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Experimental Therapeutics, Preclinical Pharmacology |
Gastrointestinal Oncology Research Laboratory, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endoreduplication has been observed in response to
MTIs3
in cells
lacking p53, pRb, and CDKIs p21 or p16 (7, 8, 9, 10, 11)
. In
response to MTIs, p53 is induced after the cells exit mitosis following
"mitotic slippage" and enter G1 with 4n DNA
content. The p53 then transactivates p21, which binds and inactivates
the cyclin E/Cdk2 complex (11)
. The inactivation of the
cyclin E/Cdk2 complex prohibits the phosphorylation of pRb (12
, 13)
and prevents the release of E2F-1, a transcription factor
essential to activate genes required for S-phase entry
(14, 15, 16)
and arrest cells in G1. In
contrast, p53-/- cells do not arrest at the pseudo
G1 state, and they endoreduplicate, resulting in
the accumulation of cells with >4n DNA content (polyploidy; Refs.
7, 8, 9, 10, 11
). p16, a member of the INK family of CDKIs, inhibits
cyclin D/Cdk 4 and 6 kinase and prevents the pRb phosphorylation
(17)
. Therefore, loss of p16 also initiates MTI-induced
endoreduplication. Therefore, inactivation of Cdks in a situation where
the previous cell cycle phase has been defective is a crucial step in
preserving the genomic integrity because polyploid cells are
genetically unstable and lose chromosomes randomly and generate
aneuploidy (18
, 19)
. Because some of the routinely used
chemotherapy drugs interfere with microtubule dynamics and many of
human cancers have a compromised G1 checkpoint,
it is essential to explore ways to reduce the risks of development of
polyploidy in response to these drugs. One of the possible ways can be
to combine the MTI treatment with agents that can inhibit Cdks
directly, thus preventing the polyploidy and hence preserving the
genomic stability. Additionally, neoplastic progression in Barrett’s
esophagus and pancreatic tumors in transgenic mice expressing SV40 TAg
has been shown to arise through the development of the
tetraploid
polyploidaneuploid sequence (20, 21, 22)
. In
that respect, MTI-induced polyploidy may provide a model to test the
utility of Cdk inhibitors for the prevention of neoplasia.
In the National Cancer Institute drug screen, flavopiridol, a synthetic flavone presently undergoing clinical trial (23) , has been identified as an agent that inhibits Cdks 1, 2, 4, and 6 in nanomolar concentrations (24 , 25) . On account of this, flavopiridol has been shown in vitro to inhibit tumor cell growth through the blockade of cell cycle progression at G1 or G2 (24 , 26) . We have previously reported that flavopiridol at noncytotoxic, nanomolar concentrations significantly enhances the induction of apoptosis by the chemotherapeutic agent mitomycin C and paclitaxel in gastric and breast cancer cell lines (27) . Synergism between flavopiridol and paclitaxel has also been observed against A549 non-small cell lung cancer cells (28) . We have recently reported that flavopiridol enhances paclitaxel-induced apoptosis in a sequential-dependent manner such that paclitaxel should precede flavopiridol to achieve this synergistic effect (29) . This observation has been translated into a Phase I clinical trial of sequential paclitaxel and flavopiridol at our center, and promising clinical activity has been reported (30) .
In the present study, we show that in response to MTIs, human breast and colon cancer cells with intact G1-S checkpoints arrest in G1 with 4n DNA content (pseudo G1), whereas cells with defective G1-S checkpoints endoreduplicate and become polyploid. Flavopiridol at nanomolar concentrations, when administered after MTIs, prevents the endoreduplication and development of polyploidy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MPM-2/PI Bivariate Flow Cytometry.
In a 100-mm dish, 1.4 x 106 cells
were cultured for 48 h and treated with paclitaxel, nocodazole,
and flavopiridol as a single agent or sequentially. The cells were
harvested at specific time points by trypsinization, pooled with
floating cells, and fixed overnight with ice-cold 70% ethanol. After
washing with PBS containing 0.05% Tween 20 and 1% fetal bovine serum,
cells were labeled with MPM-2 antibody (final concentration of 1
µg MPM-2 antibody/ml; Upstate Biotechnology) for 1 h at
4°C. Cells were washed twice with PBS and incubated with goat
antimouse FITC (Boehringer Mannheim) for 1 h at room temperature
in the dark. After washing twice with PBS, cells were resuspended in 5
µg/ml PI containing 50 µg/ml RNase A. Samples were analyzed
on a Becton Dickinson FACScan, and data of 20,000 events for each
sample were plotted with CellQuest software (Becton Dickinson). The
MPM-2-positive (mitotic) cells will show increased green
fluorescence and shift above the baseline of the dot plot (shown
in the box in Figs. 1
and 2
).
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32p) ATP], 15 µM
ATP, and 50 µg/ml Histone-H1 (Boehringer Mannheim). The reaction was
allowed to proceed for 20 min at 30°C and was terminated by adding 10
µl of Laemmli sample buffer and boiling for 5 min. Products were
resolved by 10% SDS-PAGE. The activity levels on autoradiographs were
quantified using a densitometric scanning system.
Western Analysis.
Protein lysates prepared for kinase assays were used for Western
analysis. Fifty µg of soluble protein were resolved by SDS-PAGE and
transferred to Immobilon-P membranes (Millipore). The equal
loading of proteins was confirmed by amido black staining (Sigma). The
membranes were probed with mouse monoclonal antibodies specific to pRb,
p53, p21, cyclin E (Santa Cruz Biotechnology), and mouse monoclonal
cyclin B1 (kindly provided by Dr. Tim Hunt, Imperial Cancer Research
Fund Clare Hall Laboratories, United Kingdom). The primary
antibodies were detected with sheep antimouse horse radish peroxide
secondary antibody (Amersham Life Sciences) and subjected to enhanced
chemiluminescence reagents (DuPont NEN Life Science Products, Boston,
MA). The levels of expression were quantified using a densitometric
scanning system.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Twenty-four h
after the removal of paclitaxel, cells exit mitosis as MPM-2 labeling
decreases; however, the 4n peak remains constant. The exited cells are
multinucleated and have a decondensed nuclear morphology as assessed by
DAPI staining (29)
, suggesting that cells enter the
interphase without undergoing cytokinesis, a state we refer to as
"pseudo G1." The expression of p53 and its
direct transcriptional target p21 was induced 3- and 4-fold,
respectively in pseudo G1 cells compared to
untreated controls (ND24). At the
similar time period, most of the pRb was in its faster migrating
hypophosphorylated form (Fig. 1B
). The p21 induction also
corresponds to the decrease in cyclin E/Cdk2 kinase activity after the
removal of paclitaxel from the MCF-7 cells (Fig. 1C
). Next, we examined whether p53 and p21 induction was associated with prevention of the next round of DNA synthesis. The percentages of cells with >4n DNA content did not increase 24 and 48 h after the removal of paclitaxel (data not shown). Similar results were observed with cells treated with nocodazole.
Cells with Compromised G1 Checkpoint Endoreduplicate
and Become Polyploid in Response to MTIs, and Flavopiridol Suppresses
the Polyploidy.
The MDA-MB-468 breast cancer cells are p53- and pRb-negative and posses
little or no Cdk4 kinase activity. To determine the endoreduplication
in these cells in response to MTIs, cells were first synchronized at
the G1-S boundary by a thymidine double block.
This resulted in >90% cells in the G1-S phase
of the cell cycle. As shown in Fig. 2A
, after 24 h of
nocodazole treatment (Noc24), the majority
of cells accumulated with 4n DNA content. A small 8n population was
also observed, which increased steadily after 24, 48, and 72 h of
additional nocodazole treatment
(Noc24Noc24–72).
More than 65% of cells contained >4n DNA content after 96 h of
total nocodazole treatment
(Noc24Noc72).
We did not observe a 16n or 32n population. Addition of 300
nM flavopiridol for 24, 48, and 72 h after
24 h of initial nocodazole treatment
(Noc24[Noc +
F]24–72) prevented the cells from
synthesizing additional DNA because the number of cells containing >4n
DNA content dropped by >80% compared to nocodazole treatment
(Noc24Noc24–72;
Fig. 2A
). The MPM-2 labeling shows that 8n cells also
undergo mitosis by nocodazole treatment, and the addition of
flavopiridol to nocodazole-treated cells prevents the entry of 8n cells
to undergo mitosis (Fig. 2B
).
The p21-deficient HCT116, a human colon cancer cell line, also undergoes MTI-induced endoreduplication (11) . Flavopiridol at concentrations of 150 nM prevented MTI-induced endoreduplication in these cells, with a dramatic decrease in the number of cells containing >4n DNA content (data not shown). Taken together, these data suggest that treatment with MTIs induce endoreduplication in human tumor cells with compromised G1 checkpoint, and flavopiridol prevents the endoreduplication.
Inhibition of Cyclin E/Cdk2 Kinase Activity by Flavopiridol
Prevents Endoreduplication.
Cyclin D/Cdk4 (6)
and cyclin E/Cdk2 (32
, 33)
carry out the transition from G1 to S and
progression in the S phase. These kinases phosphorylate pRb
sequentially, rendering the pRb inactive. Previous reports indicate
that inhibition of cyclin E/Cdk2 and not cyclin D/Cdk4 kinase activity
prevents endoreduplication in HCT116 cells (11)
. Loss of
p21 in these cells by homologous recombination results in persistent
activation of cyclin E/Cdk2 kinase activity and endoreduplication. We
examined the expression of cyclin E, an essential cyclin partner for
activating Cdk2 kinase and cyclin E-associated Cdk2 kinase activity in
nocodazole and nocodazole-plus-flavopiridol-treated MDA-MB-468 cells.
As shown in Fig. 3A
, after the
thymidine double block (ND0), the
expression of cyclin E was 3-fold higher compared to 24 h after
release from the thymidine double block
(ND24). This is in consistent with the
cell cycle data indicating an accumulation of cells at the
G1-S transition by thymidine double block (Refs.
34
and 35
; data not shown). Twenty four h
after the flavopiridol addition, there was a slight decrease in cyclin
E expression compared to the untreated control
(ND24), indicating a loss of some
G1 cells. Similarly, treatment of thymidine
blocked cells with nocodazole for 24 h
(Noc24) resulted in a 2-fold decrease in
cyclin E expression compared to the untreated control
(ND24), indicating a loss of
G1 cells and an accumulation of cells in the M
phase. As the cells entered into the pseudo G1
phase after 30 h
(Noc24Noc6),
the cyclin E levels increased again and remained unchanged thereafter
in nocodazole-treated cells
(Noc24Noc24–72).
Addition of flavopiridol after 24 h of initial nocodazole
treatment resulted in a time-dependent decrease in cyclin E protein
expression (Noc24[Noc +
F]24–72).
|
). In cells treated with nocodazole only, cyclin E/Cdk2
kinase activity was deregulated and persistently active, suggesting
that cells were cycling. Treatment with flavopiridol alone resulted in
an inhibition of cyclin E/Cdk2 kinase activity by 40% compared to the
untreated control. Addition of flavopiridol after 24 h of initial
nocodazole treatment resulted in a time-dependent decrease in kinase
activity. At 72 h after the addition of flavopiridol
(Noc24[Noc +
F]72), 
30% kinase activity was
remaining compared to the untreated control
(ND24). In view of the prevention of
endoreduplication by flavopiridol on nocodazole-treated cells, we
anticipated a greater degree of inhibition in cyclin E/Cdk2 kinase
activity. However, flavopiridol is a competitive inhibitor of Cdks with
respect to ATP and inhibits the kinase activities of Cdks 1, 2, 4, and
6 by directly binding to the ATP-binding site of Cdks (24
, 25)
. As previously reported, during immunoprecipitation,
flavopiridol gets displaced from the ATP-binding site of Cdk2 and
competes with the ATP added during the in vitro assays,
rendering this complex an active kinase during in vitro
kinase assays (24)
. Thus, the actual Cdk2 kinase activity
in intact cells should be substantially less than that observed from
immunoprecipitates of flavopiridol-treated cells. We confirmed this
observation by adding the flavopiridol directly to the Cdk2 kinase
reaction. This resulted in a 500–600% inhibition of kinase activity
(data not shown). This would indicate that the dramatic loss of >4n
DNA content cells and decrease in cyclin E/Cdk2 kinase activity after
the addition of flavopiridol to nocodazole-treated MDA-MB-468 cells
could be by direct binding of flavopiridol to Cdk2 or indirectly due to
decreased availability of cyclin E.
Flavopiridol Treatment Results in Hypophosphorylation of pRb in
Nocodazole-treated HCT116 p21-/- Cells.
Phosphorylation status of pRb dictates the cell cycle clock
(36)
. pRb is found in the hypophosphorylated form in the
preceding hours of G1 and needs to undergo
phosphorylation to initiate the G1-to-S-phase
transition. Previous reports have indicated that MTI-induced
endoreduplication in p21-deficient HCT116 cells is due to persistent
activation of cyclin E/Cdk2 kinase activity. This is associated with
hyperphosphorylated pRb in nocodazole-treated p21-deficient HCT116
cells (11)
. To demonstrate further that flavopiridol
prevents endoreduplication by inhibiting Cdks, the phosphorylation
status of pRb was examined in nocodazole and
nocodazole-plus-flavopiridol-treated p21-deficient HCT116 cells (Fig. 3C
). In untreated (ND24)
and nocodazole-treated (Noc24 and
Noc24Noc6–72)
HCT116 p21-/- cells, pRb remained in the slower migrating
hyperphosphorylated form. Treatment of cells with flavopiridol alone
results in the loss of the hyperphosphorylated band of pRb. After
24–72 h of the addition of flavopiridol to nocodazole-treated cells
(Noc24[Noc +
F]24–72), pRb was mostly present in
its faster migrating hypophosphorylated form. The appearance of the
hypophosphorylated form of pRb in nocodazole-plus-flavopiridol-treated
cells also coincides with arrest or maintenance of cells with 4n DNA
content. This data supports the fact that inhibition of Cdks by
flavopiridol prevents the endoreduplication and polyploidy in
G1 checkpoint-compromised cells.
Flavopiridol Potently Inhibits Cyclin B1/cdc2 Kinase Activity on
Nocodazole-treated MDA-MB-468 Cells.
The activation of cyclin B1/cdc2 kinase is required for the
G2-M transition and progress in mitosis
(37)
. As discussed earlier, the MTI-induced
endoreduplicated cells underwent mitosis, whereas flavopiridol
completely prohibited the entry of 8n cells to mitosis (Fig. 2B
). This would suggest that cyclin B1/cdc2 kinase activity
is persistently active in nocodazole-treated cells, and flavopiridol
inhibits its activity. To evaluate this, MDA-MB-468 cells were either
treated with nocodazole alone or nocodazole plus flavopiridol. In
keeping with the cell cycle data, after 24 h of nocodazole
treatment (Noc24), cyclin B1 protein was
increased 2–3 fold compared to untreated cells
(ND24; Fig. 4A
). At subsequent time points
for nocodazole or nocodazole-plus-flavopiridol-treated cells, the
protein levels of cyclin B1 remained constant (Fig. 4A
).
Flavopiridol alone did not alter cyclin B1 expression substantially. To
evaluate the cyclin B1/cdc2 kinase activity under these conditions in
MDA-MB-468 cells, cyclin B1 was immunoprecipitated, and associated
kinase activity was measured by determining the ability of
immunoprecipitate to phosphorylate histone H1 (Fig. 4B
).
After 24 h of nocodazole treatment
(Noc24), cyclin B1/cdc2 kinase activity
increased by 35-fold compared to control untreated cells
(ND24), which is consistent with the
arrest of cells in mitosis (29)
. Thereafter, with
additional 6 h of nocodazole treatment
(Noc24Noc6),
the kinase activity decreased to 50% of the 24-h nocodazole treatment
(Noc24) as cells exited mitosis and entered
the pseudo G1 state. The cyclin B1/cdc2 kinase
remained active at subsequent time points
(Noc24Noc24–72),
although it decreased to 50–35% of the 24-h nocodazole treatment
alone (Noc24), indicating continuous
recruitment of cells into mitosis. Addition of flavopiridol
dramatically inhibited the kinase activity, and as early as 6 h
after the addition of flavopiridol, a significant 700–800% drop in
kinase activity was observed compared to nocodazole treatment alone at
similar time points (e.g.,.
Noc24[Noc +
F]6 versus
Noc24Noc6).
After 72 h of adding flavopiridol
(Noc24[Noc +
F]72), only 1% of kinase activity
was left compared to the 24-h nocodazole treatment alone
(Noc24), whereas at a similar time point
with nocodazole alone
(Noc24Noc72),
50% kinase activity was remaining (Fig. 4B
). The
decrease in cyclin B1/cdc2 kinase activity in nocodazole followed by
flavopiridol-treated cells
(Noc24[Noc +
F]72) can be due to either direct
binding of flavopiridol to cdc2 or indirectly due to the arrest of the
majority of the cells in a pseudo G1 state, thus
limiting the number of cells entering into the M phase.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In response to MTIs, cells enter, transiently arrest, and exit mitosis without undergoing cytokinesis. The protein expression of p53 and p21 is induced after exit from mitosis and entry in the pseudo G1 phase. The cyclin E/Cdk2 kinase activity is inhibited presumably due to binding of p21 to the cyclin E/Cdk2 complex, which prevents the phosphorylation of pRb and arrests the cells in the pseudo G1 phase. In contrast, cells with a defective G1 checkpoint (MDA-MB-468 and p21-deficient HCT116) endoreduplicate in response to MTIs. This is associated with continuous activation of cyclin E/Cdk2 kinase. Furthermore, the 8n population also undergoes mitosis as determined by positive labeling of 8n cells with MPM-2. The cyclin B1/cdc2 kinase activity was also persistently activated after treatment with MTIs. Addition of flavopiridol to MTI-treated cells dramatically decreases the number of cells containing >4n cells. Although in vitro cyclin E/Cdk2 kinase activity shows a modest decrease in its activity after the flavopiridol treatment, we believe that in intact cells, the decrease in cyclin E/Cdk2 kinase activity is higher because after immunoprecipitation, flavopiridol dissociates from the ATP binding site of Cdk2 and gets washed out during in vitro kinase assays. This has been indicated by adding flavopiridol to the lysates of either untreated or nocodazole-treated MDA-MB-468 and HCT116 p21-/- cells during in vitro kinase assays. Furthermore, the pRb was hypophosphorylated in p21-deficient HCT116 cells after flavopiridol treatment, indicating the stronger inhibition of Cyclin E/Cdk2 kinase activity in intact cells than observed by in vitro kinase assays. The other possibility is that flavopiridol selectively induces apoptosis in 8n cells. This may be only partially true because cell death was 30–40% higher after flavopiridol addition to nocodazole-treated cells compared to nocodazole treatment alone, whereas the decrease in polyploidy was >80% in similar treatment conditions. Even if flavopiridol selectively kills the polyploid cells, it would not negate the importance of a chemical means by which we can selectively remove highly polyploid cells. Recent studies have also shown that flavopiridol transcriptionally down-regulates cyclin D1 and arrests cells in G1 with intact pRb (39) . This would suggest that flavopiridol has the potential to arrest the cells in G1 and prevent polyploidy, in addition to down-regulating cyclin E/Cdk2 kinase activity. The higher in vitro inhibition of cyclin B1/Cdc2 can be due to other cellular events, including alterations in cdc25C, Chk-1, or Wee1 besides direct inhibition. It is also possible that the decrease in cyclin B1/cdc2 kinase activity is due to a decrease in the number of cycling cells that enter into 8n because they are already arrested in 4n.
The formation of tetraploid cells has been indicated as the first step
in the development of aneuploidy (21)
. These cells develop
as a result of missing cell cycle checkpoints that usually control the
coupling between completion of mitosis and DNA synthesis. The formation
of tetraploid and polyploid cells in response to MTIs can also occur in
physiological conditions in vivo. For example, Levine
et al. (21)
have shown that loss of p53 in
transgenic mice expressing SV40 TAg under the control of the elastase I
gene causes mice to develop pancreatic tumors through the
diploidtetraploidpolyploidaneuploid sequence. Tetraploid cells
have also been identified in the development of Barrette’s esophagus
as the preceding step to the aneuploidy (20)
. Our results
demonstrate that experimentally (MTIs) induced polyploidy can be
effectively inhibited by nanomolar concentrations of flavopiridol.
These results provide the foundation for the use of flavopiridol as a
chemopreventive agent. Because the progression to malignancy from
normal cells requires a stepwise progression from polyploidy to
aneuploidy, any intervention at this step may provide the basis to
inhibit malignant transformation. There are presently high risk groups,
including women with Brca1 mutations, who may develop breast cancer.
Recent in vitro data indicate that Brca1 mutations can
result in abnormal mitotic exit, and cells can endoreduplicate if the
G1 checkpoint is not intact due to absence of p53
(40
, 41)
. Oral preparations of flavopiridol are now under
development. Therefore, flavopiridol, by inducing
G1 arrest in cells with a compromised
G1 checkpoint after aberrant mitotic exit, may
provide a convenient means to inhibit progression to malignancy in
specific high risk populations.
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FOOTNOTES |
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1 Supported by a grant (R01CA67819) from the
National Cancer Institute. 
2 To whom requests for reprints should be
addressed, at Memorial Sloan Kettering Cancer Center, 1275 York Avenue,
New York, NY 10021. Phone: (212) 639-8324; Fax: (212) 717-3320; E-mail: schwartg{at}mskcc.org
3 The abbreviations used are: MTI,
microtubule inhibitor; CDKI, cyclin-dependent kinase inhibitor;
Cdk, cyclin-dependent kinase; PI, propidium iodide; DAPI,
4',6'-diamidino-2-phenylindole.
Received 9/ 9/99; revised 11/30/99; accepted 12/ 1/99.
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