Effects of ionizing radiation and caffeine treatment on cyclin dependent kinase complexes in V79 hamster cells

CellularSignallingVol. 6, No. 5, pp. 539-550, 1994 ElsevierScienceLtd PrinteAinGreatBritain 089~6568/94 $7.00+ 0.00

Pergamon 0898-6568(94)E0015--3

EFFECTS OF IONIZING RADIATION AND CAFFEINE TREATMENT ON CYCLIN DEPENDENT KINASE COMPLEXES IN V79 HAMSTER CELLS JENS HAIN,*? ROLF JAUSSI* and FRIEDRICH E. WURGLER:~ *Institute of Medical Radiobiology, Paul Scherrer Institute and University of ZUrich, CH-5232 Viligen-PSI, Switzerland and :Hnstitute for Toxicology, Swiss Federal Institute of Technology and University of Ztirich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland (Received 21 December 1993; and accepted 3 February 1994)

Abstract--Exponentially growing V79 Chinese hamster lung fibroblasts irradiated with 7 Gy X-rays undergo cell cycle arrest in the S and G2 phases. These arrests are released, probably on completion of DNA repair. A premature release occurs after treatment of irradiated cells with caffeine. This release is accompanied by increased activity of the p34cdc2serine/threonine protein kinase complex [Hain et al. (1993) Cancer Res. 53, 1507-1510]. We have investigated in V79 cells whether the association of p34cdc2with its regulatory subunits cyclin A and B is affected by irradiation and subsequent caffeine treatment and found that this was not the case. The phosphorylation of p34cdc2as assayed by mobility shift on SDS polyacrylamide gels was increased as early as 0.5 h after irradiation and decreased after subsequent caffeine treatment. A novel protein p40, detected with antiPSTAIRE antibodies, appeared several fold more abundant than p34cdcz.Its phosphorylation state also changed after irradiation and after subsequent caffeine treatment. Key words: p34coo/X-rays, signal transduction, protein kinase, cell cycle arrest.

onine kinases is complex and is characterized by phosphorylations and altering association of the kinase subunits with specific regulatory subunits called cyclins [9-18]. After association with a cyclin, the kinase becomes active through the coordinated phosphorylation and dephosphorylation of the residues T14, Y15 and T161, respectively, of p34 cdc2 in human cells (for review, cf. [15, 17]). The p34 cac2 kinase (also called CDK1) mediates primarily mitotic entry. During the S phase of the cell cycle it associates with the regulatory subunit cyclin B (a part associates with cyclin A) and becomes phosphorylated at the above mentioned residues to form an inactive complex (for review, cf. [19]). The mammalian enzymes which are homologous to the fission yeast mikl and weel gene products phosphorylate Y15 [20-23]. At the Gz/M border the p34/cyclin B complex is activated by dephosphorylation at Y15 and T14 by the m a m m a l i a n e n z y m e which is h o m o l o g o u s to the fission yeast cdc25 phos-

INTRODUCTION THE cell cycle arrests of exponentially growing eukaryotic cells after exposure to DNA damaging agents in S and G2 phase depend on the activity of cyclin dependent kinases (CDKs) like p34 ~d~2 [1-5]. The passage of the S phase and the transition f r o m G 2 p h a s e to mitosis in cells f r o m eukaryotic species as different as yeast and man are mediated by the same family of CDKs [6-8]. The regulation of the activity of these serine/thre-

tTo whom correspondence should be addressed at: Bundesamt fiir Strahlenschutz, Abteilung Strahlenhygiene, D80762 Oberschleissheim, Germany. Abbreviations: CDK-----cyclin dependent kinase; p13 Suc~fission yeast sucl gene product, a component of the p34 cdc2 protein-kinase complex; p34°dc2--the protein serine/threonine kinase p34Cd~2; PAGE--polyacrylamide gel electrophoresis; PB S--phosphate-buffered saline; PVDF--polyvinylidene difluoride; SDS--sodium dodecyl sulphate; TBS--trisbuffered saline; V79 cells---chinese hamster lung fibroblast line V79.

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phatase [24-27]. The phosphorylation of T161, which is required for activity, remains during G2/M transition [17, 28, 29]. The bulk of cyclin A is complexed with the S-phase specific CDK2 in human cells [5, 30-33]. In synchronized human HeLa cells, irradiated in S phase, the concentration of cyclin A rises as in unirradiated controls. The concentration of cyclin B 1 remains low until the first cells enter mitosis following arrest [2-4]. V79 Chinese hamster lung fibroblasts have been extensively characterized by radiobiologists [1, 34-38] and thus were used for this study of mecha n i s m s i n v o l v e d in the s i g n a l t r a n s d u c t i o n between radiation-induced D N A damage and cell cycle arrests. Another reason to use hamster cells for our experiment was the fact that, in contrast to all eukaryotic cells tested so far, a hydroxyureainduced replication block is also releasable by caffeine, and not only the radiation induced arrest [39]. This peculiarity of hamster cells provides a valuable tool to investigate the molecular mechanisms of cell cycle arrests. The first step was to test whether the cyclins in V79 cells behaved similarly to the cyclins in HeLa cells. The second step was to precipitate complexes of p34 c~c2and investigate whether the abundance of both cyclins in these complexes changed concomitantly with the overall cyclin protein concentration in the irradiated V79 cells. It was possible to simultaneously determine the regulatory phosphorylation state of p34 cdc2 in these complexes. Finally, the influence of caffeine treatment, which can be used to release the cell cycle arrests, was investigated. During these studies a novel protein p40 was found, reacting with anti-PSTAIRE serum which recognizes the conserved ctl helix with the "PSTAIRE" consensus region of CDKs (three-dimensional structure, see [40]). Its f u n c t i o n and its p o s s i b l e involvement in the cell cycle arrests is discussed.

I.tg/ml streptomycin in a 5% CO2 atmosphere. At about 50% confluence the cultures were irradiated with 7 Gy X-rays (Philips MCN 321 X-ray tube, 320 kV, l0 mA, 4 Gy/min). Aluminium (1 mm) was used for filtering. Doses were determined with a Farmer ionization chamber (NE 2571). Flow cytometry

To monitor the amount of G~, S and Gz/M phase cells, samples of the cultures used for extract preparation were stained with the fluorescent DNA dye ethidium bromide as previously described [41]. The DNA content of each of 3 x 104 cells was determined in a flow cytometer (FACScan, Becton Dickinson) and recorded as a histogram of cell number against DNA content. The cell cycle phase distributions were analysed using the computer program "MultiCYCLE version 2.5" (Phoenix Flow Systems). Preparation of crude cell extracts

All preparation steps were carded out on ice or at 4°C. The culture dishes were rinsed twice with phosphate buffered saline (PBS: 6.5 mM Na2HPO#, 1.5 mM KH2PO4, 2.7 mM KC1, 137 mM NaC1), freshly supplemented with 1 mM phenylmethylsulphonyl fluoride and 0.4 mM Na3VO4. Approximately 107 cells were harvested with a rubber policeman, collected in 6 ml PBS and centrifuged for 10 min at 1000 x g. The pellet was resuspended in 0.5 packed cell volumes of isolation buffer (20 mM Tris--chloride, 137 mM NaC1, 1 mM MgCI2, 1 mM CaC12, 10% glycerol, 0.1% Nonidet P40, 0.4 mM Na3VO4, 10 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, pH 8.0). The cells were sonified three times with 10 pulses (Branson cell disruptor B15: 50% duty cycle, output control setting = 2). After 30 min the homogenate was centrifuged for 10 rain at 15,000 x g. Aliquots of the supernatant were frozen in liquid nitrogen. The DNA content of the homogenates was determined based on Hoechst 33258 DNA dye fluorescence [42]. Protein concentrations were measured using the Coomassie Blue assay from BioRad according to the instructions of the supplier. For each experimental setup three cell extracts were prepared on different days.

M A T E R I A L S A N D METHODS Cell culture and irradiations

V79 hamster lung fibroblasts were obtained from Dr M. Fox, Paterson Laboratories, Christie Hospital, Manchester, England. Cells were maintained at 37°C in MEM Alpha Medium (GIBCO) supplemented with 10% fetal bovine serum and 25 IU/ml penicillin and 25

Western immunoblotting

Proteins from cell extracts were separated on sodium dodecylsulphate (SDS)-10% polyacrylamide gels in Mini Protean II cells (BioRad) according to the method of Laemmli [43]. The separated proteins were blotted onto polyvinlidene difluoride (PVDF) membranes (Millipore) with half strength blotting buffer (10%

Cyclin dependentkinases and cell cycle blocks

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methanol, 96 mM glycine, 10 mM Tris Base) using a semi-dry electrotransfer apparatus (Millipore). The membranes were blocked for 1 h with 0.5% casein in Tris-buffered saline (TBS: 20 mM Tris--chloride, 500 mM NaC1, pH 7.5), washed three times for 10 min in TBS and incubated overnight at 4°C with the primary antiserum (dilutions according to the indications of the suppliers). The blots were then washed three times for 10 min in TBS and incubated for 3 h at room temperature with a secondary goat anti-rabbit-antibody alkaline phosphatase conjugate (BioRad). For alkaline phosphatase colour development nitroblue tetrazolium plus 5-bromo-4-chloro-3-indolyl phosphate were used .as substrates according to the instruction of the supplier (BioRad).

G2/M phase cells (Fig. 1A). The cell cultures showed the expected cell cycle arrests after irradiation monitored by flow cytometry as indicated by the disappearing peak which corresponds to cells in G~ phase (Fig. 1B). Four hours after irradiation, cultures had been released from the S+G2 arrest by 0, 30, 60 and 80 rain caffeine treatment and displayed a premature re-entry of arrested cells into the cell cycle as expected and indicated by the rapidly reappearing G~ peak (Fig. 1C).

Antisera

In order to find out how these effects translate into changes in key regulatory components of the cell cycle, levels of cyclin A and cyclin B1 in whole cell extracts were measured. Additionally, their levels were determined in complexes with p34 cdc2 after precipitation with p13 ~°~1 agarose beads. After irradiation the concentration of cyclin B 1 in whole cell extracts stayed as low as in exponentially growing cells for about 3 h after irradiation, increased around 4 h and remained high up to 8 h when the first cells escaped from the G2 arrest. The cyclin A concentration increased up to 4 h after irradiation and seemed then to remain constant up to 8 h after irradiation (Fig. 2A). Treatment of V79 cells with caffeine between 4 and 5 h after irradiation neither influenced the concentration of cyclin A nor of cyclin B 1 (Fig. 2B). Caffeine treatment between 1.5 and 2.5 h after irradiation led to the same result (data not shown). In the V79 cells the concentration of cyclin B1 was equal in G~, S, and S+G2 phase cells and increased significantly in mitotic cells, whereas cyclin A concentration was highest in S phase and S+G 2 phase cells (Fig. 2B).

Anti-cyclin A and anti-cyclin B1 antisera were kindly provided by Dr T. Hunter from the Salk Institute, San Diego, CA, U.S.A. The anti-PSTAIRE antiserum (a synthetic peptide corresponding to the residues 42-57 of the human 34,000 Mr cdc2 kinase: EGVPSTAIREISLLKE had been used as the immunogen) was purchased from UBI Upstate Technologies. The anti-p34 ~2 antiserum against a carboxy-terminal peptide of p34:d¢2was obtained from Life Technologies Inc.

p13sue1precipitation of p34m~2complexes One hundred microgrammes of fission yeast p13~ud protein coupled to agarose beads (Oncogene Science, Uniondale, NY, U.S.A.) were used to precipitate p34cdc2 complexes from cell extracts containing 300 ~tg protein in 350 ~tl precipitation buffer (10 mM Na2HPO4 adjusted to pH 7.2 with HC1, 154 mM NaC1, 30 mM NaN3, 1 mM NaF, 1 mM Na3Vo4, 1 mM phenylmethylsulphonyl fluoride, pH finally adjusted to 7.25 with NaH2PO4). The p34 complexes were precipitated in 0.5 ml Eppendorf tubes which were rotated slowly for 0.5 h at 4°C. The pellet was washed three times in 400 ~tl precipitation buffer, boiled at 95°C for 5 rain in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, and then processed as described under "western immunoblotting". All experiments were performed in triplicate. RESULTS

Flow cytometry Exponentially growing V79 cell cultures have a doubling time of approximately 9 h and contain 25% G~ phase cells, 60% S phase cells and 15%

Cyclin A and cyclin B1 levels in whole cell extracts

Cyclin A and cyclin B1 content and phosphorylation changes of p34 cdC2in p13 s"d recipitations The content of cyclin B1 in p13 suC2 precipitations of p34 ~dc2complexes remained constant up to 8 h after irradiation (Fig. 3A). The dominant band ( M r 62,000 on SDS-PAGE) found in whole cell

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Fig. 1. Typical age distribution histograms of all cell cultures to be used to prepare extracts were measured by flow cytometry. Each plot shows 30,000 cells (except Fig. 1A histograms 2 and 3, which show 10,000 cells each). Abscissa, DNA content of cells; ordinate, cell frequency. (A) Ceils enriched in: exponential growth (1); G1 phase (2); S phase (3); S+G2 phase (4); mitosis (5). Extracts of synchronized cells were prepared in G~ and S phase after release of a G 2 phase staurosporine block by substituting the medium with staurosporine free medium [52, 61]. Exponentially growing cells were harvested from nonconfluent cultures. Mitotic cells were obtained by mitotic shake-off of cultures treated with 1 ~g/ml nocodazole for 4 h. The remaining fraction of cells in the culture flasks was trypsinized and used to prepare the S+G~ phase cell extract. (B) Ceils which were irradiated with 7 Gy X-rays; time after irradiation: 0.5 h (1); 1.5 h (2); 3 h (3); 4 h (4); 5 h (5); 8 h (6). (C) Cells which were released 4 h after irradiation from the cell cycle block by caffeine for 30 min (1); 60 min (2); 80 min (3).

extracts was essentially not present in p13 ~u~t precipitations. However, the lower molecular weight bands recognized by cyclin B1 antiserum (/14, 50,000-56,000 on S D S - P A G E ) were enriched. The content of cyclin A also stayed constant up to 8 h after irradiation in the same p13 sucl precipita-

tions of p34 cd~2 complexes (Fig. 3A). During all parts of the cell cycle, except in mitosis, p34 cat2 appeared as a double band in the immunoblots. The lower band corresponds to the low phosphorylated active form of p34 cec2 and the upper band to the highly phosphorylated inactive form [39,

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Fig. 2. Western immunoblots of crude V79 cell extracts. Per lane 100 ktg cell extract were separated by SDS-PAGE. Cyclin A and cyclin B 1 of each cell extract were probed with anti-cyclin A and anti-cyclin B 1 antiserum. (A) Cyclin A and cyclin B 1 between 0 h and 8 h after irradiation (7 Gy X-rays). 0 h = exponentially growing cells; N.D. = not determined. (B) Cyclin A and cyclin B1 after caffeine release: 4 h after irradiation (1); 4 h after irradiation + 30 min 4 mM caffeine (2); 4 h after irradiation + 60 min 4 mM caffeine (3). Control extracts: G~ phase (4); S phase (5); S+G z phase (6); mitosis (7).

543

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Fig. 3. Western immunoblots of p13 S°cl precipitations of p34 complexes. Anti-cyclin A, anti-cyclin BI and antiPSTAIRE antisera were used. Per lane the total p13 '~u°~precipitation obtained from 300 ~tg cell extract protein was loaded. (A) p13 ~°c~ precipitations from extracts of cells between 0 h and 8 h after irradiation (7 Gy X-rays). 0 = exponentially growing cells; C = control: 100 ~tg crude cell extract of exponentially growing cells, not precipitated. (B) p13 ~oc~precipitations from cells: 4 h after irradiation; (1) 4 h after irradiation + 30 rain 4 mM caffeine; (2) 4 h after irradiation + 60 min 4 mM caffeine (3); C = control: 100 ~g crude cell extract of exponentially growing cells, not precipitated; in S+G2 phase (4); in mitosis (5).

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i! i!¸¸ !~!'ii~iiil !ii!i¸!!i!!iiiii~i i ¸¸¸!il~!i ~!iii ~ i i ~ ii Fig. 4. Western immunoblots of crude V79 cell extracts. Per lane 100 ~tg cell extract were separated by SDS-PAGE and probed with the anti-PSTAIRE antiserum. 0.5 h after irradiation (1); 1.5 h after irradiation (2); 3 h after irradiation (3); 5 h after irradiation (4); 4 h after irradiation (5); 4 h after irradiation + 30 rain 4 mM caffeine (6); 4 h after irradiation + 60 min 4 mM caffeine (7); 4 h after irradiation + 80 min 4 mM caffeine (8); exponentially growing culture (9); G~ phase (10); S phase (11); S+G2 phase (12); mitosis (13).

Cyclindependentkinases and cell cycleblocks 44--46]. Mitotic cells with the highest p34 cd~2 activity [1] displayed only the lower band (Fig. 3B, lane 5 and Fig. 4). In the p34 double bands there was a clear shift in intensity from the lower to the upper band as early as 0.5 h after irradiation. This shift persisted up to 8 h after irradiation (Fig. 3A). Caffeine treatment had no influence on the content of cyclin A or cyclin B 1 but the radiation induced phosphorylation shift within the p34 double band was reversed (Fig. 3B). Apparently, the activation of the p34 ~dc2 complex due to caffeine treatment after irradiation correlates with the dephosphorylation of the p34 subunit and the radiation induced inactivation seems to result from maintaining it phosphorylated.

Changes in phosphorylation of p34 cuteand p40 from whole cell extracts In crude extracts of V79 hamster lung fibroblasts a specific protein was detected as a double band by the antiserum against the conserved PSTAIRE region of CDKs (Fig. 4, upper double band) but not by the anti-human p34 cdcz carb__oxyterminal antiserum which only recognized the p34 double band (data not shown). This protein of approximately 40,000 Mr was not present in p13 s°cl precipitations (Fig. 3). Proteins with similar properties from mice and humans [47, 48] are protein kinases called PCTAIRE or PLSTIRE, the functions of which are not yet determined. In crude cell extracts p40 appeared more abundant than p34 (Fig. 3 Control, Fig. 4). After irradiation the phosphorylation state of the novel protein behaved qualitatively similar to that of p34 (Fig. 4). However, the kinetics of its phosphorylation were different, phosphorylation being induced with a delay of approximately 3 h (Fig. 4, lanes 1-5). The protein appeared to be depbosphorylated by caffeine treatment with kinetics similar to those of p34 (Fig. 4, lanes 5-8). The normal cell cycle related activity of the novel p40 protein seemed to be high in G1 and S phase and was carried on into Gz phase (Fig. 4, lanes 10-12). In these cell cycle phases the low phosphorylated form was more abundant. In mitosis the highly phosphorylated form predominated (Fig. 4, lane 13).

547 DISCUSSION

Based on experiments with synchronized human HeLa cells, the radiation induced S and G 2 arrests are at least in part due to delayed synthesis of cyclin B. The concentration of cyclin B mRNA stays low after irradiation of cells in S phase and cyclin B does not accumulate in cells irradiated in G z phase [2-4]. These findings agree well with our demonstration, that the cyclin B 1 concentration in whole cell extracts of irradiated V79 hamster cells stays low during the first 3 h and rises only later when the cells are accumulated in the S and G2 phase. However, there seem to be differences between hamster cells and cells from humans or mice with respect to the presence of cyclin B/p34 complexes. During an S-phase arrest induced by preventing completion of replication with hydroxyurea, cyclin B is synthesized only in hamster cells and is complexed with p34 in its highly phosphorylated form [39]. This hydroxyurea-induced S phase block can be released by caffeine, 2-aminopurine, 6-dimethyl-aminopurine (two protein kinase inhibitors) or okadaic acid (a phosphatase inhibitor). However, the corresponding S-phase block in mouse or human cells can not be released with caffeine [39]. Thus, the release by caffeine appears to depend on preexisting cyclin B/p34 complexes. The radiation induced arrest occurs mainly in G 2 phase, where cyclin B/p34 complexes exist in all cells tested so far, and indeed, the release of this arrest by the above mentioned agents was observed [39]. In the hamster cells the abundance of cyclin B1 in p13 suc~ precipitations of p34 complexes does not change during the S and G2 arrest and is not influenced by caffeine treatment. With a molecular weight of approximately 50,000-56,000 Mr the cyclin B 1 subforms in p 13sud precipitations were smaller than the major 62,000 Mr cyclin B 1 form from whole cell extracts. The phenomenon of a smaller size of cyclin B in p13 suc~ precipitations than in whole cell extracts has already been described for sea urchin eggs [49]. Additionally, a cyclin B antiserum recognized a 62,000 Mr protein from HeLa cells [44], although the value predicted from nucleic acid sequence data was 49,000 Mr [50]. It is still unclear whether the

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phosphorylation state of cyclin B is responsible for these size differences or whether the protein is subject to other modifications. Nevertheless, our results show that the fractions of cyclin A and cyclin B 1 which are associated with p34 are unaffected by irradiation and by subsequent caffeine treatment in V79 hamster cells. The 50,00056,000 Mr cyclin B 1 isoforms present in the complex could be due to alterations in the phosphorylation state of the p34-associated cyclin B 1 fraction. Clearly, the phosphorylation state of p34 from the same p13 ~u¢~precipitations is changed at least as early as 0,5 h after irradiation (Fig. 3A). After caffeine treatment, dephosphorylation occurs, which coincides with a rapid increase of p34 kinase activity against a peptide substrate specific for p34 and closely related protein kinases [1,51]. Based on the findings with the novel p40 it seems reasonable that this protein is also involved in the radiation induced cell cycle arrests. It seems to be a cyc!in dependent kinase involved in Sphase transition of hamster cells. The p40 from the V79 hamster cells behaves similarly to CDK2/cyclin A complexes during the nitrogen mustard induced G2 arrest in human lymphoma cells. These complexes remain active after DNA damage has occurred, in contrast to p34/cyclin B 1 and p34/cyclin A complexes whose activity is suppressed [5]. The putative low phosphorylated form of p40 dominates during the first 3 h after irradiation. Four hours after irradiation the two differently phosphorylated forms of this protein are present in similar amounts (Fig. 4). Provided that p40 acts as a CDK-related kinase, the predicted activity of the enzyme would stay high after irradiation. This could in turn explain the high background level of activity in measurements with the specific peptide substrate [1]. Further evidence for this interpretation is given by the fact that the background activity of a population of V79 cells blocked mainly in G2 phase by staurosporine (a protein kinase inhibitor) is lower than that observed 4 h after irradiation with 60% of the cells arrested in S phase [52] when p40 seems to have its highest activity. The kinetics of the caffeine release of the

kinase activity using a peptide substrate were biphasic and indicated that both enzymes (p34 and p40) could be activated by caffeine and may be able to phosphorylate the substrate peptide [1]. This idea is further supported by the result that after caffeine release there is a shift to a higher abundance of the active form in the p34 double band as well as in the p40 double band. Only the hamster homolog of p34 cdcz is precipitated by p13 sue1 beads, in contrast to Xenopus laevis eggs and human cells where CDK2 coprecipitates [6, 47]. Clearly, p40 differs from CDK1 and CDK2 and is probably not controlled by the p13 sue1 homolog like other members of the CDK family [18,53]. In summary, the results indicate that in V79 cells ionizing radiation influences signal transduction pathways involving protein kinases and phosphatases, which affect p34 [54-58] and related kinases (e.g. p40) and mediate the S and G2 arrests. The association of cyclins with p34 is another mechanism involved in the radiation induced S and G2 phase arrest and is relevant in mouse and human cells [2-4, 59]. For V79 hamster cells, the main signal for the radiation induced G 2 arrest seems to be transmitted by a kinase/phosphatase pathway, probably because cyclin B is already associated with p34 in S phase [39]. This particularity of hamster cells provides a useful tool for further investigation of the mechanism of the radiation induced S and G2 phase arrest. Up to now, a relatively simple assay for simultaneous, but independent, monitoring of the activity of "S-phase" and "GE-phase" CDKs was not available. The newly found p40 appears to be a CDK-related kinase which is active from Gl through S to Gz phase. It remains to be analysed if and with which cyclin p40 associates and to determine its role in the cell cycle. The phosphatase(s) and kinase(s) acting on p34 and p40 are attractive candidates for future investigations aiming at a deeper understanding of the signal pathway(s) from DNA damage to cell cycle arrests. In fission yeast, the weel kinase which phosphorylates Y15 of p34 seems not to be important for the DNA damage-dependent cell cycle arrests [60]. The abundance of the hamster homolog of the cdc25

Cyclin dependent kinases and cell cycle blocks phosphatase responsible for the dephosphorylation of this residue does not change in whole cell extracts during the radiation induced cell cycle arrests and caffeine induced release (Hain et al., unpublished results). Therefore, it seems plausible that unique checkpoint kinases and phosphatases mediate the D N A damage-dependent cell cycle arrests or that a kind of "short cut" signal transduction occurs, which bypasses some of the known signals regulating mitotic events. Acknowledgements--We gratefully acknowledge Dr I. NOVAK for critical reading of the manuscript, DR N. E. A. CROMPTON for discussion and technical support, and G. EMERY for technical assistance. This work was supported by Swiss National Science Foundation Grant No. 31-26617.89.

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