Oncogene (1997) 15, 749 ± 758 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis BG Gabrielli, JM Clark, AK McCormack and KAO Ellem Queensland Cancer Fund Research Laboratory, Joint Oncology Program, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Queensland 4029, Australia In response to low doses of ultraviolet (U.V.) radiation, kinase), and is essential for the catalytic activity of cells undergo a G2 delay. In this study we have shown cdc2, whereas phosphorylation of Thr14 and Tyr15 is that the G2 delay results in the accumulation of inactive inhibitory, and catalysed by a member of the wee1/ forms of cyclin B1/cdc2 and both the G2 and mitotic mik1 family of kinases (Morgan, 1995). The latently complexes of cyclin A/cdk. This appears to be through a active form of cyclin B/cdc2 is maintained in the block in the cdc25-dependent activation of these cytoplasm until prophase, when the majority is complexes. The expression and localisation of cyclin A translocated into the nucleus (Pines and Hunter, and cyclin B1/cdk complexes are similar in U.V.-induced 1991; Bailly et al., 1992). At the same time, the G2 delay and normal early G2 phase cells. Cdc25B and inhibitory Thr14 and Tyr15 phosphates are removed cdc25C also accumulate to normal G2 levels in U.V. by a member of the cdc25 family of dual speci®city irradiated cells, but the mitotic phosphorylation asso- phosphatases, and fully active cyclin B/cdc2 is able to ciated with increased activity of both cdc25B and cdc25C perform its many mitotic functions. is absent. The cdc25B accumulates in the nucleus of U.V. In yeast only a single form of cdc25 exists, but in irradiated cells and in normal G2 phase cells. Thus the mammalian systems three isoforms have been identi®ed block in cyclin B/cdc2 activation is in part due to the (Gabrielli, 1994). Cdc25A has been demonstrated to physical separation of cyclin B/cdc2, localised in the regulate progression into S phase, while both cdc25B cytoplasm, from the cdc25B and cdc25C phosphatases and cdc25C have roles in the G2/M transition (Millar localised in the nucleus. The data positions the U.V.- et al., 1991; Jinno et al., 1994; Gabrielli et al., 1996). induced G2 checkpoint at either the S/G2 transition or The activity of cdc25 is regulated at both a early G2 phase, prior to the activation of cyclin A/cdk2. translational and post-translational level. Cdc25C is expressed at a constant level throughout the cell cycle, Keywords: cdc25; cell cycle checkpoint; U.V. while the expression of cdc25A and cdc25B has been demonstrated to be cell cycle dependent in some cell lines (Jinno et al., 1994; Gabrielli et al., 1996). The activity of both cdc25A and cdc25C is further Introduction modulated by phosphorylation (Homan et al., 1993; Homann et al., 1994). Progression through the cell cycle is regulated by the A further level of regulation of the cyclin/cdks has ordered activation of cyclin dependent kinases (cdks). been identi®ed, through the binding of low molecular In yeast, a single cdk, cdc2/CDC28, associates with weight protein inhibitors of these complexes. Two multiple cyclin partners to regulate both the START families of these proteins have been identi®ed, the and G2/M checkpoints, whereas in higher eukaryotes, p21Cip1 family including p21Cip1,p27Kip1 and p57Kip2, and a large family of cdks have so far been identi®ed, each the p16CDKN2A family including p16CDKN2A,p15CDKN2B, of which appears to regulate discrete cell cycle p18CDKN2C and p19CDKN2D (Sherr and Roberts, 1995). functions (Meyerson et al., 1992; van den Heuvel and The members of the p21 family bind to a wide range of Harlow, 1993). The best characterised in terms of cyclin/cdks, whereas the p16 family proteins have a function and regulation is cyclin B/cdc2, the prototypic much more restricted speci®city, binding only to cyclin cyclin/cdk. This complex was ®rst puri®ed from D/cdk4 and cdk6 (Sherr and Roberts, 1995). These Xenopus eggs as maturation promoting factor (MPF), proteins appear to respond to extracellular in¯uences which was the only factor required to initiate mitosis to produce cell cycle delays, for example, increased when injected into prophase arrested frog oocytes expression of p21 is induced by exposure to ionising (Lohka et al., 1988; Gautier et al., 1988). The histone radiation and is involved in a G1 delay (Dulic et al., kinase activity of cyclin B/cdc2 is regulated through a 1994), while p16 induced by irradiation with ultraviolet complex series of protein associations, phosphoryla- (U.V.) light is correlated with a G2 delay (Wang et al., tions and dephosphorylations. Cyclin B is synthesised 1996). during S and G2 phases, and immediately binds to the Exposure of cells to low doses of U.V. light catalytically inactive cdc2 monomer in the cytoplasm. produces delays in G1, S and G2/M phases of the Upon association with cyclin B, the cdc2 subunit cell cycle, depending on the dose and cell type used (Lu undergoes a series of phosphorylations. The phosphor- and Lane, 1993; Wang and Ellem, 1994; Barker et al., ylation of Thr 161 is catalysed by CAK (cdk activating 1995; Petrocelli et al., 1996; Poon et al., 1996). A role for p53-induced expression of p21, and the redistribu- Correspondence: B G Gabrielli tion of p27 among G1 and S phase cyclin/cdks in U.V.- Received 21 November 1996; revised 6 May 1997; accepted 6 May induced G1 and possibly S phase delays has been 1997 demonstrated (Lu and Lane, 1993; Barker et al., 1995; U.V. induced G2 delay BG Gabrielli et al 750 Poon et al., 1995; Petrocelli et al., 1996). The inhibitory was complexed with cyclin B1 to the same extent as in tyrosine phosphorylation of cdk4 has also been normal S/G2 cells, and that the cdc2 was predomi- implicated in the U.V.-induced G1 delay (Terada et nantly the slower migrating, Thr14 Tyr15 phosphory- al., 1995). The U.V.-induced G2 delay is independent lated form, which was converted to the faster of p53 function (Herzinger et al., 1995; Wang et al., migrating, dephosphorylated, active form at mitosis 1996), and involves a block in the cdc25-dependent (Figure 3a). This was con®rmed by phosphotyrosine activation of cyclin B/cdc2 (Herzinger et al., 1995; immunoblotting of the same cyclin B1 immunopreci- Poon et al., 1996). In this report we show that the pitates, which showed high levels of phosphotyrosine U.V.-induced G2 delay is mediated through a block in on cdc2 in both U.V. irradiated G2 delayed and the cdc25-dependent activation of cyclin B1/cdc2, normal S/G2 cyclin B1/cdc2 complexes (Figure 3b). cyclin A/cdk2 and cyclin A/cdc2, and that this block This suggested that the G2 delay was due to a block in occurs at the S/G2 transition or early G2. The block in the cdc25-dependent activation of the cyclin B1/cdc2 cdc25 function is not due to a reduction in the levels of either cdc25B or cdc25C proteins, but rather to the nuclear accumulation of cdc25B, and the inhibition of mitotic phosphorylation and activation of both cdc25B and cdc25C. Results Activation and nuclear translocation of the cyclin B1/ cdc2 complex is blocked by U.V. irradiation In response to low doses of U.V.C., HeLa cells are delayed in S and G2/M phases of the cell cycle (Wang and Ellem, 1994). Irradiation of an asynchronously growing population of the melanoma cell line A2058 also resulted in an accumulation of cells in G2/M (Figure 1a). No signi®cant accumulation or delay of cells in S phase was observed with this cell line, which was in contrast to HeLa cells irradiated under identical conditions. Following a delay of up to 20 h in G2/M, the cells recommenced progression through the cell cycle at 36 h after irradiation, which can be seen by a sharp increase in G1 and concomitant decrease in G2/ M phase cells at this time point (Figure 1b). We have investigated further the nature of the G2/M delay in HeLa and A2058 cell lines. The expression and activity of cyclin B1/cdc2, a key regulator of entry into mitosis, was analysed by immunoblotting and assaying the immunoprecipitated kinase from lysates of asynchronously growing HeLa cells (AS) which are 60% G1 phase cells, U.V. irradiated G2 delayed cells (U.V.), and late S/G2 (G2) and G2/M (M) cells from a thymidine synchrony (see Materials and methods). Immunoblotting with an anti-cyclin B1 antibody showed that cyclin B1 accumulated to normal G2 levels in the U.V. irradiated G2 delayed cells (Figure 2a), but only the G2/M phase cyclin B1/cdc2 was catalytically active (Figure 2b). Analysis of samples of similarly treated populations of A2058 cells again showed normal G2 phase accumulation of cyclin B1 and block in activation of the cyclin B1/cdc2 complex in the U.V. irradiated G2 delayed cells as was seen in HeLa (Figure 2c and d). Indirect immuno¯uorescence showed that the U.V. irradiated G2 delayed HeLa cells accumulated high levels of cyclin B1 in the cytoplasm, as in normal G2 phase cells (Pines and Hunter, 1991; Bailly et al., 1992), although due to the delay in transit through G2 in the irradiated sample up to 60% of the population showed bright cytoplasmic staining for cyclin Figure 1 U.V. irradiation of A2058 cells produces a G2 delay.