DRO-Oncology, Pharmacology Department, Pharmacia Corp., Via Pasteur 10, 20014 Nerviano, Italy
* Author for correspondence (e-mail: corrado.santocanale{at}pharmacia.com )
Accepted 6 January 2001
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Summary |
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Key words: Cdt1, MCM, Pre-replicative complexes, DNA licensing
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Introduction |
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The human Cdt1 gene was recently isolated; hCdt1 protein fluctuates during
the cell cycle in highly proliferative HeLa cells and interacts with geminin
(Wohlschlegel et al., 2000;
Nishitani et al., 2001
).
Moreover, addition of recombinant hCdt1 protein to a geminin-poisoned
Xenopus egg extract was shown to restore the extract's ability to
replicate exogenous DNA (Wohlschlegel et
al., 2000
). Whether this occurs simply by titrating out geminin,
rather than providing activity itself, is not yet known. In this study, we
investigated the role of hCdt1 in DNA replication in intact cells. We report
that hCdt1 protein is present in G0 and early G1 phases of cell cycle in
normal cells and that ablation of hCdt1 function causes inhibition of cellular
DNA replication by blocking the licensing reaction.
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Materials and Methods |
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Cell culture and synchronization
U2OS osteosarcoma cells were cultivated in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal calf serum (FCS). U2OS were
synchronized in early mitosis by nocodazole (50 ng/ml) for 16 hours, in G1/S
boundary by hydroxyurea (5 mM) or aphidicolin (5 µg/ml) for 24 hours.
Normal dermal human fibroblasts (NHDF) were maintained in fibroblast basal
medium (FBM, Promocell) with 10% FCS and 1 ng/ml of human fibroblast growth
factor (FGF). NHDF cells were serum starved for 72 hours in FBM plus FGF and
0.1% FCS.
Antibody microinjection and immunofluorescence
Synchronized U2OS cells on glass coverslips were microinjected with GFP
encoding DNA (20 ng/µl) and either affinity-purified anti-Cdt1 antibodies
(1 mg/ml) or the same antibodies preincubated for 1 hour with antigen peptide
(0.5 mg/ml). An automated capillary system (Zeiss AIS 2) at a pressure of
50-100 hPa was used for nuclear microinjection. Cells were fixed in 4%
paraformaldehyde.
For MCM2 binding experiments, a Histone 2B-GFP fusion expressing plasmid instead of GFP vector was used as a marker because it is resistant to Triton washes. Extraction of unbound MCM2 from nuclei was achieved with a 20 minute wash in cold CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) supplemented with 0.5% Triton X-100 and protease inhibitors before methanol fixation.
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Results |
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Cdt1 protein is expressed in quiescent cells and accumulates before S
phase entry
It was previously shown that hCdt1 protein fluctuates during the cell cycle
in HeLa cells (Wohlschlegel et al.,
2000; Nishitani et al.,
2001
). We further extended this observation by examining hCdt1
expression in normal non-transformed primary cells. NHDF were serum starved
and stimulated by addition of fresh medium containing 10% serum, samples were
taken at 4 hour intervals. FACS analysis together with cyclin A accumulation
indicates that NHDF entered S-phase around 16 hours after serum stimulation
and most cells had completed DNA replication after 28 hours. G0 NHDF cells
contain low but detectable levels of hCdt1. hCdt1 protein increases two- to
threefold in late G1, and then decreases in S-phase
(Fig. 2). Geminin, a Cdt1
inhibitor, began to accumulate in S phase following the peak of hCdt1 and
concomitantly with cyclin A, suggesting that hCdt1 function may be limited to
a window of the cell cycle that precedes DNA synthesis. This result is
consistent with observations that geminin in HeLa cells accumulates in S and
G2 phases (Wohlschlegel et al.,
2000
; Nishitani et al.,
2001
). In fission yeast Cdt1 is cell cycle co-regulated with Cdc
18/Cdc6 (Nishitani et al.,
2000
). Moreover, in Drosophila DUP mRNA, the Cdt1
homologue is controlled by E2F transcription factor
(Whittaker et al., 2000
), as
is Cdc6 mRNA in human cells (Hateboer et
al., 1998
; Leone et al.,
1998
; Yan et al.,
1998
), suggesting that hCdt1 and Cdc6 may also be co-regulated in
human cells. In these experimental conditions we observed that Cdc6
accumulates with slightly delayed kinetics compared with hCdt1. Further
experiments will be necessary to determine whether hCdt1 cell cycle regulation
occurs at the transcriptional or post-transcriptional level or both.
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Ablation of Cdt1 function prevents DNA replication initiation
Expression timing suggests that hCdt1 might play a role in early stages of
DNA replication similar to what has been observed in model organisms
(Nishitani et al., 2000;
Maiorano et al., 2000
). We
used the nuclear antibody microinjection approach to test this hypothesis.
Nocodazole-treated U2OS cells were injected with highly affinity-purified
anti-Cdt1 antibodies together with green flourescent protein (GFP) encoding
DNA. After microinjection, nocodazole was removed and cells were incubated in
fresh medium in the presence of 5'-bromodeoxyuridine (BrdU) for a
further 20 hours. Microinjected cells, which expressed GFP, were assayed for
their capability to incorporate BrdU into DNA by indirect immunostaining. As a
control the same anti-Cdt1 antibodies preincubated with antigen peptide were
injected. hCdt1 ablation clearly caused inhibition of S-phase. Indeed, almost
80% of cells microinjected with anti-Cdt1 antibodies failed to initiate DNA
synthesis. By contrast, only 28% of control cells microinjected with anti-Cdt1
antibodies prebound to peptide did not replicate their DNA
(Fig. 3). Inhibition of DNA
synthesis in cells microinjected with anti-Cdt1 antibodies could be caused by
the incapability of these cells to properly execute mitosis. However, we
observed that at the time of cell fixation, 20 hours post release, the
chromatin of microinjected cells was decondensed; thus excluding the
possibility of a late mitotic block. Moreover, in a parallel experiment we
found that at 12 hours post-release, cells microinjected with anti-Cdt1
antibodies and cells microinjected with anti-Cdt1 antibodies neutralized with
antigen peptide had degraded cyclin B1 with approximately the same efficiency
as surrounding cells that were not injected (data not shown).
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Ablation of protein function through antibody microinjection can be used to
define the execution point of a given protein during cell cycle
(Pagano et al., 1992;
Ohtsubo et al., 1995
). DNA
replication is a two-step process: first, pre-replicative complexes are
assembled at origins and subsequently converted into active replication forks
(Diffley, 1996
). To determine
whether hCdt1 function is required for either one of these two mechanisms,
cells that had been blocked in early S-phase with hydroxyurea (HU) were
microinjected with anti-Cdt1 antibodies. HU-treated cells arrest with a nearly
G1 DNA content when measured by FACS (data not shown); however, origin
licensing has already occurred and MCM proteins are tightly bound to chromatin
(Krude et al., 1996
;
Aparicio et al., 1997
).
Moreover, in HU-treated cells, a number of early firing origins have already
been activated and replication origin function is no longer required for the
bulk of DNA synthesis (Vassilev and
Russev, 1984
; Bousset and
Diffley, 1998
; Santocanale and
Diffley, 1998
). As shown in
Fig. 3B, 82% of cells
microinjected with anti-Cdt1 antibodies and 80% of cells microinjected with
control antibodies efficiently recover DNA replication after HU release. The
pattern of BrdU staining was similarly uniform in microinjected and control
cells. These data, together with the finding that the same reagents fully
block DNA synthesis when microinjected into mitotic cells indicate that hCdt1
executes its function for DNA replication sometime between mitosis exit and
the G1/S boundary. Consistent with the observation from fission yeast
(Nishitani et al., 2000
) our
results demonstrate that hCdt1 is not required for DNA replication elongation
in human cells.
Cdt1 is necessary for chromatin licensing
Execution point experiments, expression pattern and genetic evidence from
model organisms hinted that hCdt1-dependent inhibition of DNA synthesis might
occur by preventing chromatin licensing. This can be monitored as the binding
of MCM proteins to DNA; in particular MCM2 protein, a subunit of the MCM
complex, is always nuclear but its binding to DNA is cell cycle regulated.
Treatment with low concentrations of non-ionic detergents discriminates
between a chromatin engaged and a free MCM2 form
(Krude et al., 1996;
Okuno et al., 2001
). We
confirmed this finding since 83% of cells blocked in G1/S showed a
Triton-resistant staining for MCM2, while only 16% of cells blocked in early
mitosis showed a similar staining pattern
(Fig. 4A). We coupled this
assay to the microinjection approach to determine the requirement of hCdt1 for
licensing. Mitotic U2OS cells were microinjected with anti-Cdt1 antibodies
plus or minus blocking peptide, allowed to go through G1 and trapped at the
G1/S border with aphidicolin in order to avoid loss of MCM2 binding due to
completion of DNA replication. Microinjection of anti-Cdt1 antibodies, plus or
minus competing peptide, did not change the normal nuclear MCM2 staining
(Fig. 4B, grey bars). However,
when a Triton wash was performed before fixation, inhibition of hCdt1 function
caused MCM2 to be released from the nucleus in 77% of injected cells. MCM2
chromatin binding was almost fully rescued when anti-Cdt1 antibodies were
pre-incubated with antigen peptide (Fig.
4B, black bars).
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Discussion |
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This work provides direct evidence that human Cdt1 is required for DNA
replication in intact cells. The striking correlation between the number of
microinjected cells incapable of replicating their DNA and the number of cells
that do not load MCMs argues that hCdt1 executes its function in DNA
replication by licensing the chromatin. Further studies will be necessary to
understand how hCdt1 cooperates with the ORC complex and Cdc6 in building a
licensed human origin. Our results indicate that, in the human cell cycle,
hCdt1 function is required before the duplication of DNA. In highly
proliferative tumor cells licensing occurs late in mitosis possibly at the
anaphase/telophase transition (Mendez and
Stillman, 2000; Okuno et al.,
2001
). Nonetheless, when normal cells exit the cell cycle to enter
a quiescent state, some licensing proteins such as Cdc6 and MCM5 are actively
degraded (Williams et al.,
1998
; Petersen et al.,
2000
; Stoeber et al.,
2001
); because of this active degradation in resting cells, their
detection represents a valuable assay for identification of malignant cells in
tissue samples (Williams et al.,
1998
; Stoeber et al.,
1999
). With respect to this, we observed that in serum-deprived
cells hCdt1 protein is still detectable whereas Cdc6 is not present,
suggesting that different mechanisms controlling the levels of these two
licensing factors may exist. The presence of hCdt1 protein in G0 and early G1
cells is also consistent with the notion that addition of Cdc6 protein is
sufficient per se to cause premature MCM loading and entry into S-phase of G1
nuclei in a mammalian cell-free system
(Stoeber et al., 1998
).
Differences in the regulation of Cdc6 and Cdt1 proteins in cycling cells were
recently described by Nishitani and co-workers
(Nishitani et al., 2001
). In
particular, they have shown that in S-phase hCdt1 is unstable and targeted for
proteolysis whereas in the same period of the cell cycle most of the Cdc6
protein is inactivated by a CDK2-dependent nuclear export mechanism
(Petersen et al., 1999
). CDKs
play a crucial role in regulating licensing activity. Intriguingly,
deregulation of CDK activity causes genomic instability both in model
organisms and in cancer cells (Brown et
al., 1991
); Spruck et al.,
1999
; Noton and Diffley,
2000
). In budding yeast, this correlates with a lower efficiency
of origin usage (Palmer et al.,
1990
; Hogan and Koshland,
1992
; Noton and Diffley,
2000
). Ultimately it will be important to define how
cyclindependent kinases affect the activity of each component of the licensing
system, including hCdt1, in normal human cells, and whether genomic
instability observed in some cancers might be caused solely by impairment of
pre-RCs formation and defective origin usage.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aparicio, O. M., Weinstein, D. M. and Bell, S. P. (1997). Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91,59 -69.[Medline]
Bousset, K. and Diffley, J. F. (1998). The Cdc7
protein kinase is required for origin firing during S phase [published erratum
appears in Genes Dev. 1998 12, 1072]. Genes
Dev. 12,480
-490.
Brown, M., Garvik, B., Hartwell, L., Kadyk, L., Seeley, T. and Weinert, T. (1991). Fidelity of mitotic chromosome transmission. Cold Spring Harb. Symp. Quant. Biol. 56,359 -365.[Medline]
Cairns, J. (1966). Autoradiography of HeLa cell DNA. J. Mol. Biol. 15,372 -373.[Medline]
Diffley, J. F. (1996). Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. Genes Dev. 10,2819 -2830.[Medline]
Diffley, J. F. (2001). DNA replication: building the perfect switch. Curr. Biol. 11,R367 -R370.[Medline]
Dutta, A. and Bell, S. P. (1997). Initiation of DNA replication in eukaryotic cells. Annu. Rev. Cell Dev. Biol. 13,293 -332.[Medline]
Harlow, E. and Lane, D. P. (1988). Antibodies: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Hateboer, G., Wobst, A., Petersen, B. O., Le Cam, L., Vigo, E.,
Sardet, C. and Helin, K. (1998). Cell cycle-regulated
expression of mammalian CDC6 is dependent on E2F. Mol. Cell.
Biol. 18,6679
-6697.
Hofmann, J. F. and Beach, D. (1994). cdt1 is an essential target of the Cdc10/Sct1 transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO J. 13,425 -434.[Abstract]
Hogan, E. and Koshland, D. (1992). Addition of extra origins of replication to a minichromosome suppresses its mitotic loss in cdc6 and cdc14 mutants of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89,3098 -3102.[Abstract]
Huberman, J. A. and Riggs, A. D. (1968). On the mechanism of DNA replication in mammalian chromosomes. J. Mol. Biol. 32,327 -341.[Medline]
Krude, T., Musahl, C., Laskey, R. A. and Knippers, R.
(1996). Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind
chromatin uniformly before S-phase and are displaced locally during DNA
replication. J. Cell Sci.
109,309
-318.
Leone, G., DeGregori, J., Yan, Z., Jakoi, L., Ishida, S.,
Williams, R. S. and Nevins, J. R. (1998). E2F3 activity is
regulated during the cell cycle and is required for the induction of S phase.
Genes Dev. 12,2120
-2130.
Maiorano, D., Moreau, J. and Mechali, M. (2000). XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404,622 -625.[Medline]
McGarry, T. J. and Kirschner, M. W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93,1043 -1053.[Medline]
Mendez, J. and Stillman, B. (2000). Chromatin
association of human origin recognition complex, cdc6, and minichromosome
maintenance proteins during the cell cycle: assembly of prereplication
complexes in late mitosis. Mol. Cell. Biol.
20,8602
-8612.
Nishitani, H., Lygerou, Z., Nishimoto, T. and Nurse, P. (2000). The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404,625 -628.[Medline]
Nishitani, H., Taraviras, S., Lygerou, Z. and Nishimoto, T.
(2001). The human licensing factor for DNA replication Cat1
accumulates in G1 and is destabilized after initiation of S-phase.
J. Biol. Chem. 276,44905
-44911.
Noton, E. and Diffley, J. F. (2000). CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis. Mol. Cell, 5,85 -95.[Medline]
Ohtsubo, M., Theodoras, A. M., Schumacher, J., Roberts, J. M. and Pagano, M. (1995). Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol. Cell. Biol. 15,2612 -2624.[Abstract]
Okuno, Y., McNairn, A. J., den Elzen, N., Pines, J. and Gilbert,
D. M. (2001). Stability, chromatin association and functional
activity of mammalian pre-replication complex proteins during the cell cycle.
EMBO J. 20,4263
-4277.
Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. and Draetta, G. (1992). Cyclin A is required at two points in the human cell cycle. EMBO J. 11,961 -971.[Abstract]
Palmer, R. E., Hogan, E. and Koshland, D.
(1990). Mitotic transmission of artificial chromosomes in cdc
mutants of the yeast, Saccharomyces cerevisiae.
Genetics 125,763
-774.
Petersen, B. O., Lukas, J., Sorensen, C. S., Bartek, J. and
Helin, K. (1999). Phosphorylation of mammalian CDC6 by cyclin
A/CDK2 regulates its subcellular localization. EMBO J.
18, 396-410
Petersen, B. O., Wagener, C., Marinoni, F., Kramer, E. R.,
Melixetian, M., Denchi, E. L., Gieffers, C., Matteucci, C., Peters, J. M. and
Helin, K. (2000). Cell cycle- and cell growth-regulated
proteolysis of mammalian CDC6 is dependent on APC-CDH1. Genes
Dev. 14,2330
-2343.
Santocanale, C. and Diffley, J. F. (1998). A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature, 395,615 -618.[Medline]
Spruck, C. H., Won, K. A. and Reed, S. I. (1999). Deregulated cyclin E induces chromosome instability. Nature 401,297 -300.[Medline]
Stoeber, K., Mills, A. D., Kubota, Y., Krude, T., Romanowski,
P., Marheineke, K., Laskey, R. A. and Williams, G. H. (1998).
Cdc6 protein causes premature entry into S phase in a mammalian cell-free
system. EMBO J. 17,7219
-7229.
Stoeber, K., Halsall, I., Freeman, A., Swinn, R., Doble, A., Morris, L., Coleman, N., Bullock, N., Laskey, R. A., Hales, C. N. and Williams, G. H. (1999). Immunoassay for urothelial cancers that detects DNA replication protein Mcm5 in urine. Lancet 354,1524 -1525.[Medline]
Stoeber, K., Tlsty, T. D., Happerfield, L., Thomas, G. A.,
Romanov, S., Bobrow, L., Williams, E. D. and Williams, G. H.
(2001). DNA replication licensing and human cell proliferation.
J. Cell Sci. 114,2027
-2041.
Tada, S., Li, A., Maiorano, D., Mechali, M. and Blow, J. J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107-113.[Medline]
Vassilev, L. and Russev, G. (1984). Hydroxyurea treatment does not prevent initiation of DNA synthesis in Ehrlich ascites tumour cells and leads to the accumulation of short DNA fragments containing the replication origins. Biochim. Biophys. Acta 781, 39-44.[Medline]
Whittaker, A. J., Royzman, I. and Orr-Weaver, T. L.
(2000). Drosophila double parked: a conserved, essential
replication protein that colocalizes with the origin recognition complex and
links DNA replication with mitosis and the down-regulation of S phase
transcripts. Genes Dev.
14,1765
-1776.
Williams, G. H., Romanowski, P., Morris, L., Madine, M., Mills,
A. D., Stoeber, K., Marr, J., Laskey, R. A. and Coleman, N.
(1998). Improved cervical smear assessment using antibodies
against proteins that regulate DNA replication. Proc. Natl. Acad.
Sci. USA 95,14932
-14937.
Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C.,
Walter, J. C. and Dutta, A. (2000). Inhibition of eukaryotic
DNA replication by geminin binding to Cdt1. Science
290,2309
-2312.
Yan, Z., DeGregori, J., Shohet, R., Leone, G., Stillman, B.,
Nevins, J. R. and Williams, R. S. (1998). Cdc6 is regulated
by E2F and is essential for DNA replication in mammalian cells.
Proc. Natl. Acad. Sci. USA
95,3603
-3608.
Yanow, S. K., Lygerou, Z. and Nurse, P. (2001).
Expression of Cdc18/Cdc6 and Cdt1 during G(2) phase induces initiation of DNA
replication. EMBO J. 20,4648
-4656.