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2 Parada, L. and Misteli, T. (2002) Chromosome positioning in the interphase nucleus. Trends Cell Biol. 12, 425 3 Croft, J.A. et al. (1999) Differences in the localization and morphology of chromosomes in the human nucleus. J. Cell Biol. 145, 1119 – 1131 4 Sun, H.B. et al. (2000) Size-dependent positioning of human chromosomes in interphase nuclei. Biophys. J. 79, 184 – 190 5 Parada, L. et al. (2002) Conservation of relative chromosome positioning in normal and cancer cells. Curr. Biol. 12, 1692 6 Walter, J. et al. (2003) Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages. J. Cell Biol. 160, 685 – 697 7 Gerlich, D. et al. (2003) Global chromosome positions are transmitted through mitosis in mammalian cells. Cell 112, 751 – 764 8 Vazquez, J. et al. (2001) Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus. Curr. Biol. 11, 1227– 1239 9 Zink, D. et al. (1998) Structure and dynamics of human interphase chromosome territories in vivo. Hum. Genet. 102, 241 – 251 10 Manders, E. et al. (1999) Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. J. Cell Biol. 144, 813 – 821 11 Bridger, J.M. et al. (2000) Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts. Curr. Biol. 10, 149 – 152 12 Csink, A.K. and Henikoff, S. (1998) Large-scale chromosomal movements during interphase progression in Drosophila. J. Cell Biol. 143, 13 – 22 13 Tumbar, T. and Belmont, A.S. (2001) Interphase movements of a DNA

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chromosome region modulated by VP16 transcriptional activator. Nat. Cell Biol. 3, 134 – 139 Shelby, R.D. et al. (1996) Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J. Cell Biol. 135, 545– 557 Boyle, S. et al. (2001) The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells. Hum. Mol. Genet. 10, 211 – 219 Tanabe, H. et al. (2002) Evolutionary conservation of chromosome territory arrangements in cell nuclei from higher primates. Proc. Natl. Acad. Sci. U. S. A. 99, 4424 – 4429 Misteli, T. (2001) Protein dynamics: Implications for nuclear architecture and gene expression. Science 291, 843 – 847 Chubb, J.R. and Bickmore, W.A. (2003) Considering nuclear compartmentalization in the light of nuclear dynamics. Cell 112, 403 – 406 Levsky, J.M. and Singer, R.H. (2003) Gene expression and the myth of the average cell. Trends Cell Biol. 13, 4 – 6 Manuelidis, L. (1984) Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences. Proc. Natl. Acad. Sci. U. S. A. 81, 3123 – 3127 Borden, J. and Manuelidis, L. (1988) Movement of the X chromosome in epilepsy. Science 242, 1687 – 1691

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Cycling without CDK2? Jonathan E. Grim and Bruce E. Clurman Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, and Department of Medicine, University of Washington School of Medicine, Seattle, WA 98109, USA

Cyclin-dependent kinase 2 (CDK2) regulates diverse aspects of the mammalian cell cycle. Most cancer cells contain mutations in the pathways that control CDK2, and CDK2 activity has received much attention as a target for cancer therapy. However, a recent report demonstrating that some cancer cells can proliferate without CDK2 activity questions the essential role of CDK2 in cell-cycle control, as well as its suitability as a therapeutic target. Cyclin-dependent kinases (CDKs) catalyze cell-cycle transitions [1 – 3]. In budding yeast, a single CDK subunit is sequentially activated by a group of cyclins that exhibit cell-cycle-specific expression. In contrast, mammalian cells contain many CDKs and cyclins, and oscillations in the expression and activity of these enzymes regulate cell division. It takes two to cycle: CDK4 and CDK2 regulate G1 progression An enormous body of work supports the model that two types of cyclin-CDKs, cyclin D-CDK4 and cyclin E-CDK2, play essential roles in mammalian cell cycles that are overlapping but non-redundant. For example, CDK4 inhibitors, including INK4 proteins (specific Corresponding author: Bruce E. Clurman ([email protected]). http://ticb.trends.com

inhibitors which bind to and prevent CDK4 activity), microinjected anti-cyclin D antibodies (which prevent CDK4 activation), and pharmacologic CDK4 inhibitors, cause cells to arrest in G1 phase [4]. CDK2 inhibition also causes G1 arrest [2,4,5]. Overexpression of catalytically inactive, dominant-negative CDK2 (dn-CDK2), or the p21cip1 and p27kip1 proteins (which directly bind and inhibit cyclin E-CDK2), microinjection of antibodies directed against cyclin E or CDK2, or chemical CDK2 inhibitors, all block G1 progression. Conversely, enforced cyclin D-CDK4 or cyclin E-CDK2 activity enhances cell proliferation and speeds G1 progression. Moreover, these effects are additive, suggesting that CDK4 and CDK2 have unique functions [6]. Deregulated cyclin E-CDK2 activity also causes genetic instability, further supporting its role in coordinating cell division [7,8].

CDK2 substrates: Rb and beyond Determining the protein substrates through which CDKs regulate cell division remains a major and uncompleted effort in cell-cycle research. However, the substrates that have been identified suggest that CDK2 and CDK4 play distinct roles in cell-cycle control (Fig. 1). The retinoblastoma protein (Rb) is the most exhaustively characterized CDK substrate.

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Mitogens Rb E2F CycD P

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Fig. 1. CDK4 and CDK6 regulate cell-cycle progression by phosphorylating protein substrates. The retinoblastoma protein (Rb) is phosphorylated by cyclin D-CDK6 and cyclin E-CDK2 as cells progress through G1 phase. Rb phosphorylation liberates E2F transcription factors, which then activate S-phase gene transcription. Rb phosphorylation is believed to be the only required CDK4 function. In contrast, cyclin E-CDK2 phosphorylates substrates in addition to Rb, including nucleophosmin, p27, and NPAT, which regulate centrosome duplication, S-phase entry, and histone transcription, respectively. In S-phase, cyclin A-CDK2 phosphorylates DP1, which inactivates E2F, and cdc6, which in turn alters its subcellular localization.

The Rb pathway regulates G1 progression [9,10]. In non-dividing cells, Rb is unphosphorylated and binds E2F-DP1 transcription factors. Rb prevents cell-cycle progression by sequestering E2F (thus preventing E2Fdependent transcription), and Rb-E2F also represses other genes. Mitogens promote the expression, assembly, and activity of cyclin D-CDK4/6 complexes early in G1, and cyclin E-CDK2 is activated as G1 progresses. Cyclin D-CDK 4/6 and cyclin E-CDK2 each phosphorylate Rb, and this releases E2F, which then executes its transcriptional program. Because cyclin E is an E2F target, E2F liberation reinforces cyclin E-CDK2 activity through increased cyclin E transcription. Cyclin E-CDK2 activation is an important downstream consequence of Rb pathway signaling, and many Rb pathway mutations found in cancer cells deregulate CDK2. In fact, replacing the endogenous mouse cyclin D1 gene with cyclin E suppresses the cyclin D1-null phenotype, suggesting that cyclin E-CDK2 activation is the essential function of CDK4 activity [11]. Cells with disrupted Rb pathways no longer require cyclin D-CDK4 activity for S-phase entry [10,12]. These data suggest that Rb is the only essential CDK4 substrate. By contrast, CDK2 inhibition arrests Rb-null cells, indicating that it has cell-cycle targets in addition to Rb. Several CDK2 substrates have been described, and their functions imply that CDK2 governs diverse processes. For example, nucleophosmin is a cyclin E-CDK2 target that regulates centrosome duplication in a phosphorylation-dependent manner [13]. Cyclin E-CDK2 also http://ticb.trends.com

phosphorylates the transcription factor NPAT and this controls replication-dependent histone gene transcription [14,15]. P27 is another cyclin E-CDK2 target, and phosphorylation triggers p27 proteolysis and promotes S-phase entry [4]. Finally, cyclin E-CDK2 is thought to regulate DNA replication by coordinating the loading of replication proteins such as mcm2 and cdc45 onto origins. Cyclin A activates CDK2 in S-phase and cyclin A-CDK2 phosphorylates DP1, which disables E2F transactivation [16]. Cyclin A-CDK2 also phosphorylates cdc6, a prereplication complex protein involved in licensing replication origins, whose subcellular localization is altered by phosphorylation [17]. In summary, whereas CDK4 is thought to be required primarily for Rb phosphorylation, CDK2 coordinates multiple cell-cycle processes, including G1 progression, centrosome duplication, and DNA synthesis. CDK2: essential no more? This tidy model of G1 control might need revision in the face of current work indicating that CDK2 is not essential for cell proliferation. Indeed there were early rumblings in this direction. For example, enforced E2F activity sustained cell division in rodent fibroblasts in which all CDK2 activity had been inhibited [18]. However, because this relied upon excess E2F activity, its physiologic relevance was unclear. Tetsu and McCormick have now provided evidence that some cancer cells can proliferate without CDK2 activity [19]. These studies began with the observation that pharmacologic MEK/MAP kinase inhibitors arrest cancer

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cell lines in G1. Because this was associated with loss of both CDK4 and CDK2 activity, they investigated the contribution of each to the G1 arrest. They found that specifically inhibiting cyclin D-CDK4 activity recapitulated the G1 arrest. Strikingly, however, they found that CDK2 inhibition by overexpression of either p27 or dnCDK2 did not arrest these cells. Specific CDK2 depletion by small interfering RNA or antisense oligonucleotides also resulted in cell proliferation without detectable CDK2 activity. These authors concluded that decreased CDK4 activity arrested these cells after MEK/MAP inhibition and that CDK2 was dispensable for cell division. Although these results appear to be directly at odds with previous studies indicating that CDK2 inhibition prevents cell-cycle progression, these discrepancies might be due, in part, to differences inherent in the various approaches employed. For example, the finding that short-term cyclin E-CDK2 inhibition by microinjected antibodies in G1 cells prevents S-phase entry, whereas CDK2 short interfering (si)-RNA expression in asynchronous cells does not, might reflect the unique features of these assays as much as the distinct subsets of cyclin E activities targeted [20]. However, CDK2 knockdown by si-RNA would seem to be the least pleiotropic approach, and thus the most likely to reveal specific CDK2 functions. How might cancer cells proliferate without CDK2? Tetsu and McCormick speculate that elevated CDK4 activity renders CDK2 redundant in these tumor cell lines and demonstrate that phosphorylation of preferred CDK2 sites in Rb still occurs, and might be due to CDK4 [19]. However, this suggests that phosphorylation of CDK2 substrates other than Rb is also dispensable, since they are unlikely to be phosphorylated by CDK4. Because they contain mutations that relax normal constraints on cell division (such as p53 mutations, which disrupt checkpoints that detect abnormalities that might result from CDK2-loss), Tetsu and McCormick propose that cancer cells might have reduced requirements for CDK2 in processes such as centrosome duplication. However, unpublished work from Barbacid and colleagues indicates that CDK2 might not be required for normal cell cycles either (M. Barbacid, personal communication), in that CDK2-null mice are viable and appear to develop normally. Although this work is ongoing and defects related to CDK2 deletion might be found, these observations strongly suggest that, not only can cell cycles occur in normal mammalian cells without CDK2, but also that cell-cycle regulation is largely intact as well. In contrast, CDK2 is an essential gene in Drosophila, although its required functions are not known [21]. Redundancy runs rampant through the cell cycle, and the finding that a particular protein is dispensable for a given pathway is not evidence that it does not ordinarily perform that function. Indeed, many genes with clearly defined roles in cell-cycle control have only minimal impact on cell division when deleted in mice (e.g. Rb, p130, p107, p21, p27, p16, p15, p18, p19, cyclin D1, -2, and -3, and CDK4). The observation that cancer cells proliferate without CDK2 can be interpreted in two ways: 1) CDK2 is not required, and that cell division occurs in the absence http://ticb.trends.com

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of CDK2 substrate phosphorylation, or 2) CDK2 is redundant, and CDK2 substrates are phosphorylated by other kinases. In fact, redundancy among CDKs is well known. For example, in fission yeast a single mitotic cyclin can efficiently run both S- and M-phases [22]. The fact that Tetsu and McCormick found no detectable cyclin A-associated kinase activity in the CDK2 knockdown cells would seem to argue against redundancy. However, it is not clear why cyclin A activity was absent, since cyclin A- cdc2 would not be expected to be inhibited by the CDK2 ablation. Furthermore, other CDKs that were not assayed (e.g. cyclin B-cdc2), or even non-CDK kinases, might substitute for the missing CDK2. Thus, although tantalizing, more work is needed before we know if these findings represent a new paradigm, or are instead another example of the redundancy that pervades cell-cycle control. Concluding remarks Ultimately, these studies might tell us more about how one can (or can not) manipulate the cell cycle than about normal pathways of cell-cycle regulation. Because most cancer cells contain mutations that deregulate CDK2 activity, CDK2 has been considered a ripe therapeutic target. In fact, CDK inhibitors have reached the clinical trial stage, albeit with unclear benefits to date [23,24]. Based on their current work, Tetsu and McCormick suggest that the substantial efforts aimed at targeting cdk2 might be in vain, and that CDK2 inhibition will not be an effective cancer treatment strategy. Perhaps the most promising work on CDK2 inhibition comes from Kaelin and colleagues, who reported that cyclin A-CDK2 inhibitory peptides kill cells with deregulated E2F activity (but not normal cells) [25]. These studies provide a basis for the hypothesis that cancer cells are hypersensitive to CDK2 inhibition. It is thus somewhat disappointing that the same cells that were killed by the Kaelin peptides (U2OS osteosarcoma cells) were unaffected by CDK2 depletion. The reasons for the discrepancies between these studies are unclear, and the possible explanations range from differences in cell lines to the different sets of CDK2 substrates that are targeted by the two approaches. Furthermore, Tetsu and McCormick studied transient inhibition of CDK2 expression in all but one case. However, if cancer cells with deregulated E2F activity are hypersensitive to CDK2 inhibition, perhaps long-term CDK2 inhibition might have clinically relevant consequences not revealed by these types of assays. Thus, although Tetsu and McCormick raise an important cautionary note, it might be premature to completely dismiss the therapeutic potential of CDK2 inhibition. RNA interference in human cells is rapidly generating data about the functions of cell-cycle proteins, some of which might immediately question established dogma. CDK2 is a prime example, having apparently been demoted from master G1 regulator to ancillary cell-cycle protein in one fell swoop. However, despite its exquisite specificity, RNA interference might also lead to complex cellular responses, particularly in redundant networks such as the cell-cycle machinery. The challenge now lies in integrating these data to yield the mechanistic insights that we expect will result

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from applying the power of reverse genetics to the study of cell-cycle regulation.

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Acknowledgements Work in our laboratory is supported by the National Institutes of Health and the W.M. Keck Foundation.

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1 Morgan, D.O. (1995) Principles of CDK regulation. Nature 374, 131 – 134 2 Sherr, C.J. (1996) Cancer cell cycles. Science 274, 1672– 1677 3 Hunter, T. and Pines, J. (1994) Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79, 573– 582 4 Sherr, C.J. and Roberts, J.M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13 (12), 1501 – 1512 5 van den Heuvel, S. and Harlow, E. (1993) Distinct roles for cyclindependent kinases in cell cycle control. Science 262, 2050– 2054 6 Resnitzky, D. and Reed, S.I. (1995) Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol. Cell. Biol. 15, 3463– 3469 7 Spruck, C.H. et al. (1999) Deregulated cyclin E induces chromosome instability. Nature 401, 297– 300 8 Minella, A.C. et al. (2002) p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation. Curr. Biol. 12, 1817 – 1827 9 Bartek, J. and Lukas, J. (2001) Pathways governing G1/S transition and their response to DNA damage. FEBS Lett. 490, 117– 122 10 Sherr, C.J. and McCormick, F. (2002) The RB and p53 pathways in cancer. Cancer Cell 2, 103 – 112 11 Geng, Y. et al. (1999) Rescue of cyclin D1 deficiency by knockin cyclin E. Cell 97, 767 – 777 12 Lukas, J. et al. (1995) Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature 375, 503– 506 13 Okuda, M. et al. (2000) Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell 103, 127 – 140 14 Zhao, J. et al. (2000) NPAT links cyclin E-Cdk2 to the regulation of

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replication-dependent histone gene transcription. Genes Dev. 14, 2283– 2297 Ma, T. et al. (2000) Cell cycle-regulated phosphorylation of p220(NPAT) by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription. Genes Dev. 14, 2298 – 2313 Krek, W. et al. (1994) Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase. Cell 78, 161 – 172 Petersen, B.O. et al. (1999) Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization. EMBO J. 18, 396– 410 DeGregori, J. et al. (1995) E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity. Genes Dev. 9, 2873– 2887 Tetsu, O. and McCormick, F. (2003) Proliferation of cancer cells despite CDK2 inhibition. Cancer Cell 3, 233– 245 Pagano, M. et al. (1993) Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. J. Cell. Biol. 121, 101 – 111 Lane, M.E. et al. (2000) A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2. Genetics 155, 233– 244 Fisher, D.L. and Nurse, P. (1996) A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins. EMBO J. 15, 850 – 860 Senderowicz, A.M. (2000) Small molecule modulators of cyclindependent kinases for cancer therapy. Oncogene 19, 6600– 6606 Tan, A.R. and Swain, S.M. (2002) Review of flavopiridol, a cyclindependent kinase inhibitor, as breast cancer therapy. Semin. Oncol. 29 (Suppl. 11), 77 – 85 Chen, Y.N. et al. (1999) Selective killing of transformed cells by cyclin/ cyclin-dependent kinase 2 antagonists. Proc. Natl. Acad. Sci. U. S. A. 96, 4325 – 4329

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Secretory granules: and the last shall be first… Michele Solimena1 and Hans-Hermann Gerdes2 1

Experimental Diabetology, ‘Carl Gustav Carus’ Medical School, University of Technology Dresden, Fetscherstrasse 74, 01307 Dresden, Germany 2 Interdisciplinary Center for Neurosciences, Department of Neurobiology, University Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

By tagging secretory granules with the fluorescent protein dsRed-E5, which changes its emission from green to red over time, Duncan et al. analysed the agedependent distribution of secretory vesicles within chromaffin cells. This elegant study illustrates as never before how age is a critical factor that segregates granules with respect to their localization and mobility and the probability of them undergoing exocytosis in response to different stimuli. The field of regulated exocytosis is always ‘brighter’. So it appears, as the coupling of each new variant of green Corresponding authors: Michele Solimena ([email protected]), Hans-Hermann Gerdes ([email protected]). http://ticb.trends.com

(GFP) and red (Discosoma sp. Red, dsRed) fluorescent proteins to cell cargoes is exploited to better track the biogenesis, transport and exocytosis of secretory granules in living neuroendocrine cells. Before the advent of GFP, investigators relied on loading secretory vesicles with fluorescent dyes such as FM1– 43 [1] and acridine orange [2], which, however, can also be taken up by the endosomal –lysosomal system. It was first shown in 1997 that regulated secretory proteins fused to GFP variants were properly stored in the neurosecretory granules of rat phaeochromocytoma PC12 cells [3,4]. The use of such GFP chimaeras has thus improved the possibility of labelling granules specifically rather than other membranous compartments. Immature secretory granules (ISGs) in PC12 cells, for example, could be selectively viewed by confocal microscopy after conjugation of the