Generation of clones.
To further define the role of Cdk2 in the human cell cycle, we generated cell lines in which expression of Cdk2-wt and Cdk2-dn could be rapidly and strongly induced. We cloned cDNAs for Cdk2-wt and Cdk2-dn into a Tet-regulated expression vector (the Tet-off system [22
]). In the Cdk2-dn protein, an asparagine residue is substituted for the aspartic acid residue at position 145 (74
). This aspartic acid residue is conserved in all protein kinases and has been implicated in orienting the beta and gamma phosphates of ATP for the phospho-transfer reaction (9
). Each cDNA encoded a carboxy-terminal influenza HA epitope tag, to permit identification of the exogenous enzymes.
We transfected these constructs into a U2-OS clone (U24 [29
]) that expressed a Tet-sensitive transcription activator. We chose U2-OS cells because they have been shown in transient transfection studies to be efficiently arrested by Cdk2-dn (74
). In addition, these cells have been shown to support efficient regulation of transcription using the Tet-off system (24
). Puromycin-resistant colonies were selected in the presence of Tet, to repress transcription from the target vectors. Similar numbers of colonies were recovered from parallel plates transfected with target vector lacking an insert, suggesting that any leaky expression of the exogenous enzymes that may have occurred did not generally confer a selective disadvantage on clone growth or selection pressure for mutation of the host cells (data not shown). Consistent with this observation, we found little or no expression of the exogenous proteins in the uninduced state (Fig. ). We selected clones with a range of expression of the exogenous proteins relative to the endogenous enzyme (Fig ) and identified clones with comparable levels of expression of exogenous wt and dn proteins (e.g., dn.4 and wt.2 [Fig. ]). Note that induction of Cdk2-dn (Fig. , dn.5 lanes) appeared to inhibit formation of the rapidly migrating, active form of Cdk2, suggesting inhibition of endogenous Cdk2 activity (25
). The effect of Cdk2-dn on endogenous Cdk2 activity is further addressed below.
FIG. 1 Induction of Cdk2-wt and Cdk2-dn in U2-OS clones. The designated clones were cultured in the presence (+) or absence (−) of Tet for 3 days. Protein extracts were subjected to immunoblotting with monoclonal antibody 12CA5 directed (more ...) Induction of Cdk2-dn imposes S and G2/M phase arrests.
In preliminary studies, we chose a clone with a moderate level of expression (dn.3) to assess whether induction of Cdk2-dn inhibited DNA synthesis, as judged by pulse tritiated thymidine incorporation. Tet withdrawal had no discernible effect on tritiated thymidine incorporation in vector-transfected clones (data not shown) (8
). In contrast, Tet withdrawal for 3 days in dn.3 yielded an 85% inhibition of tritiated thymidine incorporation relative to uninduced cells (data not shown). Next, we assessed by flow cytometry the effects of Tet withdrawal on total cellular DNA content in this clone over the same time course. Surprisingly, we observed an increase in the fraction of cells in S and G2
/M phases in cells maintained without Tet (data not shown) rather than the expected accumulation of cells in G1
). The reduced thymidine incorporation that followed Cdk2-dn induction in dn.3 argued against acceleration of G1
progression and indicated that the accumulation of cells in S and G2
/M phases was likely caused by cell cycle inhibition. Similar S and G2
/M arrests were obtained from a second clone (dn.2 [data not shown]).
We then examined whether this was a reproducible response in Cdk2-dn-expressing clones. We derived a new set of clones from an independent transfection and selected representative low- and high-expressing clones (dn.5 and dn.4, respectively) for analysis (Fig. ). Tet withdrawal in each clone again yielded S and G2/M arrests (Fig. ). (We use the term “arrest,” as opposed to “delay,” throughout this report, without implying that the cells are necessarily permanently arrested.) In contrast, induction of Cdk2-wt had no discernible cell cycle effect (Fig. ).
FIG. 2 Cdk2-dn induction preferentially imposes S and G2/M arrests. Dn.5, dn.4, and wt.2 cells were grown in the presence (left) or absence (right) of Tet for 3 days, and the DNA content of the cells was assayed by flow cytometry. DNA content is displayed (more ...) Cdk2-dn-mediated G1 arrest.
We sought to reconcile these results with the previously observed G1
arrest mediated by transient transfection of Cdk2-dn in U2-OS cells (74
). Because the S and G2
/M arrests were seen in every Cdk2-dn-expressing clone and the exogenous enzymes were consistently of the expected size, it seemed unlikely that the Cdk2-dn coding region had undergone rearrangement or mutation during plasmid amplification or integration into genomic DNA. We further excluded these possibilities by performing Southern blotting, PCR amplification, and DNA sequencing of the integrated plasmids from two clones (data not shown).
We noted that simply replating U2-OS cells, which was also done prior to the transient transfections, induces a moderate synchronization in G1
) (data not shown). In addition, transient transfection is somewhat growth inhibiting (data not shown) and may contribute to cell synchronization in G1
. Another potential difference in these experimental settings is that analysis of the transiently transfected cells was gated to the 1 to 5% of cells, with strongest staining for a cotransfected marker protein (74
). We therefore reasoned that a G1
arrest might be observed in the stable clones if Cdk2-dn was expressed to high levels during M and/or G1
phases, perhaps mimicking the setting of transient transfection.
To test this notion, we synchronized cells from clones dn.5 and dn.4 in early mitosis, using the microtubule inhibitor nocodazole. We induced expression of the mutant protein during this period, then released the nocodazole block, and analyzed cellular DNA content at subsequent time intervals by flow cytometry. Cdk2-dn induction yielded a G1
arrest in each clone (Fig. ). The arrest in the high-expressing clone (dn.4) was as strong as that obtained following transient transfection (Fig. and reference 74
). Note that Cdk2-dn did not nonspecifically delay passage through all phases of the cell cycle, because there was no delay in progression through mitosis following release from the nocodazole-mediated arrest (Fig. ). Furthermore, no mitotic delay was detected in cells examined 1.5 and 3 h after nocodazole release (data not shown). Immunoblotting confirmed that Cdk2-dn was expressed during the nocodazole block (data not shown).
FIG. 3 Inhibition of progression through G1 phase following induction of Cdk2-dn during a nocodazole block. Dn.5 (low expressor; A) and dn.4 (high expressor; B) cells were incubated with nocodazole for 24 h in the presence or absence of Tet. The mitotic (more ...)
Finally, we tested the effect of transient transfection on the cell cycle distribution of dn.5 cells. We found that cells successfully expressing the transfected marker CD20 were slower to progress through G1 and S phases than either CD20-negative cells exposed to the transfection procedure or untransfected cells (data not shown). This phenomenon was observed in the presence or absence of Cdk2-dn induction, but it particularly enhanced the G1/S arrest imposed by induction of Cdk2-dn (data not shown). We conclude that whereas transient transfection of Cdk2-dn revealed a bona fide role for Cdk2 in the G1/S transition, these experimental conditions masked a more general propensity of Cdk2-dn to arrest progression through S and G2/M phases in U2-OS cells.
Inhibition of S and G2/M phase progression.
To demonstrate directly that expression of Cdk2-dn results in S and G2/M arrests and to identify favorable settings for biochemical analysis of Cdk2-dn's effects in synchronized cells, we performed experiments using cells synchronized in late G1 and S phases with HU. We determined cellular DNA content at intervals following release of dn.4 cells from this block, with and without induction of Cdk2-dn. The results showed that induced cells were inhibited in passage through S and G2/M phases (Fig. ). In addition, we noted that 15% of cells with Cdk2-dn induction retained a G1 DNA content, while fewer than 5% of uninduced cells did so following release, further confirming the ability of Cdk2-dn to inhibit G1/S progression. In similar experiments, induction of the wt protein again had no marked effect on cell cycle progression (data not shown).
FIG. 4 Inhibition of cell cycle progression following induction of Cdk2-dn during an HU block. Dn.4 cells were incubated with HU for 24 h in the presence or absence of Tet. Cells were collected for flow cytometry at 6-h intervals after removal of HU (more ...)
We considered the possibility that the apparent G2/M arrest following induction of the mutant resulted from slow progression through S phase. However, in exponentially growing cells, cells of clones displaying low induced levels of the mutant showed little S-phase arrest, but still accumulated in G2/M (data not shown). We therefore repeated the HU experiment using dn.5, a clone with a lower expression level. As expected, induction of Cdk2-dn in this clone yielded only a modest S-phase arrest, compared to dn.4, but a prominent G2/M arrest (Fig. ). Likewise, a G1 arrest was not seen in the clone with lower expression of Cdk2-dn (Fig. ). We conclude that G2/M is the phase most sensitive to Cdk2-dn expression.
FIG. 5 Induction of lower levels of Cdk2-dn preferentially yields a G2/M arrest, whereas higher levels also yield S and G1 arrests. Dn.5 (low expressor) and dn.4 (high expressor) cells were incubated with HU for 24 h in the presence or absence of Tet. (more ...) DNA damage checkpoint pathways.
Cdk2 is believed to be essential for the firing of DNA replication origins (2
). Some regions of the mammalian genome are typically replicated early in S phase, and others are replicated late. It has been documented that in yeast, some origins preferentially fire in early S phase, whereas others fire late (68
). The S-phase arrest observed upon induction of Cdk2-dn may therefore reflect a need for Cdk2 within S phase, to fire late-replicating origins (see Discussion). It is less clear what event(s) may be responsible for the accumulation of cells in G2
/M phase. Moreover, this is the cell cycle phase most sensitive to expression of Cdk2-dn. We therefore focused on characterizing further the point at which the G2
/M arrest occurs.
Given that Cdk2 has been implicated in initiation of DNA synthesis and that Cdk2-dn expression inhibits S-phase progression, we first asked whether the observed G2
/M arrest might result from activation of checkpoint pathways that block cell division in response to damaged or unreplicated DNA (16
). Some cell death was seen after 3 days of induction of Cdk2-dn, but there was hardly any cell death during the experiments described here (data not shown). p53 is functional in U2-OS cells and is commonly induced by DNA damage (71
). We found that p53 levels were unaffected by Cdk2-dn expression (data not shown). p21 levels were moderately increased, but no more so than by induction of Cdk2-wt (data not shown). Chk2 undergoes a shift in mobility on polyacrylamide gels in response to DNA damaging agents in some cells (44
). A protein of the expected size that reacts with anti-Chk2 antibodies showed no mobility shift in response to Cdk2-dn expression but also showed no shift with irradiation (data not shown). Thus, these experiments did not provide evidence for Cdk2-dn- induced activation of checkpoint pathways that monitor DNA integrity or replication.
Caffeine is known to be a potent antagonist of checkpoint pathways that monitor damaged and unreplicated DNA in mammalian cells (48
). We therefore examined whether caffeine could rescue the S and G2
/M arrests mediated by Cdk2-dn. We focused on clones with high levels of Cdk2-dn expression for this analysis, because they display more distinct S and G2
/M arrests. As a positive control for caffeine's effects, we exposed uninduced cells to gamma irradiation. Representative flow cytometry results are shown in Fig. . Caffeine efficiently prevented the G2
/M arrest imposed by irradiation (Fig. , Tet +, Irr). In contrast, caffeine only slightly reduced the S and G2
/M fractions in cells with Cdk2-dn induction (Fig. , Tet −), less so than in control cells without induction or irradiation (Fig. , Tet +). Thus, the S and G2
/M arrests imposed by Cdk2-dn appear to be relatively resistant to the effects of caffeine.
FIG. 6 Caffeine fails to rescue the S and G2/M arrests imposed by Cdk2-dn. Dn. 4 (A) and dn.2 (B) cells were incubated in the presence or absence of Tet for 48 h. A third culture maintained in Tet was subjected to 5 Gy of gamma irradiation (Irr) at (more ...)
In addition to its effects on DNA content, caffeine is capable of inducing premature DNA condensation in cells with unreplicated DNA (64
). Such an effect would not be evident by flow cytometry. To address this issue, we synchronized cells with HU, with or without Cdk2-dn induction, released the HU block, and examined whether caffeine addition during S phase could increase the fraction of cells with condensed nuclear DNA, assessed by fluorescence microscopy. The results are summarized in Table . Caffeine was able to induce premature DNA condensation in 15% of uninduced cells but only 2% of cells with Cdk2-dn induction. We conclude that the S and G2
/M arrests imposed by Cdk2-dn are not solely due to activation of caffeine-sensitive checkpoint pathways but may reflect disruption of events required for normal cell cycle progression.
Caffeine does not mediate premature DNA condensation in cells with Cdk2-dn inductiona
Cdk2-dn associates efficiently with endogenous cyclins.
To further characterize the mechanism by which Cdk2-dn exerted its effects, we analyzed the induced protein's association with known cyclin partners. Cyclin E expression is not markedly cell cycle regulated in U2-OS cells (49
) (data not shown). We therefore used unsynchronized cells 24 h after induction of wt or dn enzymes to assay association with cyclin E. Immunoblotting with antibodies directed against the HA epitope tag demonstrated that the exogenous proteins were induced to similar levels (Fig. A). We immunoprecipitated the exogenous proteins through their HA tags and assayed their associated kinase activity using histone H1 as a substrate. The results confirmed that the wt enzyme was catalytically active whereas the mutant was not (Fig. A) (74
FIG. 7 Induced Cdk2-wt and Cdk2-dn each associate with the majority of endogenous cyclin A and E, but Cdk2-dn is catalytically inactive. (A) Induced Cdk2-dn is catalytically inactive, whereas induced Cdk2-wt retains catalytic activity. Cells from clones (more ...)
Next, we compared the abilities of the induced enzymes to associate with endogenous cyclins. To estimate the fraction of endogenous cyclin E bound by the induced enzymes, we immunodepleted the induced proteins from the lysates using antibody directed against the HA tag and assayed the level of cyclin E remaining in the supernatant. Immunodepletion effectively removed most of the induced protein (Fig. B; compare lane 4 with lane 3 and lane 8 with lane 7). Although some cyclin E was nonspecifically lost from the supernatants due to the procedure itself (lanes 2 and 6), most cyclin E appeared to be bound to the exogenous proteins (lanes 4 and 8). These results suggest that the exogenous wt and dn proteins, respectively, associated with most of the endogenous cyclin E, consistent with their abundance relative to endogenous Cdk2. Consistent with this, cyclin E-associated kinase activity was inhibited by Cdk2-dn induction (data not shown). Because Cdk2-wt induction was without cell cycle effect, the results provide further evidence that the cell cycle inhibition mediated by Cdk2-dn is not due to sequestration of endogenous cyclins per se but also reflects a lack of Cdk2 kinase activity.
We then sought to extend these results by analyzing association of the exogenous enzymes with cyclin A and effects on cyclin A-associated kinase activity. Cyclin A expression and kinase activity are strongly cell cycle regulated in U2-OS cells, peaking in S and G2
), and the increase in cyclin A-associated Cdk2 activity is primarily responsible for the peak in Cdk2 activity that occurs in late S/G2
). To control for cell cycle position effects, we induced Cdk2-dn during synchronization of dn.4 cells with HU and prepared cell extracts at intervals following release from this block (see Fig. for flow cytometry profiles of cells treated in this manner). Synchrony was lost in the uninduced cells beyond 20 h, and so we focused our analysis on earlier time points. Induction of Cdk2-dn had little impact on cyclin A expression, assessed either by direct immunoblotting (data not shown) or by immunoprecipitation of cyclin A followed by immunoblotting (Fig. C, top). However, cyclin A-associated kinase activity was strongly inhibited (Fig. C, middle). This correlated with binding of cyclin A to the exogenous enzyme at the expense of the endogenous (Fig. C, bottom). In contrast, induction of Cdk2-wt in wt.2 under similar conditions had no substantial effect on cyclin A-associated kinase activity (data not shown). In conclusion, both Cdk2-wt and Cdk2-dn compete with the endogenous Cdk2 for cyclin binding, but Cdk2-dn lacks kinase activity and blocks the accumulation of cyclin A-associated kinase activity during S and G2
G2/M arrest occurs prior to DNA condensation.
To characterize further the point at which cells are arrested in the G2
/M period, we sought to determine whether Cdk2-dn-expressing cells arrested before or after nuclear DNA condensation, a robust marker for prophase, the first stage of mitosis. We synchronized cells with HU, released cells from this block, and added nocodazole, to trap and quantitate cells reaching the mitotic spindle checkpoint. We fixed the cells 24 h after release from HU and stained nuclear DNA with bisbenzimide (Hoechst). A large fraction of U2-OS cells that reach prophase are arrested by nocodazole and show condensed nuclear DNA (65
). Flow cytometry showed that most cells without induction were indeed arrested in the presence of nocodazole with a G2
/M DNA content (Fig. A, left) and that 30 to 40% of cells had fully condensed nuclei (Fig. B and C, left). In contrast, even though nearly half of the dn.4 cells and most of dn.5 cells with Cdk2-dn induction achieved a G2
/M DNA content (Fig. A, right), only 4 and 8%, respectively, had fully condensed nuclear DNA (Fig. B and C, right). These data indicate that the cells with induction of Cdk2-dn were arrested prior to prophase.
FIG. 8 Cdk2-dn arrests cells in G2, prior to DNA condensation. Dn.5 and dn.4 cells were synchronized at the G1/S border by incubation with HU for 24 h in the presence (left) or absence (right) of Tet. HU was then removed, and nocodazole was added; 24 (more ...)
We next used this experimental format to examine whether the delay in progression to mitosis imposed by Cdk2-dn could be abrogated by overexpression of Cdk2-wt. Dn.4 cells were cotransfected with a vector expressing β-Gal and either a vector without insert or a vector expressing Cdk2-wt. As discussed previously, this experiment was complicated by the fact that the successfully transfected cells showed an increased fraction of cells arrested at G1 phase and generally slower cell cycle progression; fewer cells progressed to fully condensed DNA under these experimental conditions (data not shown). We therefore scored the fraction of vector- or Cdk2-wt-transfected cells that showed any distinct nuclear condensation, identified by an examiner blinded to the treatment conditions as a marked reduction in nuclear size and/or loss of an oval shape. Cdk2-dn induction reduced the fraction of cells with nuclear condensation by 40% in the vector-transfected population, whereas inclusion of a Cdk2-wt cDNA insert in the transfection vector at least partially abrogated this effect (Table ). Flow cytometry analyses of similarly treated cells indicated that Cdk2-wt transfection also modestly decreased the fraction of cells that retained G1 and S phase DNA contents and increased the fraction of G2/M cells (data not shown).
Transfection of Cdk2-wt rescues the inhibition of DNA condensation mediated by Cdk2-dn inductiona
Regulation of Cdk1.
We next examined the effect of Cdk2-dn induction on cyclin B levels and associated kinase activity. Cells were synchronized at the G1
/S and then G2
/M borders as before (Fig. ), to minimize cell cycle position effects. We observed that cyclin B levels were reproducibly lower at each stage in cells with Cdk2-dn induction, consistent with recent evidence that hypophosphorylation of pRb and/or inhibition of Cdk2-cyclin A activity at the G1
/S transition may decrease cyclin B stability in U2-OS cells (42
) (Fig. A). However, cyclin B levels actually fell as cells progressed toward G2
/M in the absence of Cdk2-dn induction, and the difference in cyclin B levels between uninduced and induced cells narrowed as cells progressed toward G2
/M (Fig. A). pRb was hyperphosphorylated, as judged by its migration on polyacrylamide gels, with or without Cdk2-dn induction in the G1
/S synchronized cells but migrated slightly more rapidly in the extract with Cdk2-dn induction (data not shown). pRb remained largely hyperphosphorylated, with or without Cdk2-dn induction, as the cells progressed toward G2
/M (data not shown).
FIG. 9 Cdk2-dn-expressing cells arrest in G2 phase with moderately reduced levels of cyclin B and greatly reduced activation of Cdk1. Dn.4 cells were synchronized with HU and then nocodazole, with or without Cdk2-dn induction, as described in the legend (more ...)
In addition to the modestly lower cyclin B levels in the cells with Cdk2-dn induction, we observed accumulation of Cdk1 in a form that migrated more slowly on polyacrylamide gel electrophoresis (Fig. B). Inhibitory phosphorylation on threonine 14 and tyrosine 15 is known to reduce the electrophoretic mobility of Cdk1 (58
). We therefore examined whether the slower-migrating form seen in cells with Cdk2-dn induction reacted with an antibody generated against peptide K1Y15-P. Immunoblotting with K1Y15-P antibody yielded a single major band that comigrated with the slower-migrating Cdk1-reactive species (Fig. B). Endogenous Cdk2 has an electrophoretic mobility greater than that of Cdk1 (58
) (data not shown), but the HA tag on Cdk2-dn causes this protein to migrate at rate similar to that of Cdk1. To confirm that the slowly migrating species reactive with both anti-Cdk1 and anti-K1Y15-P antibodies was not derived from cross-reactivity with Cdk2-dn, we repeated the experiments following Cdk2-dn immunodepletion, using an antibody directed against the HA tag. Depletion of more than 90% of Cdk2-dn (see below) had no effect on the intensity of either the slower-migrating Cdk1-reactive band or the K1Y15-P-reactive band (data not shown). We conclude that these bands represent tyrosine-phosphorylated Cdk1.
The results suggested that Cdk1 activation was likely inhibited in cells with Cdk2-dn induction. Cdk1 was difficult to immunoprecipitate directly, as has been observed by others, but could be precipitated through associated cyclin B. We immunoprecipitated cyclin B from extracts prepared with and without Cdk2-dn induction. Immunoblotting with anti-Cdk1 and anti-K1Y15-P antibodies demonstrated the slowly migrating species, further confirming its identity as tyrosine-phosphorylated Cdk1 (Fig. B). We assayed kinase activity associated with the immunoprecipitates using histone H1 as a substrate. Cdk2-dn induction resulted in a strong reduction in cyclin B-associated kinase activity, even after normalizing for the amount of cyclin B immunoprecipitated (Fig. B). Similar results were obtained without prior synchronization with HU (data not shown). Somewhat less Cdk1 was present in the immunoprecipitates from Cdk2-dn-expressing cells than would be expected from the level of the protein present in the extracts. Because most of this Cdk1 appears to be tyrosine phosphorylated, an event requiring prior cyclin binding, we infer that the relative defect in immunoprecipitating Cdk1 is likely due to changes in recovery of the complexes or accessibility of the cyclin B epitope. Nonetheless, it is evident that the majority of Cdk1 associated with cyclin B in the cells with Cdk2-dn induction is in the slower migrating form (Fig. B). We conclude that the reduced levels of cyclin B-associated kinase activity result both from reduced cyclin B levels and from inhibitory phosphorylation of Cdk1.
We considered the possibility that sequestration of cyclin B by direct binding to Cdk2-dn might contribute to the defect in cyclin B-associated kinase activity. This scenario seemed unlikely, because Cdk2-wt induction should also compete with Cdk1 for binding, yet Cdk2-wt induction had no demonstrable cell cycle effect. Moreover, induction of Cdk2-dn during a nocodazole block had no effect on progression through mitosis, a process dependent on cyclin B-Cdk1 activity. Consistent with this reasoning, we found that immunodepletion of Cdk2-dn from the extracts did not significantly reduce the level of cyclin B remaining in the supernatant, providing further evidence against sequestration of cyclin B by Cdk2-dn (Fig. C).
Taken together, these experiments indicate a requirement for Cdk2 in progression through S and G2 phases of the human cell cycle, in addition to its previously described role at the G1/S transition.
Other cell types.
We then examined whether Cdk2-dn could impose S and/or G2
arrests in other cell types. These experiments were complicated by the fact that transient transfection of Cdk2-dn appears to predispose to G1
arrest, potentially outweighing S and G2
arrests mediated in asynchronous cultures. We therefore performed experiments in 3T3 cells synchronized in G1
, allowing us to directly assess effects of Cdk2-dn on progression into replicative phases. 3T3 cells were deprived of serum for 66 h, yielding greater than 90% G1
cells (data not shown). Serum was restored, and the cells were cotransfected with a marker plasmid expressing β-Gal and either an empty vector or one expressing Cdk2-dn. Continuous BrdU labeling was used to monitor S-phase progression. Nocodazole was added at 30 h (late S phase [data not shown]) and maintained until 48 h, to trap and quantitate cells reaching the spindle checkpoint. 3T3 cells arrest prior to prophase in response to spindle disruption, apparently due to an intact Chfr checkpoint (65
). We therefore released cells from the nocodazole trap for 2 h prior to fixation and assessed the fraction of cells that were able to condense their DNA. Cdk2-dn yielded a dose-dependent reduction in the ability of BrdU-positive cells to undergo DNA condensation (Table ). In similarly designed flow cytometry experiments in which nocodazole was omitted and cells were fixed 40 h after serum stimulation, Cdk2-dn-transfected cells showed a trend toward increased S-phase fractions and a statistically significant 50% increase in the G2
/M fraction (four independent experiments [data not shown]). These results suggest that Cdk2-dn can inhibit S- and G2
-phase progression in a nontransformed cell type. Preliminary experiments suggest that Cdk2-dn also mediates S and G2
arrests in HCT 116 colorectal carcinoma cells synchronized with HU (data not shown).
Transfection of Cdk2-dn in serum-starved and restimulated 3T3 cells blocks DNA condensation after BrdU incorporationa