoxygenated cells: The medium of two cultures with starved T24 cells was renewed and both were incubated hypoxically for a fur- ther 7 h. One culture was subsequently reoxygenated for 30 min. The immunoprecipitates of cytosolic, NP40-extractable nucleosolic, and salt eluted chromatin bound proteins were analysed for protein kinase activ- ity as described in Eperimental procedures. The result- ing autoradiograph (Fig. 1A) convincingly shows that the highest concentration (relative to total protein pre- sent) of kinase activity occurs in the chromatin bound fraction and (slightly less) in the cytosolic fraction. The nucleosol exhibits the lowest activity. There is obvi- ously no difference between Cdk2 precipitated from hypoxic and 30-min reoxygenated cells. Western blot analysis of the same membrane (Fig. 1B), exhibited analogous differences of general signal intensities, but revealed two bands of different electrophoretic mobil- ity, most probably reflecting two different phosphoryla- tion states. We therefore suspect that chromatin bound Cdk2 differs from the cytosolic form by its preferential phosporylation state. However, both forms obviously exhibit comparable protein kinase activity in vitro. Cdk2 is dispensable for replicon initiation D. Stabenow et al. 5624 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS We next examined the inhibitory action of the Cdk2 inhibitors olomoucine, roscovitine and staurosporine, all of which compete for the ATP-binding domain of the kinase [29,30], and the Cdk2 ⁄ cyclin inhibitory pep- tide II which is reported to inhibit specifically the phosphorylation of substrates by Cdk2⁄ cyclin A and Cdk2 ⁄ cyclin E complexes [31] in an in vitro kinase assay. All four inhibitors were used at medium concen- trations reported by others to reliably inhibit Cdk2 [31–34]. Starved T24 cells incubated normoxically after medium stimulation were lysed. Subsequently, Cdk2 was immunoprecipitated. The kinase assay was per- formed both in the absence and in the presence of the inhibitors. Figure 1C shows that Cdk2 activity is signi- ficantly decreased in the presence of the inhibitors. Furthermore, to demonstrate that the Cdk2 ⁄ cyclin inhibitory peptide II is cell permeable and localizes at the sides of Cdk2, we used the Cdk2 ⁄ cyclin inhibitory peptide II in a form carrying a fluorescence label at the amino end (Fig. 1D, middle column). Starved T24 cells were stimulated by medium renewal (except for the culture designated N–), and hypoxic or normoxic gassing was started concurrently. The labelled peptide was added (without interruption of the hypoxic incu- bation) 4 h after the start of gassing. A further 3 h later, one hypoxic culture was reoxygenated for 30 min. After this, cells were processed for Cdk2 immunostaining as described in Experimental proce- dures. Finally, we found a strong fluorescence within the cells colocalized with the Cdk2 immunostaining (Fig. 1D, last column). Starved T24 cells (N–) expres- sing barely detectable amounts of Cdk2 protein A B C D Fig. 1. Cdk2 activity does not decrease under hypoxia. (A) Phos- phorylation of histone H1 by immunoprecipitated Cdk2 obtained from the cytosolic, nucleosolic and salt eluted chromatin bound pro- teins, after 7 h hypoxia or reoxygenation, respectively. Immuno- precipitations and subsequent kinase assays were performed as described. The kinase reaction was stopped by boiling in protein buffer, and proteins were separated by SDS ⁄ PAGE (12% polyacryl- amide). After blotting, the membrane was autoradiographed. (B) Western blot analysis of immunoprecipitated Cdk2 from (A). H, 7 h hypoxic incubation after medium renewal; 30¢, reoxygenated for 30 min after 7 h hypoxic incubation. (C) Phosphorylation of histone H1 by immunoprecipitated Cdk2 from cell lysate prepared after 7 h normoxic incubation following medium stimulation. Immunoprecipi- tation was performed as described. Subsequently, the kinase assay was performed in the absence or presence of olomoucine (20 l M), roscovitine (7 l M), staurosporine (100 nM) or the Cdk2 ⁄ cyclin inhibi- tory peptide II (50 l M). The kinase reaction was stopped by boiling in protein sample buffer, and the proteins were separated by SDS ⁄ PAGE (12% polyacrylamide). After blotting, the membrane was autoradiographed (upper row). Western blot analysis of the membrane of immunoprecipitated Cdk2 was performed afterwards (lower row). (D) Immunofluorescence staining of Cdk2 under norm- oxic, hypoxic and reoxygenated incubation conditions in the pres- ence of FITC labelled Cdk2 ⁄ cyclin inhibitor peptide II. T24 cells were grown on coverslips for 44 h. The FITC labelled Cdk2 ⁄ cyclin inhibitory peptide II was added 4 h before the end of the respective incubation conditions. After fixation of the cells, Cdk2 was visual- ized by using a Cdk2 antibody, followed by an anti-mouse Ig labelled with Alexa FluorÒ 568. Total DNA was stained with bis- benzimide. The respective incubation conditions are indicated below the images. N–, 7 h normoxic incubation without medium renewal; N+, 7 h normoxic incubation after medium renewal; H, 7 h hypoxic incubation after medium renewal; R 30¢, reoxygenated for 30 min after 7 h hypoxic incubation. D. Stabenow et al. Cdk2 is dispensable for replicon initiation FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5625 showed significantly less fluorescence of the inhibitor and as mentioned above Cdk2 immunofluorescence. Both increased dramatically after feeding the cells. Cdk2 inhibitors fail to prevent replicon initiation after reoxygenation As mentioned, our starvation–feeding ⁄ hypoxia proto- col arrests T24 cells very close before the effective entry into S-phase. During the hypoxic period, they accumulate in a state separated by a few minutes only from initiation of the replicons scheduled to be repli- cated first [27]. Readmission of O 2 to a thus pretreated T24 culture triggers a subsequent synchronous burst of replicon initiations. Up to about 1 h thereafter, the replicative activity in the reoxygenated culture is gov- erned mainly by synchronous daughter strand growth within the replicon cohort activated in response to the reoxygenation event. Afterwards, initiations of repli- cons scheduled to be activated later in the S-phase succeed, followed by G 2 , mitosis and a further syn- chronous cell cycle [27]. Alkaline sedimentation analy- sis of the length distribution of pulse labelled daughter strand DNA is suited to demonstrate the initial burst as well as the succeeding synchronous daughter strand elongation. On the basis of numerous prior studies [26,35–40], 8-min [ 3 H]dThd pulses applied 20 and 40–50 min after reoxygenation, proved to be most con- venient for demonstrating initiation and succeeding elongation. We studied the influence of olomoucine, roscovitine and the Cdk2 ⁄ cyclin inhibitory peptide II on the sedi- mentation profiles of pulse labelled daughter strand DNA 20 and 50 min after reoxygenation (Fig. 2). Starved T24 cells were stimulated by medium renewal, and hypoxic gassing was started concurrently. The inhibitors were added (without interruption of the hyp- oxic incubation) 4 h after the start of gassing. A fur- ther 3 h later, all cultures were reoxygenated. After continuing normoxic incubation for 20 or 50 min, respectively, the cells were labeled by [ 3 H]dThd pulses. In controls, inhibitor addition was omitted. The controls presented in the inset of Fig. 2 serve to confirm the expected effect of hypoxia in the present experiment. These alkaline sedimentation profiles represent pulse labelled DNA of cells which were Fig. 2. Inhibition of Cdk2 does not affect replicon initiation after reoxygenation. Alkaline sedimentation patterns of pulse-labelled T24 DNA after lysis on top of the gradients. Eight minutes before the end of the respective incubation conditions, nascent daughter strand DNA chains were pulse-labelled with 7 lCi [ 3 H]dThdÆmL )1 . Inset, Starved T24 cells were stimulated by medium renewal and incubated normox- ically (n) or hypoxically (.)for7h;x, 14 C-labelled matured bulk DNA. Main figure, T24 cells were stimulated by medium renewal, incubated hypoxically for 7 h and then reoxygenated for 20 min (open symbols) and 50 min (filled symbols), respectively. Olomoucine (20 l M), roscovi- tine (7 l M) or the Cdk2 ⁄ cyclin inhibitory peptide II were added 3 h before reoxygenation. s, d, Reoxygenated for 20 min and 50 min, respectively, after 7 h hypoxic incubation; h, n, reoxygenated for 20 min and 50 min, respectively, after 7 h hypoxic incubation in presence of olomoucine; e, r, reoxygenated for 20 min and 50 min, respectively, after 7 h hypoxic incubation, in presence of roscovitine; n, m, reoxy- genated for 20 min and 50 min, respectively, after 7 h hypoxic incubation, in presence of the Cdk2 ⁄ cyclin inhibitory peptide II. Cdk2 is dispensable for replicon initiation D. Stabenow et al. 5626 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS incubated 7 h after medium renewal normoxically (open triangles) and hypoxically (filled triangles). The mature DNA of these cells contained a prelabel result- ing from [ 14 C]Thd added 44 h before. The 14 C profile obtained (crosses) typically exhibits a peak in the last third of the gradient. The 3 H-profile of the normox- ically incubated culture exhibits a sedimentation profile attributable to asynchronously acting replicons and reflects the normal steady-state of asynchronous initi- ation, elongation, and termination [26,38,41]. The gra- dient of hypoxic cells contains practically no 3 H-label, indicating almost total absence of replicative activity. Twenty minutes after reoxygenation (Fig. 2, main, open symbols), four almost coincident prominent radioactivity peaks at fraction 7–8 (35–41 S 20w ) indi- cate the presence of growing daughter strands of about 35–50 kb length in both inhibitor treated and control cells. Assuming a bidirectional strand elongation rate of 1.5 kbÆmin )1 at either end of strands, this size is compatible with daughter strands of replicons initiated 10–20 min before. Minor shoulders around fractions 11–12 (51–67 S 20w ) indicate the additional presence of small portions of longer growing strands. This shoul- der was more or less prominent in different independ- ent experiments of this type (not shown). As in prior studies, we attribute them to replicons in few cells which could no more escape the late S-phase during starvation and are hit by hypoxia in a still active state. Reoxygenation probably reactivates these late S-phase replicons. However, the shoulder is no more present in the profiles obtained by analysing the cells 50 min after reoxygenation (Fig. 2, filled symbols). All four profiles exhibit nearly identical narrow single peaks around fraction 13 (about 62 S 20w or 140–150 kb). Relative to the main peaks obtained at 20 min, the observed shift is again compatible with an elongation rate of about 1.5 kbÆmin )1 . Thus, the inhibitors had no detectable influence, neither on the replicon initiation burst trig- gered by reoxygenation, nor on the succeeding growth of daughter strands. Changes of chromatin bound pRb and Cdc6 after reoxygenation Starved T24 cells were stimulated by medium renewal (except for the culture designated N–) and incubated normoxically or hypoxically. Further cultures were reoxygenated after hypoxic incubation. After fraction- ation of the cells, chromatin bound proteins Cdc6 and pRb were examined by western blot analysis. Western blot analysis of pRb using a pRb antibody which recognizes an epitope between amino acids 332– 344 (Fig. 3A, upper row) and an antibody specific for underphosphorylated pRb (Figs 3A, row 2 from top) concurrently suggested that pRb is hypophosphorylated in starved cells and becomes phosphorylated within 7 h after medium stimulation and further normoxic incubation (Fig. 3B shows the effect of phosphatase A B C Fig. 3. Phosphorylation of Cdc6 and pRb after reoxygenation. (A) Western blot analysis of chromatin bound pRb, Cdc6, and PCNA from 7 h normoxic, hypoxic, and 30 min reoxygenated T24 cells. (B) Lambda protein phosphatase digestion of immunoprecipitated Cdc6 and pRb. (–), Immunoprecipitated Cdc6 and pRb prior phos- phatase digestion; (+), immunoprecipitated Cdc6 and pRb after phosphatase digestion according to the manufacturer’s instructions (New England Biolabs: Lambda protein phosphatase). (C) Western blot and subsequent immunological detection of chromatin bound PCNA and Cdc6. Staurosporine (10 l M) was added 30 min before reoxygenation where indicated. N–, 7 h normoxic incubation with- out medium renewal; N+, 7 h normoxic incubation after medium renewal; H, 7 h hypoxic incubation after medium renewal; 5¢, reoxy- genated for 5 min after 7 h hypoxic incubation; 30¢, reoxygenated for 30 min after 7 h hypoxic incubation. D. Stabenow et al. Cdk2 is dispensable for replicon initiation FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5627 digestion on pRb immunoprecipitated from N+ T24 cells). In comparison with normoxic cells, a larger portion seems to remain hypophosphorylated in the hypoxic cells. Reoxygenation apparently causes no detectable change of pRb phosphorylation, at least during the following 30 min. Cdc6 protein was not detectable in starved cells and appeared after medium stimulation, whereby more Cdc6 accumulated during hypoxic than under normox- ic incubation conditions (Fig. 3A, row 3 from top). After reoxygenation, the amount of chromatin bound Cdc6 decreased within 30 min below the level of the normoxic control. Chromatin bound Cdc6 obtained from hypoxic cells seems to migrate slightly faster compared with that occurring in reoxygenated cells. Confirmed by the effect of phosphatase digestion (Fig. 3B), this points to a phosphorylation after re- oxygenation. The suspected phosphorylation could (nonspecifically) be prevented by adding 10 lm staurosporine before reoxygenation (Fig. 3C). The lat- ter experiment again clearly shows that the amount of chromatin bound Cdc6 is high at the end of the hyp- oxic period and distinctly decreases within 30 min after reoxygenation. The remainder is apparently converted into the slower migrating form. This significant decrease and conversion to the slower migrating form is inhibited by 10 lm staurosporine. The presence of substantial amounts of chromatin- bound proliferating cell nuclear antigen (PCNA) [27,28] can, in conformity with the function of PCNA as processivity factor of polymerase delta, serves as an indicator of active replicative DNA strand elongation which, for its part, depends on successful replicon initi- ation. Therefore, its increase following reoxygenation as shown in Fig. 3A,C indicates successful replicon ini- tiation. Treatment with 10 lm staurosporine (Fig. 3C) abolishes this increase. This suggests that, in accord- ance with earlier findings of our group [42], replicon initiation was suppressed by the elevated concentration of staurosporine. Effects of Cdk2 inhibition on pRb, Cdc6 and PCNA in vivo In Fig. 4 we analysed the effects of the Cdk2 inhibitors olomoucine, roscovitine and staurosporine (at 100 nm) on Cdk2 activity and its substrates pRb and Cdc6 in vivo. Starved T24 cells were stimulated by medium renewal (except for the culture designated N–). Subse- quently, the cells were incubated normoxically or hypoxically, or were reoxygenated with or without prior addition of the inhibitors. A concentration of 100 nm staurosporine suffices to inhibit Cdk2 accord- ing to [43], but does not suppress replicon initiation in cultured cells [42]. Accordingly, chromatin bound PCNA increased after reoxygenation under 100 nm staurosporine as well as under olomoucine and rosco- vitine. As already found in the experiment shown in Fig. 3A, the hypophosphorylated form of pRb (migra- ting faster) is prominent in starved cells and mostly changes to a higher phosphorylation state after feeding with fresh medium (Fig. 4A, top row). As before (Fig. 3) this change is also suppressed by hypoxic gas- sing started concurrently with feeding. Equally, the changes in appearance and mobility of Cdc6 were reproduced. The decrease of Cdc6 in response to reoxy- genation is strengthened until 30 min, whereas chro- matin bound PCNA increases, reflecting increasing DNA strand elongation (Fig. 4A, bottom row). A B Fig. 4. Western blot analysis of pRb, Cdc6 and PCNA after inhibi- tion of Cdk2 activity by olomoucine, roscovitine and staurosporine in vivo. (A) Western blot analysis of chromatin bound pRb, Cdc6, and PCNA from 7 h normoxic, hypoxic, 5 min, and 30 min reoxy- genated T24 cells. Cdk2 inhibitors were added 3 h before reoxygen- ation where indicated. (B) Western blot analysis of total cellular pRb, Cdc6, and PCNA from 7 h normoxic, hypoxic, 5 min, and 30 min reoxygenated T24 cells. Cdk2 inhibitors were added 3 h before reoxygenation where indicated. N–, Normoxic incubation without medium renewal; N+, 7 h normoxic incubation after med- ium renewal; H, 7 h hypoxic incubation after medium renewal; 5¢, reoxygenated for 5 min after 7 h hypoxic incubation; 30¢, reoxygen- ated for 30 min after 7 h hypoxic incubation. Cdk2 is dispensable for replicon initiation D. Stabenow et al. 5628 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS However, neither addition of olomoucine, roscovitine or staurosporine (at 100 nm) before reoxygenation pro- duced, until 30 min thereafter, any change of chroma- tin bound pRb, Cdc6 or PCNA in comparison to the untreated reoxygenated sample. Application of the Cdk2 ⁄ cyclin inhibitory peptide II in a separate experi- ment according to the same schedule (data not shown) yielded identical results. This suggests that initiation and further replication proceeded normally despite the presence of the Cdk2 inhibitors. Figure 4B, on the other hand, shows that the differ- ent treatments of the cells in this experiment caused no visible quantitative differences of total cellular pRb and PCNA. Both proteins, apparently, are relatively abundant among total cellular proteins. The change of the electrophoretic mobility of pRb in response to feeding with fresh medium (attributed by us to phos- phorylation) obviously concerns the total cellular pro- tein including the chromatin associated proteins. On the other hand the relative intensity of the Cdc6 signal in Fig. 4B is very low. Because all wells of the gel for Fig. 4 were loaded with strictly equal amounts of pro- tein and the fraction of chromatin associated proteins represents only 8.2% of total proteins, the distinctly higher Cdc6 signal intensities in Fig. 4A indicate that, in contrast to PCNA and pRb, a substantial portion of Cdc6 was concentrated in the chromatin fraction. The high content of PCNA in the starved cells (N– in Fig. 4B) indicates that these cells rather reside in a G 1 -arrest than in a G 0 -phase [44]. Discussion This study continues prior work on molecular mecha- nisms governing the initiation of replication units in mammalian cells and its regulation in vivo. The dis- covery of the fast acting O 2 -dependent control of mammalian replicon initiation [26,39] and the suc- ceeding detailed evaluation of the relevant conditions (ranges of O 2 partial pressures, etc. [38–40,45]) provi- ded a useful tool for this purpose: exposing cultures of growing mammalian cells for several hours to hyp- oxic conditions accumulates replicons (scheduled to be initiated within the hypoxic period), in a state from which they can easily be released within a few minutes into a synchronous wave of initiations, by abruptly restoring atmospheric pO 2 . The state of repl- icon origins accumulated under hypoxia was opera- tionally called by us ‘hypoxic preinitiation state’. Having characterized the changes occurring at the level of the viral DNA replication in response to reoxygenation [46], we developed a protocol for analogous studies on cellular mammalian DNA replication and its regulation, based on human T24 bladder carcinoma cells [27]. Because we followed this protocol exactly in this study, we explicitly refer to the latter communication with respect to data charac- terizing cell cycle, DNA synthesis and cell fraction- ation into cytosolic, nucleosolic and chromatin associated material. Clearly, T24 is an abnormal can- cer cell line having a permanently active H-ras [47] which conceivably could bypass some regulatory checkpoints of normal cells. We choose these cells because they, best of all cell lines examined, permitted a selective accumulation of a defined cohort of repli- cons (i.e. that is scheduled to be activated as first of the S-phase) in the ‘hypoxic preinitiation state’, in preferable absence of other activated replicons [27]. A main intention of this work was to study the mechan- ism of the transition of the latter state to actual initi- ation. We emphasize that some of the results obtained, possibly, could only be valid for T24 cells and not for normal mammalian cells. On the other hand, the hypoxic suppression of replicon initiation (situated very close before actual initiation), obvi- ously, is a widespread property of proliferating mam- malian cells, possibly serving basal functions, such as protection against metabolic catastrophes during embryonic development or wound healing and, unfor- tunately, also in tumour growth [38]. So far, we found it in all types of cells examined in this respect during the past 20 years, ranging from cells replica- ting a virus (SV40) over a diversity of tumour cell lines to normal human primary explanted from umbi- lical cord vein (HUVEC) and nasal epithelium (HNEpC) (G. Probst, H. Probst & V. Gekeler, unpub- lished observations). We suggest, therefore, that the data presented here have more general significance. The ‘hypoxic preinitiation complex’ as defined by us [27] appears very similar to the preinitiation complex described by Dutta and Bell [2,48]. We suspected that the mechanisms activating the ‘hypoxic preinitiation complex’ and the ‘classical prereplication complex’ are similar, and that hypoxia interferes with the switch from preinitiation to initiation. The molecular mode of action of the suspected switch is, so far, completely obscure. However, as mentioned, fast recruitment of regulatory proteins such as protein kinases to the ori- gins of replicons and ⁄ or modifications of other pro- teins associated with them, by the still inactive initiation complex are involved. A likely candidate was thus Cdk2. Numerous reports suggested that Cdk2 activity is essential for growth, and that the absence of Cdk2 activity leads to growth arrest. In addition, hyp- oxia has been reported to suppress Cdk2 activity through elevated expression of the Cdk inhibitor p27, D. Stabenow et al. Cdk2 is dispensable for replicon initiation FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5629 thus preventing pRb phosphorylation and activation [49]. We found that Cdk2 associates with cellular chro- matin within 4–6 h, when cells arrested in G 1 are re- stimulated by feeding under normoxic as well as under hypoxic conditions. Cdk2 protein kinase activity was immunoprecipitable from the chromatin and cytosolic fraction of hypoxic as well as of reoxygenated cells. Total cellular Cdk2 protein kinase activity was found to be susceptible to inhibition by olomoucine, roscovi- tine, staurosporine or the Cdk2 ⁄ cyclin inhibitory peptide II in vitro. However, administration of the inhibitors in vivo before reoxygenation to the hypoxic cells had virtually no influence on the DNA replication occurring after reoxygenation, neither on the synchron- ous burst of replicon initiations, nor on the succeeding daughter strand elongation (Fig. 2). The inhibitors had no detectable influence on the behaviour of total cellu- lar and chromatin associated pRb, Cdc6 and PCNA after the reoxygenation either. Hypoxia, started concurrently with feeding with fresh medium, suppressed pRb phosphorylation at least until 30 min after reoxygenation (Figs 3 and 4), despite the initiation burst occurring within these 30 min. However, because the pRb phoshorylation appearing after cell feeding without imposed hypoxia emerges in the course of 6–7 h, it is possible through- out that a pRb phoshorylation, susceptible to the inhibitors, occurs some hours later after reoxygenation, but then clearly cannot be causal for triggering the ini- tiation burst already passed by. Obviously, increased Cdc6 accumulation occurred in the chromatin associ- ated protein fraction when, after feeding, hypoxia was imposed on the cells. Subsequent reoxygenation caused, within 30 min, a significant decrease of chro- matin associated Cdc6 accompanied by a phosphoryla- tion of a part of it. The decrease of chromatin associated Cdc6 as well as the phosphorylation, was not prevented by 100 nm staurosporine (sufficient to inhibit Cdk2), or by olomoucine, roscovitine or the Cdk2 ⁄ cyclin inhibitory peptide II. Staurosporine at 10 lm, however, distinctly inhibited this decrease and the presumed phosphorylation. At the same time, the increase of chromatin associated PCNA, indicating emerging processive DNA strand elongation after reoxygenation, failed to appear (Fig. 3C). The high staurosporine concentration also suppressed the burst of replicon initiations released by reoxygenation in the T24 system observable by alkaline sedimentation (data not shown). In this context it is tempting to speculate that Cdc6 may be an in vivo substrate of a protein kin- ase other than Cdk2, and its phosphorylation may be involved in the eventual release of replicon initiation. This phosphorylation possibly abolishes binding of Cdc6 to the still incomplete initiation complex, thus, perhaps, allowing recruitment of still missing proteins. However, despite the recent data that Cdk2 may be dispensable for proliferation in a mouse model [24], our data may seem contradictory to published studies on the prerequisites of initiation of DNA replication starting from different states present in G 1 or G 0 nuclei. It has been reported that the initiation of DNA replication is dependent on the coordinate activity of Cdk2 ⁄ cyclin E and Cdk2 ⁄ cyclin A complexes [18,21,50]. The mentioned results are obtained with in vitro replication systems from HeLa and ⁄ or mouse 3T3 cells by mixing isolated nuclei and cytosolic extracts from different cell cycle phases. Our in vivo situation is a quite different one: the relevant intervals of time between mixing cytosolic extracts with nuclei and emergence of additional replicating nuclei are in the order of hours. In contrast, our protocol produces, in living cells, a defined state of replicons situated before origin unwinding (as shown in the SV40 system [46]) and characterized with respect to the absence of the proliferation marker PCNA in the chromatin frac- tion [27]. Moreover, in the mentioned studies, syn- chronous cells for the preparation of nuclei and cytosolic extracts were obtained by different protocols. It can be asked whether the kind of synchronization is critical for the need of Cdk2 ⁄ cyclin complexes in initi- ation of replication. It was also demonstrated that Cdk2 activity is diminished in hypoxic cells [49,51,52]. These studies examined Cdk2 preparations immunoprecipitated from whole cells. The observed decrease in Cdk2 activity was probably mainly due to elevated levels of the Cdk2 inhibitory protein p27 obviously expressed under hypoxia in the studied cells and coimmunoprecipitated with Cdk2. With T24 cells we found did neither eleva- ted levels of p27 after several hours of hypoxic incuba- tion nor mentionable amounts of p27 coprecipitated with Cdk2 under any incubation condition (data not shown). This suggests that in T24 cells inhibition of the pRb pathway is not the direct cause of the hypoxic suppression of initiation in replicons. Moreover, in cells replicating SV40 DNA pRb is inactivated by binding to the large T-antigen; nevertheless, their repli- cation of viral genomes obeys the fast O 2 dependent control of replicon initiation [53]. The elucidation of the in vivo molecular processes leading from the hypoxic preinitiation state, within a few minutes, to actual initiation was the sole objective of this work. At the time, the T24 cell based protocol, to our knowledge, accomplishes that comparatively Cdk2 is dispensable for replicon initiation D. Stabenow et al. 5630 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS well. However, we may have observed phenomena that are characteristic only of this cell line and possibly for some further (abnormal) cells carrying comparable genetic defects. The general validity of our results depends on the demonstration of the described effects in a broader range of cell types, including noncancer cells. Experimental procedures Cell culture, transient hypoxia, reoxygenation, and radioactive labelling T24 cells (ATCC No. HTB-4) were grown in plastic flasks in DMEM supplemented with 10% (v ⁄ v) fetal bovine serum and 100 UÆmL )1 penicillin ⁄ 100 lgÆmL )1 streptomy- cin. The cells were subcultured when they reached conflu- ence. For synchronization, the desired number of glass Petri dishes was seeded from an almost confluent large cul- ture with 150 000 cellsÆmL )1 (35 mm, 1.5 mL; 145 mm, 25 mL) 44 h before the start of an experiment, as reported previously [27]. As a result, most cells were arrested in G 1 due to starvation. For prelabelling of DNA, the seeding medium was supplemented with 5 nCiÆ mL )1 [ 14 C]thymidine. Experiments were started by stimulation of the cells by complete medium change, using prewarmed fresh medium, supplemented with 10% (v ⁄ v) fetal bovine serum. Then, the cultures were gassed by a continuous flow of humidified artificial air containing 5% (v ⁄ v) CO 2 in case of normoxic, and with 0.0075% (v ⁄ v) O 2 ,5%(v⁄ v) CO 2 , Ar ad 100% in case of hypoxic incubations. This hypoxic gassing protocol diminished the pO 2 in the cultures within 1.5–2 h to about 0.1% and within 6–7 h to about 0.02%. For gassing, the equipment and the procedures described by [53] were used. For reoxygenation, 0.25 vols 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 ⁄ 5% (v ⁄ v) CO 2 . Hypoxic addition of inhibitors or radioactive precursors was performed by plunging a spatula carrying the appropri- ate quantity into the culture medium without opening the gassing vessel. To stop incubations, medium was removed by aspiration, and the cells were washed once with ice-cold NaCl ⁄ P i . Alkaline sedimentation analysis of cellular DNA To analyse the length distribution of growing daughter strands of T24 DNA, cultures on 35-mm glass Petri dishes were pulse labelled for 8 min with 7 lCi [methyl- 3 H]deoxy- thymidineÆmL )1 . Labelling was stopped by washing the cells with ice cold NaCl ⁄ P i . The cells were trypsinized and layered onto the top of 15–30% alkaline sucrose gradients. After denaturation of the DNA for 6 h, centrifugation was performed at 20 000 r.p.m., at 23 °C for 10 h in a Beckman SW28 rotor (Krefeld, Germany). Fractions of 1.2 mL were collected from the top of the gradient and processed to ana- lyse acid insoluble radioactivity. Cell fractionation Fractions containing cytosolic, nucleosolic, and chromatin bound proteins of the cells were prepared as described pre- viously [27]. Modified buffers were used for immunoprecipi- tation and kinase assays. The cytosolic proteins were obtained after incubation of the cells in buffer A [50 mm Hepes pH 7.5; 20 mm b-glycerolphosphate; 1 mm dithio- threitol; containing aprotinin (1 lm); leupeptin (50 lm); 4-(2-aminoethyl)-benzenesulfonylfluoride HCl (AEBSF) (1 mm); NaF (10 mm); and Na 3 Vo 4 (1 mm)] and subsequent disruption of the cellular envelope with 20 strokes of a tight fitting pestle. Nucleosolic proteins were obtained after incu- bation in buffer B (buffer A containing 0.1% NP40). The remaining chromatin bound proteins were solubilized by incubating the chromatin in IP buffer (buffer B containing 450 mm NaCl) for 1 h on ice and subsequent centrifuga- tion. The fraction remaining after extraction of nuclei con- tains about 8.2% of total protein recovered and 96.6% of total DNA. Immunoprecipitation, kinase assay and phosphatase digestion For immunoprecipitation of Cdk2 and the subsequent kin- ase assay, solutions of cytosolic, nucleosolic, solubilized chromatin bound or total cellular proteins were adjusted to equal protein content (300 lg) and incubated with 2 lg Cdk2 antibody (Santa Cruz, Clone M2; Santa Cruz, CA, USA) for 40 min on ice. Then, 20 lL of a slurry of Protein G-agarose beads (Roche, Mannheim, Germany) equili- brated with IP buffer were added, and incubation was con- tinued overnight under continuous rotation. Subsequently, the beads were washed three times with 500 lL IP buffer and three times with 500 lL kinase buffer [50 mm Tris pH 7.5; 10 mm MgCl 2 ;1mm dithiothreitol; 20 mm b-glycerolphosphate; containing aprotinin (1 lm); leupeptin (50 lm); AEBSF (1 mm); NaF (10 mm); and Na 3 Vo 4 (1 mm)]. Excess solution was removed completely and beads were incubated for 30 min at 30 °C in a mix of 20 lL kinase buffer containing 2.5 lg H1; 0.2 mm ATP; and 10 lCi [ 32 (or33) P]ATP[cP] in the absence or presence of Cdk2 inhibitors. Incubation was stopped by adding 20 lL2· protein sam- ple buffer. After denaturation, the reaction products were separated by an SDS ⁄ PAGE on a 12% polyacrylamide gel, blotted onto a Nylon-P membrane (Amersham, Bucks, UK) and subsequently exposed to Hyperfilm MP (Amersham). For immunoprecipitation and subsequent phosphatase digestion of Cdc6 or pRb, respectively, solutions of D. Stabenow et al. Cdk2 is dispensable for replicon initiation FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5631 solubilized chromatin bound proteins, containing 300 lgof protein, were incubated with 2 lg Cdc6 antibody (MoBi- Tec, Clone DCS-180), or pRb antibody (Pharmingen, Clone G3-245; BD Biosciences Pharmingen, Canada) for 40 min on ice. Then, 20 lL of a slurry of Protein A-agarose beads (Bio-Rad, Hercules, CA, USA) equilibrated with IP buffer were added, and incubation was continued for 3 h (Cdc6) or overnight (pRb) under continuous rotation. Sub- sequently, the beads were divided in two equal parts and washed three times with IP buffer. Protein sample buffer (20 lL) was added to one part afterwards. The other part was washed three times with phosphatase buffer (New Eng- land Biolabs, Beverly, MA, USA) supplemented with prote- ase inhibitors. Phosphatase digestion was performed for 30 min at 30° with 200 U Lambda phosphatase (New Eng- land Biolabs). After a final wash with IP buffer 20 lL pro- tein sample buffer were added. After denaturation, proteins were separated on an 8% (49 : 1, Cdc6, PCNA and total pRb) or 10% (149 : 1, chromatin bound-pRb) SDS ⁄ poly- acrylamide gel, blotted onto Hybond-P (Amersham). Pro- teins were visualized as described below. Immunofluorescence staining of Cdk2 Cells grown on coverslips and incubated with the fluoresc- ein isothiocyanate (FITC) labelled Cdk2 ⁄ cyclin inhibitory peptide II during the respective incubations, washed once with ice-cold NaCl ⁄ P i . For subsequent immunostaining of Cdk2, cells were directly fixed with ice cold acetone ⁄ meth- anol (1 : 1, v ⁄ v) for 10 min at 4 °C and processed for detec- tion of Cdk2 after air drying. Cells were blocked with 1% (w ⁄ v) BSA in NaCl ⁄ P i for 20 min and incubated with the Cdk2-antibody (Pharmingen, Clone 55) in NaCl ⁄ P i ⁄ BSA for 1 h at room temperature. After washing three times with NaCl ⁄ P i for 5 min they were further incubated for 30 min with a mouse-antibody labelled with Alexa FluorÒ 586 (Molecular Probes, dilution 1 : 200; Eugene, OR, USA) 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 Cdk2, the Cdk2 ⁄ cyclin inhibitory peptide II and total DNA (bisbenzimide stain) were visualized with a Zeiss fluores- cence microscope (Axioskop; Jena, Germany) using the appropriate filter combinations. Electrophoresis of proteins and western blotting Cytosolic and nucleosolic proteins were precipitated from the respective supernatants using the procedure described by Wessel-Flu ¨ gge [54]. Chromatin bound proteins for west- ern blot analysis were either separated from the DNA by treatment with 450 mm NaCl in buffer B and subsequent Wessel-Flu ¨ gge precipitation as described above, or chroma- tin was directly denatured and solubilized with SDS electro- phoresis sample buffer. 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