Cell Cycle and Biochemical Effects of PD 0183812
A POTENT INHIBITOR OF THE CYCLIN D-DEPENDENT KINASES CDK4 AND
CDK6*
David W.
Fry
,
David C.
Bedford§,
Patricia H.
Harvey
,
Alexandra
Fritsch¶
,
Paul R.
Keller
,
Zhipei
Wu
,
Ellen
Dobrusin
,
Wilbur R.
Leopold
,
Ali
Fattaey
, and
Michelle D.
Garrett§
**
From the
Departments of Cancer Research and
Chemistry, Pfizer Global Research and Development, Ann Arbor
Laboratories, Ann Arbor, Michigan 48105, the
§ CRC Centre for Cancer Therapeutics at the Institute of Cancer
Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom,
¶ Cytokinetics, South San Francisco, California 94080, and
Onyx Pharmaceuticals, Richmond, California 94806
Received for publication, September 28, 2000, and in revised form, January 18, 2001
 |
ABSTRACT |
Progression through the
G1 phase of the cell cycle requires phosphorylation
of the retinoblastoma gene product (pRb) by the cyclin
D-dependent kinases CDK4 and CDK6, whose activity can
specifically be blocked by the CDK inhibitor
p16INK4A. Misregulation of the pRb/cyclin
D/p16INK4A pathway is one of the most common events in
human cancer and has lead to the suggestion that inhibition of cyclin
D-dependent kinase activity may have therapeutic value as
an anticancer treatment. Through screening of a chemical library, we
initially identified the [2,3-d]pyridopyrimidines as inhibitors of
CDK4. Chemical modification resulted in the identification of PD
0183812 as a potent and highly selective inhibitor of both CDK4 and
CDK6 kinase activity, which is competitive with ATP. Flow cytometry
experiments showed that of the cell lines tested, only those expressing
pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a
loss of pRb phosphorylation and a block in proliferation, which was
reversible. These results suggest a potential use of this
chemical class of compounds as therapeutic agents in the treatment of
tumors with functional pRb, possessing cell cycle aberrations in other
members of the pRb/cyclin D/p16INK4A pathway.
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INTRODUCTION |
The cyclin-dependent kinase
(CDK)1 family of
serine/threonine protein kinases are the cornerstone of the cell
division cycle. Full CDK activity requires association with a cyclin
regulatory subunit, and it is the distinct combinations of CDK-cyclin
complexes that control progression through the different phases of the
cell cycle (1). During early to mid G1, when the cell is
responsive to mitogenic stimuli, it is CDK4 and CDK6 in association
with the D-type cyclins that control advancement through the cell
cycle, by their phosphorylation of the tumor suppressor protein, pRb (2). The cyclin D-dependent kinases also act as integrators of extracellular stimuli. For example, induction of cyclin D1 transcription can be activated by the ras signaling pathway (3, 4),
whereas degradation of cyclin D1 protein is stimulated by the
serine/threonine kinase GSK-3
, which is itself regulated by the
phosphatidylinositol 3-kinase and Wnt signaling pathways (5, 6).
The importance of the cyclin D-dependent kinases and pRb in
controlling transit through G1 is further reflected by the
fact this pathway is a major target of genetic alteration in cancer. This is exemplified by the RB gene itself, which is found
mutated in a number of tumor types, and cyclin D1, which is
overexpressed in a variety of tumors (7, 8). Another major player in
this pathway is the tumor suppressor gene INK4A/CDKN2A
encoding the p16INK4A protein, which specifically inhibits
the kinase activity of the two cyclin D-dependent kinases
CDK4 and CDK6 and is found genetically altered in multiple tumor types
(9). In a number of cases, the pattern of RB mutation
appears to inversely correlate with mutations of
INK4A/CDKN2A, suggesting that a single defect of the
pRb/cyclin D/p16INK4A pathway is enough for tumor
development (10). The intensity with which this pathway appears to be
misregulated in cancer has lead to the suggestion that in situations
where cyclin D-dependent kinase activity may be
up-regulated such as by mutation of INK4A/CDKN2A or
overexpression of cyclin D1, inhibiting the kinase activity of both
CDK4 and CDK6 may be an effective anticancer treatment. Evidence for
the validity of such an approach has been provided in a number of ways.
Reintroduction of INK4A/CDKN2A into tumor cells where this
gene is defective will inhibit cell growth in culture and tumor growth
in xenograft studies, whereas a peptide mimic of p16INK4A
has demonstrated antiproliferative properties in a number of cell lines
(11-14). Interestingly, reintroduction of p16INK4A
activity into tumor lines only blocks proliferation in cells where pRb
is active (15, 16). Thus, inhibition of CDK4 and CDK6 kinase activity
may only be a useful anticancer treatment in tumors where pRb is
functional, but other members of the pathway such as
p16INK4A or possibly cyclin D1 have aberrant functions.
Further support for this model has come from studies using antisense
cDNA technology and an antibody to block cyclin D1 function, which
showed that loss of cyclin D1 action only blocks proliferation in cells
with active pRb (17, 18). Expression of antisense cDNA has also been used to show that loss of cyclin D1 function will also block pRb
phosphorylation and inhibit the growth and tumorigenicity of colon
cancer cells (19). These data provide a strong rationale for inhibition
of cyclin D-dependent kinase activity as an approach to
cancer chemotherapy in tumor cells where pRb is functional, but the
activity of other members of the pRb/cyclin D/p16INK4A
pathway has been altered.
Screening efforts have recognized the [2,3-d]pyridopyrimidines as
inhibitors of CDK4 (20). Here we describe the identification of one
such compound PD 0183812 as a potent and specific inhibitor in
vitro of the cyclin D-dependent kinases CDK4 and CDK6
(Fig. 1). This study characterizes the
biochemical properties of PD 0183812 and provides evidence that the
biological effects of this compound are consistent with cyclin
D-dependent kinase inhibition.
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EXPERIMENTAL PROCEDURES |
Kinase Assays--
CDK assays for IC50
determinations and kinetic evaluation were performed in 96-well filter
plates (Millipore, MADVN6550). All CDK-cyclin kinase complexes were
expressed in insect cells through baculovirus infection and purified as
described previously (21). The substrate for the assays was a fragment
(amino acids 792-928) of pRb fused to GST (GST·RB-Cterm, Ref. 22).
The total volume for each well was 0.1 ml containing a final
concentration of 20 mM Tris-HCl, pH 7.4, 50 mM
NaCl, 1 mM dithiothreitol, 10 mM
MgCl2, 25 µM ATP (for CDK4-cyclin D1,
CDK6-cyclin D2, and CDK6-cyclin D3) or 12 µM ATP (for
CDK2-cyclin E, CDK2-cyclin A, and CDC2-cyclin B) containing 0.25 µCi
of [
-32P]ATP, 20 ng of enzyme, 1 µg of
GST·RB-Cterm, and appropriate dilutions of inhibitor. All components
except the [
-32P]ATP were added to the wells, and the
plate was placed on a plate mixer for 2 min. The reaction was then
started by adding the [
-32P]ATP, and the plate was
incubated at 25 °C for 15 min. The reaction was terminated by
addition of 0.1 ml of 20% trichloroacetic acid, and the plate was kept
at 4 °C for at least 1 h to allow the substrate to precipitate.
The wells were then washed five times with 0.2 ml of 10%
trichloroacetic acid, and radioactive incorporation was determined with
a
plate counter (Wallac Inc., Gaithersburg, MD). Kinase assays for
PDGFr, FGFr, EGFr, SRC, and PKC kinases were performed as described
previously (23, 24).
Cell Culture and Cell Counting--
All cell lines were
maintained at 37 °C, 5% CO2 in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum (Life Technologies,
Inc). For cell counts, cells were trypsinized and counted using a
Coulter Z2 particle count and size analyzer.
Thymidine Incorporation into DNA--
Cells were seeded at
2 × 104 per well in a 96-well Cytostar T plate
(Amersham Pharmacia Biotech) and incubated overnight. Varying concentrations of PD 0183812 were added to the wells and incubated for
24 h at 37 °C. 0.1 µCi of [14C]thymidine was
added to each well, and incorporation of the radiolabel was allowed to
proceed for 48 h. Incorporated radioactivity was determined with a
plate counter.
Flow Cytometry--
Cells were washed and harvested using
phosphate-buffered saline (PBS) containing 5 mM EDTA. Cells
were then washed in PBS containing 1% FBS (1% FBS/PBS), fixed in 85%
ethanol and stored at 4 °C for at least 16 h and up to 5 days
before staining with propidium iodide (PI). For PI staining, cells were
washed in 1% FBS/PBS prior to incubation at 37 °C for 30 min in the
same solution containing 40 µg/ml PI (Molecular Probes) and 250 µg/ml RNase A (Roche Diagnostics Ltd.). Data were collected using a
Coulter EPICS Elite ESP equipped with a Spectraphysics argon-ion laser and analyzed using the WinMDI program, version 2.8. Results represent a
minimum of 15,000 cells assayed for each sample.
Cell Lysis, Western Blot Analysis, and Antibodies--
Cells
were trypsinized and 200 µl of each sample was counted in 19.8 ml of
PBS using a Coulter Z2 particle count and size analyzer. The rest of
each sample was washed once with ice-cold PBS and lysed on ice for 30 min, in lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 0.1% Nonidet P-40) containing 1 mM
DTT, 1 mM EDTA, 1 mM NaF, 10 mM
-glycerophosphate, 0.1 mM sodium orthovanadate, and a
complete mini EDTA-free protease inhibitor mixture tablet per 10 ml of
lysis buffer (Roche Diagnostics Corp). The volume of lysis buffer used
was 100 µl per 1 × 106 cells. Following lysis,
samples were clarified by centrifugation at 18,000 × g
for 10 min at 4 °C, and protein concentrations were determined using
the Bradford assay (25). Proteins were resolved by SDS-polyacrylamide
gel electrophoresis using 6% polyacrylamide gels (10 µl of cell
lysate per gel lane) and electroblotted onto ImmobilonTM-P
membranes (Millipore, Bedford, MA). Membranes were blocked overnight at
4 °C in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl,
and 0.1% Tween 20 containing 3% casein and then incubated in the same buffer with primary antibodies for 3 h at room temperature
followed by a 1-h incubation with a horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody (1:10,000 dilution, Bio-Rad).
Immunodetection was carried out using enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech). For Western analysis, pRb was detected
using the Rb C-terminal control antibody from Cell Signaling Technology at a dilution of 1:2000. The phosphorylation status of pRb at serine
780 was detected with the phospho-Rb (Ser-780) antibody (at a dilution
of 1:4000) also from Cell Signaling Technology.
 |
RESULTS |
Identification of PD 0183812 As a Potent and Specific Inhibitor of
the Cyclin D-dependent Kinases CDK4 and CDK6--
We have
recently identified the [2,3-d]pyridopyrimidines as inhibitors of
CDK4 (20). From this series we identified PD 0183812 as an extremely
potent inhibitor of CDK4-cyclin D1 with an IC50 value of
0.008 µM (Table I). Similar
IC50 values of 0.0071 and 0.013 µM were also
measured for inhibition of CDK6-cyclin D2 and CDK6-cyclin D3
respectively (Table I). In contrast, the non-cyclin D-dependent kinases CDK2 and CDC2 exhibited considerably
higher IC50 values for inhibition by PD 0183812 (Table I).
Specifically, selectivity ratios of >500-, 26-, and 20-fold were
obtained for CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E,
respectively versus CDK4-cyclin D1. PD 0183812 had very
little inhibitory activity against other protein kinases including
PDGFr, FGFr, EGFr, SRC, and PKC (Table I). Kinetic analysis shows that
PD 0183812 exhibited pure competitive inhibition with respect to ATP as
indicated by the double reciprocal plot of ATP concentration
versus catalytic activity of CDK4-cyclin D1 (Fig.
2). Thus, PD 0183812 is a potent and
selective ATP competitive inhibitor of the cyclin
D-dependent kinases CDK4 and CDK6.
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Table I
PD 0183812 is a selective inhibitor of cyclin D-dependent
kinases
Assays were carried out as described under "Experimental
Procedures."
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Fig. 2.
Double reciprocal plot of CDK4-cyclin D1
activity in the presence of PD 0183812 as a function of ATP.
CDK4-cyclin D1 activity was assayed as described under "Experimental
Procedures." Inhibitor concentrations were 0 nM
(filled circles), 10 nM (empty
circles), 20 nM (filled triangles) and 40 nM (empty triangles).
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PD 0183812 Produces a pRb-specific G1 Arrest
in Human Tumor Lines That Correlates with Inhibition of DNA
Synthesis--
As outlined in the Introduction, the major known
cellular target of the cyclin D-dependent kinases is pRb
and phosphorylation of this protein is required for progression through
G1. Therefore a compound that can inhibit the cellular
activity of both CDK4 and CDK6 in tumor cells expressing functional
pRb, but where another member of the pRb/cyclin D/p16INK4A
pathway is altered would be predicted to invoke a G1
arrest. In tumor cells in which pRb is not present or not functional
and where the pRb/cyclin D/p16INK4A pathway is not required
for G1 progression, treatment with such an inhibitor would
not be expected to cause an arrest in G1. To test the
ability of PD 0183812 to behave in this manner, we treated three human
carcinoma cell lines that express pRb but not p16INK4A,
(MDA-MB-453, breast carcinoma; H1299, lung carcinoma; U251, glioblastoma; data not shown; Refs. 26-28) and three cell lines that
do not express pRb, but do express p16INK4A (MDA-MB-468,
breast carcinoma; H2O09, lung carcinoma; SF539, glioblastoma; data not
shown; Refs. 26, 28, 29), for 24 h with 0.1-1.5 µM
PD 0183812. In all three cell lines that express pRb, a significant
increase in the G1 fraction and a concomitant decrease in
the S and G2/M fractions was observed when cells were treated with between 0.2 and 0.8 µM PD 0183812 (Fig.
3). At higher concentrations, a
significant increase in G2/M was seen in the H1299 and U251
tumor lines. No increase in G1 was noted between 0.2 and
0.8 µM for the cell lines not expressing pRb (Fig. 3). However a strong increase in G2/M starting at 0.8 µM for all three of these cell lines was noted. From
these results, we conclude that in the tumor lines tested, PD 0183812 induces a pRb-specific G1 arrest indicative of cyclin
D-dependent kinase inhibition. However, high concentrations
of the drug cause a pRb-independent increase in
G2/M.

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Fig. 3.
Cell cycle analysis of cell lines treated for
24 h with a titration of PD 0183812. Each cell line
(exponentially growing) was treated for 24 h with 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 µM PD 0183812. Cells were then
harvested, stained with propidium iodide and analyzed for DNA content
as described under "Experimental Procedures." Each graph
shows the percent of the stained population in G1
(filled circles), S (empty circles), and
G2/M (filled squares) phases of the cell cycle.
The name of the cell line is given above each
graph.
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We next went on to address whether the pRb-specific G1
arrest caused by PD 0183812 correlated with a block in DNA synthesis as
measured by thymidine incorporation. To do this we selected the two
breast cancer cell lines MDA-MB-453 and MDA-MB-468. Each cell line was
exposed to varying concentrations of PD 0183812 for 24 h, at which
point the amount of thymidine incorporated by all the samples over the
next 48 h in the presence of PD 0183812 was measured (Fig.
4). From this graph, the IC50
of PD 0183812 for inhibition of DNA synthesis was calculated and found
to be 0.32 µM for the MDA-MB-453 cell line, which
expresses pRb, and >3.0 µM for the MDA-MB-468 cell line,
which does not. For the MDA-MB-453 cell line, this is similar to the
concentration range of the compound (0.2-0.8 µM), which
causes a G1 arrest (Fig. 4). For the MDA-MB-468 cell line,
treatment of the cells with <1.0 µM of PD 0183812 had no
significant effect on DNA synthesis. Thus treatment of tumor cell lines
with PD 0183812 causes a pRb-specific G1 arrest, which
correlates with a block in DNA synthesis.

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Fig. 4.
PD 0183812 inhibits DNA synthesis in cell
culture. Exponentially growing cultures of MDA-MB-468
(filled circles) and MDA-MB-453 (empty circles)
cells were treated for 24 h with 0, 0.037, 0.11, 0.33, 1, or 3 µM PD 0183812. [14C]thymidine was then
added to each well and incorporation of the radiolabel was allowed to
proceed for 48 h. DNA synthesis is defined as the amount of
thymidine incorporated into the DNA over this time period, as a
percentage of the Me2SO control.
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PD 0183812 Blocks Phosphorylation on pRb in Tumor
Cells--
Because we had shown that PD 0183812 is a cyclin
D-dependent kinase selective inhibitor in vitro
and causes a pRb-specific G1 arrest in human tumor cells,
we went on to investigate whether this compound could also block the
in vivo phosphorylation of pRb, the cellular substrate of
the cyclin D-dependent kinases. For this experiment we
again used the human breast carcinoma cell line MDA-MB-453. Cells were
treated with 0.1-0.9 µM PD 0183812. Untreated cells or
cells treated with the drug vehicle, Me2SO, were
included as controls. Mimosine, a plant amino acid often used to
synchronize cells in G1, was incorporated into the
experiment as a drug that is known to cause a G1 arrest
(30). Expression and phosphorylation of pRb was monitored by Western
blot analysis using two antibodies, one for detection of pRb expression
and a second for detection of phosphorylation at serine 780 of pRb (P-Ser-780Rb). Phosphorylation at serine 780 on pRb was investigated as
it has been reported that CDK4 can specifically phosphorylate this site
in vitro and that serine 780 is phosphorylated in mid G1 when both CDK4 and CDK6 but not CDK2 are active (22, 31, 32). In addition, it has been shown that immunoprecipitates of the
transcription factor E2F1, which associates with pRb, contain no serine
780-phosphorylated pRb, indicating that phosphorylation of this site
may be involved in disruption of the E2F1·pRb complex (31). FACS
analysis was also carried out on a portion of each sample so that the
level of pRb phosphorylation could be compared side by side with the
fraction of cells in the G1 phase of the cell cycle. From
these studies, we found that whereas the untreated or
Me2SO-treated cells possessed mainly hyperphosphorylated
pRb, with strong phosphorylation at serine 780, samples treated with 0.3-0.9 µM PD 0183812 showed an increase in the
G1 fraction of the cells (peaking at 0.5 µM),
which correlated with loss of serine 780 phosphorylation on pRb (Fig.
5). We also noted that there was no
significant increase in the sub-G1 population of cells, which is often used as an indicator of apoptosis (33). Interestingly, the cells treated with mimosine showed an increase in their
G1 population equivalent to treatment with 0.3 µM PD 0183812, but there was no significant loss of pRb
phosphorylation. Together, these data suggest that treatment of
pRb-expressing tumor cells with PD 0183812 causes a G1
arrest, which coincides with loss of pRb phosphorylation at serine 780, indicating inhibition of cyclin D-dependent kinase
activity.

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Fig. 5.
Effect of PD 0183812 on pRb
phosphorylation. Cells were treated with either medium alone
(C1), or medium containing Me2SO
(C2), 1 mM mimosine (M), or 0.1-0.9
µM PD 0183812 for 24 h and harvested, and samples
were subjected to SDS-polyacrylamide gel electrophoresis on 6% gels
and Western blotted for detection of pRb expression (Rb),
phosphorylation at serine 780 of pRb (P-Ser780Rb), and
propidium iodide staining followed by analysis of DNA content by flow
cytometry as described under "Experimental Procedures." The
percentage of the total cell population with either sub-G1,
G1, S, or G2/M DNA content is shown
below the Western for each cellular condition.
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PD 0183812 Inhibits Cell Proliferation--
To investigate the
long term effects of PD 0183812 on cell proliferation, we treated the
MDA-MB-453 cell line with either Me2SO (vehicle control) or
0.5 µM PD 0183812, a concentration of the drug, which we
have shown will cause a G1 block and a loss of pRb
phosphorylation (Fig. 5). We then took samples for cell counts, and
examination of pRb phosphorylation by Western blot at 0-5 days after
drug addition. It should be noted that medium with either drug or
Me2SO was only added once to the cells (on day 0) and was
not replaced during the experiment. Looking at the cell counts between
1 and 5 days after treatment, it was clear that there was a significant
block in cell proliferation (Fig. 6A), which correlated with
loss of pRb phosphorylation (Fig. 6B). It should be noted
that the control cells ceased to grow after day 4, which may have been
because of their confluency and exhaustion of nutrients in the cell
medium. Thus, treatment of pRb expressing tumor cells with PD 0183812 causes a cytostatic block in proliferation which is maintained for at
least 5 days and correlates with a loss of pRb phosphorylation at
serine 780 indicative of cyclin D-dependent kinase
inhibition.

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Fig. 6.
PD 0183812 inhibits cell proliferation in
tissue culture. Exponentially growing MDA-MB-453 cells were
treated with either 0.5 µM PD 0183812 or
Me2SO on day 0. This medium remained on the cells for the
duration of the experiment and was not changed. A, cell
counts were carried out on each day of the experiment. Empty
circles represent the Me2SO control and filled
circles represent PD 0183812 treated cells. B, samples
were taken each day of Me2SO ( )- and PD 0183812 (+)-treated cells for detection by Western blot on 6% gels of pRb
expression (Rb) and phosphorylation at serine 780 of pRb
(P-Ser780Rb).
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The Block in Cell Proliferation Caused by PD 0183812 Is
Reversible--
The final question addressed in this study is whether
the block in tumor cell proliferation induced by PD 0183812 is
reversible. For this experiment the MDA-MB-453 cells were exposed to
either Me2SO (vehicle control) or 0.5 µM PD
0183812 for 24 h. After 24 h (day 1) the medium was removed,
and the cells were washed. Fresh medium containing Me2SO
was added to the Me2SO-treated cells. Fresh medium
containing 0.5 µM PD 0183812 was added to half the PD
0183812-treated cells, whereas the other half were treated with medium
containing Me2SO. All medium was changed at day 3 to try
and counteract nutrient exhaustion, especially where cells were
proliferating for the length of the experiment. Samples were then taken
for cell counts and detection by Western blot of pRb expression and
phosphorylation at serine 780. It is clear from the cell counts (Fig.
7A), that PD 0183812 causes a
sustained block in proliferation. This result is reflected in the
Western blot data, which show that phosphorylation at serine 780 of pRb is low in the cells treated with PD 0183812 for the first 5 days of the
experiment, compared with the Me2SO-treated cells (Fig. 7B). By day 6, the Me2SO-treated cells have
stopped growing, and pRb phosphorylation at serine 780 is low. As in
the previous experiment, this may be because of the confluency of the
cells and nutrient exhaustion of the medium. If however, the compound
is removed on day 1, 24 h after addition, it appears that the
cells recover and resume proliferation (Fig. 7A). It must be
noted that there does appear to be a lag in resuming proliferation
because cell counts on days 2-4 were still low. In comparison, the
Western blot data from this experiment (Fig. 7B) show that
if the compound is removed on day 1 the amount of pRb phosphorylation
on serine 780 on days 2-5 returns to the level seen for the vehicle
control-treated cells. Therefore PD 0183812 causes a block in
proliferation, which is reversible when the compound is removed 24 h after addition and is accompanied by a return of phosphorylation on
serine 780 of pRb.

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Fig. 7.
The block in cell proliferation caused by PD
0183812 is reversible. Exponentially growing MDA-MB-453 cells were
treated with either Me2SO or 0.5 µM PD
0183812 for 24 h. After 24 h (day 1), the medium was removed,
and the cells were washed. Fresh medium containing Me2SO
was added to the Me2SO-treated cells. Fresh medium
containing 0.5 µM PD 0183812 was added to half the PD
0183812-treated cells, whereas the other half were treated with medium
containing Me2SO. At day 3, all the cells had their medium
changed, and either Me2SO or PD 0183812 was added as on day
1. A, cell counts were carried out on each day of the
experiment. Empty circles represent cells treated with
Me2SO, filled circles represent cells treated
continually with 0.5 µM PD 0183812, and filled
squares represent cells treated for 24 h with 0.5 µM PD 0183812, followed by 5 days of Me2SO.
B, samples were taken each day of Me2SO-treated
cells ( ), cells treated continually with 0.5 µM PD
0183812 (+), and cells treated for 24 h with 0.5 µM
PD 0183812. They were then washed and treated with Me2SO
for 5 days (w) and subjected to Western blot analysis on 6%
gels for detection of total pRb (Rb) and pRb phosphorylation
at serine 780 (P-Ser780).
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DISCUSSION |
Within the last 5 years, enormous progress has been made in
deciphering the mechanism by which mammalian cells regulate and progress through the cell cycle. Clearly CDKs have emerged as key
regulators of cell cycle progression, and in fact movement either
proceeds or stops based on the catalytic activity of these enzymes.
CDKs have also been some of the more intensely studied enzymes in terms
of potential targets in cancer chemotherapy. The cyclin
D-dependent kinases have been of special interest because of the exceptionally high percentage of tumors that exhibit
abnormalities in the pRb/cyclin D/p16INK4A pathway. In
particular the fact that tumors that have functional pRb often harbor
an alteration in another member of this pathway, such as a defect in
the cyclin D-dependent kinase inhibitor
p16INK4A, has led to the suggestion that inhibition of
cyclin D-dependent kinase activity in these tumors may have
therapeutic value. We therefore set out to identify inhibitors of
cyclin D-dependent kinase activity by initially screening
for inhibitors of CDK4. Through this effort we recognized the
[2,3-d]pyridopyrimidines as inhibitors of this enzyme (20).
Subsequent chemical modification lead to the identification of PD
0183812 as a compound with unprecedented potency in vitro
against both the cyclin D-dependent kinases CDK4 and CDK6.
Investigation into the cellular behavior of PD 0183812 revealed that
this compound gave a pRb dependent G1 cell cycle arrest. This result is indicative of cyclin D-dependent kinase
inhibition, as this activity has previously been shown to be required
for pRb-dependent G1 progression (15, 16). The
concentration of PD 0183812 required to give this pRb-specific
G1 arrest (0.5 µM in MDA-MB-453 cells) also
caused a loss of phosphorylation on pRb at serine 780, a site
previously reported to be targeted by CDK4 for phosphorylation and
correlated with its IC50 for inhibition of DNA synthesis
(31). Together these data suggest that the cellular target of PD
0183812, which gives a pRb-specific G1 arrest in human
tumor cells, is the cyclin D-dependent kinases. At higher concentrations however, PD 0183812 caused a significant increase in the
G2/M population of cells in a non-pRb-dependent
manner. There are a number of reasons why this may have occurred. One option is that at high concentrations, PD 0183812 may be inhibiting other kinases (including other CDKs) in the cell that are rate-limiting at G2/M but not G1. A second possibility is
that a cyclin D-dependent kinase (but pRb-independent) step
may be rate-limiting in G2/M at these concentrations of the
drug. There is some evidence that CDK4 may play a role in the
G2/M transition and so for the moment, this possibility
cannot be ruled out (34).
Finally we come to the effect of PD 0183812 on cellular proliferation.
From the studies presented here it is clear that one treatment of 0.5 µM PD 0183812 is sufficient to produce a G1
arrest, loss of phosphorylation on pRb at serine 780, and significantly inhibit growth for at least 5 days. This is an extremely interesting result as there has been some discussion as to whether a catalytic inhibitor of CDK4 would be sufficient to arrest cycling cells (35). One
reason for this, was the discovery that expression of
p16INK4A, but not a dominant-negative form of CDK4 will
cause a cell cycle arrest (36). This may be because of the fact that
p16INK4A expression results in redistribution of the CDK
inhibitor p27KIP1 within the cell and inhibition of CDK2 as
well as CDK4 (36). With PD 0183812 we also found that removal from the
cells after 24 h allowed rephosphorylation of pRb and an eventual
resumption of proliferation. Thus at this stage the effects of PD
0183812 are reversible. It is interesting to note that expression of
p16INK4A is associated with replicative senescence, and it
will be of interest to investigate the long term effects of PD 0183812 on tumor cells (37).
In recent years a number of CDK inhibitors have been disclosed, which
have a wide variety of potencies and specificities (38). Many of these
existing compounds such as the purines, olomoucine and roscovitine, and
the non-purines, butyrolactone and kenpaullone, show considerable
activity against CDK2 and CDC2, but little activity against CDK4
(39-40). The flavone flavopiridol also known as L86-8275 will inhibit
CDK4 but with a similar potency to CDK2 and CDC2, and there is building
evidence that it may have other cellular targets including aldehyde
dehydrogenase, glycogen phosphorylase, and duplex DNA (39, 41-43).
Thus potent and selective inhibitors of the cyclin
D-dependent kinases CDK4 and CDK6 have been elusive.
Recently however, there have been several reports of compounds that do
inhibit CDK4-cyclin D1 in vitro and show selective growth
inhibition of tumor cells with functional pRb or defective p16INK4A (28, 44, 45) These reports therefore reinforce the
gathering evidence that the identification of pharmacological
inhibitors of cyclin D-dependent kinases could allow the
development of a novel therapeutic approach to the treatment of
patients with tumors where the retinoblastoma gene is still functional
but other members of the pRb/cyclin D/p16INK4A pathway are
defective. With PD 0183812, we have identified the most potent
CDK4-cyclin D1 inhibitor to date. We have also shown that PD 0183812 can inhibit CDK6-cyclin D2 and CDK6-cyclin D3 with a similar potency to
CDK4-cyclin D1. Therapeutically this is an important point because
tumors that, for example, have functional pRb but defective
p16INK4A and express CDK6 as well as CDK4 along with one or
more of the D-type cyclins, may require inhibition of both these CDKs
for therapeutic value. PD 0183812 would be capable of this whereas in
contrast the recently discovered CDK4-cyclin D1 inhibitor fascaplysin would not, because it specifically targets this form of the cyclin D-dependent kinases (45). Thus the identification of PD
0183812 as a small molecule inhibitor of CDK4 and CDK6 provides us with an important tool to investigate the cellular processes in which the
cyclin D-dependent kinases participate and a chemical lead in the development of a chemotherapeutic agent for the treatment of
genetically characterized tumors.
 |
ACKNOWLEDGEMENTS |
We would like to thank everyone who has
worked on the CDK inhibitor project at both Onyx Pharmaceuticals and at
Pfizer Global Research and Development, Ann Arbor Laboratories. We
would also like to thank J. Titley and M. Quinn for their assistance
with the FACS analysis.
 |
FOOTNOTES |
*
This study was supported by the UK Cancer Research Campaign
(CRC) and the Institute of Cancer Research, UK (to M. D. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed. Tel.: 44-208-722-4352;
Fax: 44-208-770-7899; E-mail: mgarrett@icr.ac.uk.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M008867200
 |
ABBREVIATIONS |
The abbreviations used are:
CDK, cyclin-dependent kinase;
PBS, phosphate-buffered saline;
FBS, fetal bovine serum;
PDGFr, platelet-derived growth factor
receptor;
FGFr, fibroblast growth factor receptor;
EGFr, epidermal
growth factor receptor;
SRC, p60src;
PKC, protein kinase C;
pRb, retinoblastoma gene product;
FACS, fluorescence-activated cell
sorter.
 |
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