©2006 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
grant CA 39930 from the National Cancer
Institute (to R.C.B.).
normally with limited phenotypes (Refs. 7, 8, and Refs. within the reviews). These mouse
knockout experiments suggest that each of the G1regulators as an individual entity is not
essential for cell cycle regulation. Double or triple knockout of D-type cyclins (D1, D2,
D3), double knockout of E-type cyclins (E1 and E2), or double knockout of CDK4 and
CDK6 are lethal for mice.8These data suggest that cyclins and CDKs in the G1phase
when acting together do play the important roles in regulating the cell cycle.
[Cell Cycle 5:15, 1654-1661, 1 August 2006]; ©2006 Landes Bioscience
1654Cell Cycle 2006; Vol. 5 Issue 15
Robert C. Bast Jr.1,*
Departments of 1Experimental Therapeutics, 2Surgical Oncology, 3Experimental
Radiation Oncology and 4Molecular & Cellular Biology; University of Texas MD
Anderson Cancer Center; Houston, Texas USA
5Department of Biomedical Genetics; University of Rochester Medical Center; New
*Correspondencce to: Xiao-Feng Le; Department of Experimental Therapeutics;
Unit 354; The University of Texas MD Anderson Cancer Center; 1515 Holcombe
Boulevard; Houston, Texas 77030-4009 USA; Tel.: 713.745.4353; Fax:
713.745.2107; Email: firstname.lastname@example.org/ Robert C. Blast; Department of
Experimental Therapeutics; Unit 354; The University of Texas MD Anderson
Cancer Center; 1515 Holcombe Boulevard; Houston, Texas 77030-4009 USA; Tel.:
713.792.7743; Fax: 713.792.7864; Email: email@example.com
Original manuscript submitted: 05/26/06
Manuscript accepted: 06/08/06
Previously published online as a CellCycleE-publication:
HER2/c-neu, trastuzumab, cell cycle, CDK2,
We sincerely thank Amy Lammayot and
Xianfeng Wen for the technical assistance. The
Media Preparation Core Facility was supported
in part by the NCI Cancer Center Core Grant
CA #16672. We are grateful to Genentech,
Inc. (South San Francisco, CA) for providing
the trastuzumab (Herceptin®) and 4D5 anti-
bodies. This work was supported in part by
Anti-HER2 Antibody Trastuzumab Inhibits CDK2-Mediated NPAT
and Histone H4 Expression via the PI3K Pathway
The anti-HER2 antibody trastuzumab (Herceptin®) has been used to treat patients with
breast cancers that overexpress HER2. We have demonstrated that p27Kip1upregulation
is one of the key events that cause G1arrest upon trastuzumab treatment. Here, we have
examined the effect of trastuzumab on expression of CDK2, Rb, E2F, NPAT and histone
H4 in breast cancer cells that overexpress HER2. Trastuzumab treatment dramatically
inhibited the kinase activity and expression of CDK2, whereas the kinase activity and
expression of CDK4 were not affected. Unlike the p27Kip1upregulation that occurs
primarily through post-translational mechanisms, CDK2 was downregulated primarily at
a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. With
a decrease in CDK2 activity, trastuzumab decreased the kinase activity of cyclin E but
had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower
molecular weight forms did not influence the response to trastuzumab. Levels and activities
of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result,
trastuzumab inhibited Rb phosphorylation that associates with CDK2, cyclin E, CDK6,
cyclin A, or cyclin D1. As predicted from these changes, trastuzumab decreased the
DNA-binding activity of E2F, decreased the level of NPAT protein, and decreased the
level of histone H4 mRNA. Blockade of the PI3K pathway with LY294002 produced
similar effects to trastuzumab treatment on expression of each of these genes. Taken
together, treatment of breast cancer cells that overexpress HER2 with the anti-HER2 antibody
trastuzumab inhibits CDK2, Rb phosphorylation, E2F activity, NPAT, and histone H4 via
PI3K signaling that are needed for both DNA and histone synthesis during progression
from G1phase to S phase of the cell cycle.
In mammalian cells, proliferation is controlled at two main checkpoints. The first
checkpoint is at the G1-S phase transition limiting the initiation and completion of DNA
replication in S phase. The second checkpoint is at the G2-M phase transition controlling
mitosis and cell division.1CDK4 or CDK6 in combination with cyclin D and CDK2 in
association with cyclin E play key roles in regulating G1-S progression. Elevation of
cyclin E/CDK2 activity in G1phase causes premature entry into S phase.2,3Inhibition of
cyclin E/CDK2 activity inhibits entry into and progression through S phase.3,4Initiation
of S phase depends on activation of a number of biosynthetic processes, including DNA
synthesis, histone synthesis, and chromatin assembly.5Both cyclin D/CDK4 or /CDK6
and cyclin E/CDK2 phosphorylate the retinoblastoma protein (Rb), a well-known tumor
suppressor. This tumor suppressor activity relies on Rb role in gating S phase entry
through its ability to repress genes activated by the E2F family of transcription factors.
Phosphorylation of Rb releases members of the E2F family that play an integral role in cell
cycle progression by inducing the expression of gene required for S phase entry.6
Cyclin D/CDK4, cyclin D/CDK6, and cyclin E/CDK2 complexes promote entry into
the cell cycle and cell cycle progression of normal, nontransformed cells, and cancer cells.
However, mice deficient in one of the three D-type cyclins, one of the two E-type cyclins,
cyclin D-dependent CDK4 and CDK6, or cyclin E-dependent CDK2 surprisingly survive
According to current models, the CDK2/cyclin E complex can
regulate DNA and histone synthesis in the S phase of the cell cycle
through activation of E2F family members and of the nuclear
protein mapped to the ATM locus (NPAT or p220).9-11Through
cyclin E/CDK2, histone synthesis coordinates tightly with cell
cycle-dependent DNA replication in both physiological and patho-
logical conditions.12NPAT, as the substrate of cyclin E/ CDK2,
localizes in nuclear organelles called Cajal bodies in a cell cycle-
regulated manner.10,12CDK2-dependent NPAT promotes histone
H2A and H4 reporter gene expression through cell cycle-regulated
cis elements within their promoters.12-14NPAT is also regulated by
E2F, whereas histone H4 genes are not directly regulated by
E2F.12,15Increased DNA replication and histone synthesis promote
the progression through S phase.
Anti-HER2 antibody trastuzumab (Herceptin®) alone and in
combination with chemotherapy has been used to treat patients with
breast cancers that overexpress HER2. However, the molecular
mechanisms by which trastuzumab inhibits tumor cancer growth
and potentiates chemotherapy are still not completely understood.
We have demonstrated that trastuzumab suppresses cancer growth
and induces G1arrest primarily through induction of a cell cycle G1
arrest16and upregulation of the CDK inhibitor p27Kip1.17,18Here
we report a novel mechanism by which trastuzumab acts through the
PI3K pathway to inhibits CDK2/cyclin E-dependent expression of
NPAT and histone H4.
MATERIALS AND METHODS
Cell culture. The human breast cancer cell lines SKBr3 and
BT474 were obtained from the American Type Culture Collection
(ATCC, Manassas, VA). SKBr3 cells were grown in complete medium
that contained RPMI 1640 supplemented with 10% fetal bovine
serum (FBS) (Sigma, St. Louis, MO), 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin in humidified air with
5% CO2at 37˚C. BT474 cells were grown in complete medium
containing DMEM supplemented with 10% FBS, 2 mM L-gluta-
mine, 1 mM sodium pyruvate (Sigma), 100 units/ml penicillin, and
100 µg/ml streptomycin. For all experiments, cells were detached
with 0.25% trypsin-0.02% EDTA. For cell culture, 3 x 105expo-
nentially growing cells were plated into 100-mm tissue culture dishes
or 3 x 103into 96-well plates in complete medium. After culture
overnight in complete medium, cells were treated with the anti-
HER2 antibody 4D5 at 5~10 µg/ml for SKBr3 or trastuzumab at
10 µg/ml for BT474 in complete medium at 37˚C for 24 hr (SKBr3)
or 48 hr (BT474). Monoclonal antibody MOPC21 (5–10 µg/ml)
served as a control for 4D5 with SKBr3 cells and human immuno-
globin G (hIgG) (10 µg/ml) served as a control for trastuzumab with
Reagents. Anti-HER2 murine monoclonal antibody 4D5 and
humanized monoclonal antibody trastuzumab (Herceptin®) were
provided by Genentech (South San Francisco, CA). MOPC21
murine myeloma cells were obtained from the American Type
Culture Collection (ATCC, Manassas, VA). MOPC21 cells were
grown in the peritoneal cavities of Balb/c mice to produce ascites
fluid and the immunoglobin was purified as previously reported.16
A control IgG1 was purchased from Calbiochem (San Diego, CA)
and further dialyzed against sterile cold PBS to eliminate sodium
azide. Antibodies reactive with phospho-Ser780 Rb and phospho-
Ser795 Rb were purchased from Cell Signaling Technology (Beverly,
MA). Antibodies against phospho-Thr821 Rb, phospho-Thr356 Rb
were obtained from Biosource International (Camarillo, CA).
Antibodies reactive with p27Kip1and total Rb were purchased from
BD Biosciences (San Diego, CA). Antibodies to CDK2, CDK4,
CDK6, cyclin E, cyclin D1, and cyclin A were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). A monoclonal antibody
to β-actin was purchased from Sigma (St Louis, MO).
Preparation of total cell lysate and immunoblot analysis. These
procedures were performed as described previously.16
Immunoprecipitation and kinase activity assay. Aliquots of total
cell lysates containing equal amounts of protein (300 µg) in lysis
buffer (137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA,
1 mM DTT, 1% nonidet P-40, 10% glycerol, and protease
inhibitors) were precleared with 2 µg normal mouse or rabbit IgG
(Santa Cruz) together with 20 µl of protein A/G agarose conjugate.
Lysates were then immunoprecipitated overnight at 4˚C with 3 µg
each of antibodies reactive with CDK2, CDK4, CDK6, cyclin D1,
cyclin A or cyclin E and 20 µl of protein A/G agarose conjugate.
CDK2-, cyclin E-, and cyclin A-associated kinase activities were
measured with a histone H1 kinase assay.16CDK4-, CDK6- and
cyclin D1-associated kinase activities were measured using a glu-
tathione S-transferase-Rb kinase assay.16After washing four times
with lysis buffer and twice with 1 X kinase buffer (20 mM Tris-HCl,
pH 7.4, 7.5 mM MgCl2and 1 mM DTT), the agarose pellets were
resuspended in 30 µl of kinase buffer containing 20 mM Tris-HCl,
pH 7.4, 7.5 mM MgCl2, 1 mM DTT, 20 µM ATP, 6 µCi of
[γγ-32P] ATP (6000 Ci/mMol, Amersham Pharmacia Biotech,
Arlington Heights, IL), protease inhibitors, and 3 µg histone H1
(Boehringer Mannheim, Indianapolis, IN) or GST-Rb fusion protein
(Santa Cruz). The mixture was incubated at 30˚C for 30 min for the
histone H1 kinase assay and at 30˚C for 60 min for the GST-Rb
kinase assay. The reaction was stopped by boiling the sample in
Laemmli SDS loading buffer for 5 min and samples were resolved on
a 12% SDS-PAGE. The gels were then stained, destained, dried
and exposed to X-ray film. For quantification, the protein bands
corresponding to histone H1 and GST-Rb were excised and the
radioactivity of each band was measured by Cerenkov counting.
Experiments were done in triplicate.
Preparation of total RNA. SKBr3 cells were treated with 4D5
(10 µg/ml) or MOPC21 (10 µg/ml) for 24 hr. BT474 cells were
treated with trastuzumab (10 µg/ml) or hIgG (10 µg/ml) for 48 hrs.
Total RNA was then extracted from the treated SKBr3 or BT474
cells using TRIZOL reagent (Invitrogen, Carlsbad, CA). Procedures
were performed according to the manufacturer’s recommendation.
The purity of RNA was assessed by absorption at 260 and 280 nm
(values of the ratio of A260/A280 of 1.9–2.1 were considered
acceptable) and by ethidium bromide (EtBr) staining of 18S and
28S RNA on gel electrophoresis. RNA concentrations were deter-
mined from the A260. Only samples of intact RNA were used for
subsequent Northern blot analysis.
Northern blot analysis. Fifteen micrograms of total RNA of each
sample were separated on 1% agarose/2.2 mol/L formaldehyde
denaturing gel and transferred onto a Hybond-N+membrane
(Amersham Biosciences, Piscataway, NJ). A full-length human
CDK2 cDNA19was used as a probe to detect CDK2 mRNA. An
RT-PCR amplified fragment of histone H4 (primer set sequences:
sense 5'-AATCCGCGATGCAGTTACCT-3'; anti-sense 5'-CCAC-
GTCCATGGCTGTGA-3') was used as a probe to detect H4. Both
probes were labeled with [α-32P]dCTP by using the Megaprimer
DNA labeling system (Amersham Biosciences, Piscataway, NJ)
according to the instructions of the manufacturer. Prehybridization
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
1656 Cell Cycle 2006; Vol. 5 Issue 15
and hybridization were done at 42˚C in a solution containing 5x
SSC (10x SSC is 1.5 mol/L NaCl and 0.15 mol/L sodium citrate),
2.5 mmol/L EDTA, 0.1% SDS, 5x Denhardt’s solution, 2 mmol/L
sodium PPi, 50 mmol/L sodium phosphate, and 50% formamide.
Membranes were washed with 0.2x SSC, 0.1% SDS at 56˚C for
1 hour, and hybridization signals were detected by autoradiography.
The agarose gel was stained with EtBr before transferring to the
membranes. 28S and 18S bands on EtBr-staining gel were used as a
Real-time reverse-transcription polymerase chain reaction (RT-
PCR) analysis.Quantitative real-time RT-PCR analysis was performed
for the CDK2 and NPAT genes with an ABI Prism 7900HT
Sequence Detection System using SYBR Green PCR Master Mix
(Cat. No. 4310179) according to the manufacturer’s specifications
(Applied Biosystems Incorporated (ABI), Foster City, CA). The
primer sequences for NPAT (sense 5'-TCCAGCCTGCTTACTGT-
CCT-3' and anti-sense 5'-AGCAAACCTTGGGGAACTTT) yielded
a 178-bp product. The primer sequences for CDK2 (sense 5'-CATT-
CCTCTTCCCCTCATCA-3' and anti-sense 5'-CATCCTGGAA-
GAAAGGGTGA-3') yielded a 572-bp product. The human 18S
ribosomal RNA (18S) gene
was used as an endogenous
control. The primer sequences
for 18S (sense 5'-CTTAGA-
and anti-sense 5'-ACGCTG -
yielded a 106-bp product.
The thermal cycler was set to
hold for 10 min at 95˚C,
followed by two-step PCR for
40 cycles of 95˚C for 15 sec,
followed by 1 min at 62˚C
(for NPAT) or 58˚C (for
CDK2). All assays were
performed in triplicate.
Amplification data were
analyzed using ABI Prism
Sequence Detection Software
version 2.1 (ABI). A dissocia-
tion curve for each experiment
was generated immediately
after amplification to assess
the specificity of amplification.
The amplification efficiencies
of the primer sets for CDK2,
NPAT, and 18S were similar
under the conditions we used.
Relative expression of CDK2
and NPAT was normalized to
the expression 18S reference
RNA using the Delta-delta
by the manufacturer.
Preparation of nuclear
extract and electrophoretic
mobility shift assay. BT474
cells were treated with
trastuzumab (10 µg/ml) or
hIgG (10 µg/ml) for 48 hrs.
Nuclear extracts were prepared by resuspending the cells in 400 µl
of lysis buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl), followed by incubation on ice for 10 min. Next, the lysates
were vortexed briefly before centrifugation for 1 min at 14,000 rpm
at 4˚C. The pelleted nuclei were resuspended in 50 µl of lysis buffer
(20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA) and left on ice for 20 min with occasional
mixing. Subsequently, the lysates were centrifuged at 14,000 rpm for
10 min at 4˚C, and the supernatants were stored at -70˚C. All
buffers were supplemented with 0.5 mM DTT and 0.2 mM phenyl-
methylsulfonyl fluoride. A synthetic oligonucleotide containing the
consensus binding site for E2F-1, 5'-ATTTAAGTTTCGCGCC-
CTTTCTCAA-3' (Santa Cruz Biotechnology), was used as a probe
for the assay. The probe was labeled with [32P]-dATP using the T4
polynucleotide kinase from Promega (Madison, WI). Eight micro-
grams of nuclear extract was used in each binding reaction. Where
appropriate, the extract was preincubated for 15 min at room
temperature with 10 pM unlabeled E2F-1 oligonucleotide.
Subsequently, binding to 1 pM of radioactive E2F-1 oligo was carried
out in a reaction mixture containing 1 µg of poly(dI·dC), 20 mM
Figure 1. Anti-HER2 antibody inhibits expression and activity of CDK2 but not CDK4. (A) Anti-HER2 antibody 4D5
decreases expression of CDK2 and CDK6, but not CDK4 proteins. SKBr3 cells were treated with different concentrations
of 4D5, or MOPC21 (10 µg/ml) as a control for 24 hr and total protein was collected for Western blot analysis with
anti-CDK2 antibody. The same blot was stripped and reprobed sequentially with anti-CDK4, CDK6, and β-actin.
(B) 4D5 decreases CDK2 enzymatic activity. SKBr3 cells were treated as described in (A) and total protein was
collected for immunoprecipitation and kinase assay. *p < 0.05; **p < 0.01. (C) 4D5 does not inhibit CDK4 enzy-
matic activity. SKBr3 cells were treated as described in (B). (D) Anti-HER2 antibody 4D5 decreases expression of cyclin
A and cyclin D1, but not cyclin E proteins. SKBr3 cells were treated with 4D5 at 10 µg/ml for different intervals and
total protein was collected for Western blot analysis with cyclin E antibody. The blot was stripped and reprobed sequen-
tially with cyclin D1, cyclin A and β-actin. (E) 4D5 inhibits cyclin E enzymatic activity. SKBr3 cells were treated as
described in (B). *p < 0.05; **p < 0.01. All experiments were repeated at least twice.
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
Hepes, pH 7.6, 70 mM KCl, 5 mM MgCl2, 0.05% Nonidet P-40,
12% glycerol, 1 mg/ml bovine serum albumin, 0.5 mM DTT for
30 min at room temperature. DNA-protein complexes were separated
on a 4.5% nondenaturing polyacrylamide gel electrophoresis
(PAGE) at 4˚C in buffer containing 25 mM Tris, 190 mM glycine,
1 mM EDTA at 25 mA. Gels were then dried and autoradiographed.
Nuclear extracts from MDA-MB-468 were used as a positive control.
Densitometric Analysis. Densitometric analysis was performed
by using SigmaGel software (SPSS Science Software, Chicago, IL).
The densities of targets (CDK2, E2F, NPAT, and histone H4) were
determined by the value of targets of interest divided by the density
of corresponding controls (28S, 18S, or HER3). The relative densi-
ty of targets of interest was calculated by using the expression level
in the control samples as 1.0. The fold change in various samples was
Statistical analysis.A two-tailed Student’s t-test was used to compare
different groups. Values with p < 0.05 were considered significant.
Anti-HER2 antibody significantly inhibits expression and activity
of CDK2 and CDK6 but not CDK4. CDK2 is one of major
physiological kinases that phosphorylates cyclin-dependent kinase
inhibitor p27Kip1in the presence of cyclin E and A.18We have
previously shown that CDK2 expression was inhibited by
anti-HER2 antibody ID5.16Here we have examined the effects of
anti-HER2 antibody 4D5, the murine precursor of trastuzumab, on
several cell cycle G1-associated kinases (CDK2, CDK4, and CDK6)
and cyclins (E, D1, and A). As shown in Figure 1A, treatment with
4D5 antibody markedly reduced both the active and inactive forms
of CDK2 protein in a concentration-dependent manner. Consistent
with these observations, the enzymatic activity of CDK2 was
markedly inhibited by 4D5 treatment (Fig. 1B). CDK6 protein was
also reduced by 4D5 treatment (Fig. 1A), as was the kinase activity
of CDK6 (data not shown). By contrast, neither the protein level
(Fig. 1A) nor the kinase activity of CDK4 (Fig. 1C) was affected by
Levels of CDK2-dependent cyclin E protein were not affected by
anti-HER2 antibody treatment (Fig. 1D), but the kinase activity of
cyclin E was significantly reduced by 4D5 treatment, consistent with
the impact of anti-HER2 antibodies on CDK2 and CDK6 (Fig. 1E).
Overexpression of wild-type cyclin E or its more active lower mole-
cular weight forms did not, however, influence the response to
trastuzumab (data not shown). Levels of CDK4- or CDK6-dependent
cyclin D1 protein and enzymatic activity were transiently inhibited
by 4D5 (Fig. 1D and data not shown). Both protein levels and
activity of CDK2-dependent cyclin A were markedly inhibited by
anti-HER2 antibody (Fig. 1D and data not shown). Thus, treatment
of breast cancer cells that overexpressed HER2 with anti-HER2
antibody reduced the kinase activities associated with cyclin E,
cyclin D1, and cyclin A.
Anti-HER2 antibody decreases the expression of CDK2 mRNA.
Among the cell cycle-associated kinases and cyclins, anti-HER2
antibody had the greatest effect on those that impacted the G1-S
Figure 2. Anti-HER2 antibody decreases the expression of CDK2 mRNA.
SKBr3 cells were treated for 24 hrs with different concentrations of 4D5 or
MOPC21 at 10 µg/ml as a control and total RNA was extracted for Northern
blot analysis (A) and real-time RT-PCR (B) as described in Materials and
Methods. EtBr-staining 28S and 18S bands were used as loading controls.
Figure 3. Anti-HER2 antibody decreases phosphorylation of Rb protein at
serine 780, serine 795, threonine 356, and threonine 821. (A) BT474 cells
were treated with different concentrations of trastuzumab, or IgG1 (10 µg/ml)
as a control for 48 hr and total protein was collected for Western blot analysis
with phospho-Thr821 Rb antibody (P-T821-Rb). The blot was then stripped
and reprobed sequentially with anti-phospho-Thr356 Rb antibody (P-T356-Rb),
anti-phospho-Ser780 Rb antibody (P-S780-Rb), anti-phospho-Ser795 Rb anti-
body (P-S795-Rb), and anti-total Rb antibody. (B) SKBr3 cells were treated
with different concentrations of 4D5 or MOPC21 (10 µg/ml) as a control for
24 hr and total protein was collected for Western blot analysis with
phospho-Thr356 Rb antibody (P-T356-Rb), phospho-Ser780 Rb antibody
(P-S780-Rb), phospho-Ser795 Rb antibody (P-S795-Rb) and total Rb antibody.
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
1658Cell Cycle2006; Vol. 5 Issue 15
transition. Even the nonactive form of CDK2 was dramatically
reduced by 4D5 treatment, suggesting that reduction of protein
expression might be due to transcriptional regulation of CDK2
mRNA. To examine this possibility, SKBr3 cells were treated with
4D5 or MOPC21 anti-HER2 antibody for 24 hrs and the mRNA
level of CDK2 was analyzed. As shown in Figure 2A, 4D5 reduced
levels of CDK2 mRNA up to 70% in a concentration-dependent
manner. Similarly, treatment of BT474 cells with trastuzumab
decreased the levels of CDK2 mRNA in a concentration-dependent
manner as demonstrated by real-time RT-PCR (Fig. 2B).
Anti-HER2 antibody decreases phosphorylation of Rb protein
at serine 780, serine 795, threonine 356 and threonine 821.
Alteration of the protein levels and kinase activities of cyclin D1/
CDK6, cyclin A/CDK2 and cyclin E/CDK2 should impact the
phosphorylation status of the Rb protein. Different cyclin/CDK
complexes preferentially phosphorylate different sites on the Rb.
Cyclin D1/CDK4 and cyclin D1/CDK6 complexes selectively
phosphorylate serine 780 and serine 795 of the Rb protein, whereas
cyclin A/CDK2 and cyclin E/CDK2 complexes selectively phospho-
rylate threonine 356 and threonine 821 of the protein.20-22As
shown in Figure 3, both trastuzumab (Fig. 3A) and 4D5 (Fig. 3B)
significantly inhibited the phosphorylation of Rb protein at serine
780, serine 795, threonine 356 and threonine 821 in a concentration-
dependent manner. As the activity and level of CDK4 are not affected
by anti-HER2 antibody, the marked decrease in phosphorylation of
the Rb protein at serine 780 and serine 795 is likely related to the
decreased activities and levels of cyclin D1 and CDK6.
Anti-HER2 antibody decreases the E2F-1 DNA-binding
Activity. Reduced Rb phosphorylation decreases the DNA binding
activities of E2F family members.6Therefore, the effect of anti-
HER2 antibody on the DNA binding activity of E2F proteins was
measured by EMSA. BT474 cells were treated with trastuzumab and
nuclear proteins were extracted for EMSA after incubation with a
probe for the E2F recognition site. Specific E2F binding activity was
inhibited about 70% by trastuzumab treatment (Fig. 4). Competitive
inhibition with unlabeled E2F probe identified a single complex that
contained E2F-Rb-DP1/2 and other proteins (Fig. 4). Trastuzumab
treatment did not alter protein levels of E2F nor its chaptones DP1
and DP2 (data not shown).
Anti-HER2 antibody decreases NPAT expression. CDK2/
cylcin E also phosphorylates and regulates NPAT protein.12Using a
specific antibody against NPAT protein,12western blot analysis
demonstrated that the protein level of NPAT was reduced by
trastuzumab treatment in a concentration-dependent manner
(Fig. 5A). The mRNA level of NPAT was also decreased by
trastuzumab treatment in a concentration-dependent manner as shown
by real-time RT-PCR (Fig. 5B). Interestingly, NPAT protein (Fig. 5A)
was reduced to a greater extent than NPAT mRNA levels (Fig. 5B)
after trastuzumab treatment, suggesting that NPAT expression might
be regulated by both transcriptional and post-transcriptional
The PI3K pathway regulates the expression of CDK2, Rb
phosphorylation, and NPAT. Based on our previous studies,16-18
inhibition of the PI3K pathway is one of the major mechanisms by
which anti-HER2 antibodies exert their growth-inhibitory effect.
Previous reports suggest that expression of CDK2, but not CDK4,
is decreased by inhibitors of the PI3K pathway.23,24Therefore, we
tested whether the inhibitory effect of trastuzumab on CDK2, Rb
phosphorylation, and NPAT might be mediated through the PI3K
pathway. As shown in (Fig. 6A), the PI3K inhibitor LY294002
reduced both the active and nonactive forms of CDK2 in a
Figure 4. Anti-HER2 antibody decreases E2F-1-DNA binding activity. E2F-DNA
complex formation was measured by electrophoretic mobility shift assay as
described in Materials and Methods. BT474 cells were treated with different
concentrations of trastuzumab for 48 hrs and nuclear protein was prepared.
For competition with nonlabeled oligonucleotides, a 10-fold excess of non-
labeled E2F-1 oligonucleotide was mixed with nuclear extracts from
MDA-MB-468 (as a positive control) before the addition of radiolabeled
E2F-1 oligonucleotide. This experiment was repeated three times with similar
Figure 5. Anti-HER2 antibody decreases NPAT expression. (A) BT474 cells
were treated with different concentrations of trastuzumab, or control IgG1
(10 µg/ml) for 48 hrs and total protein was collected for Western blot analysis
with anti-NPAT antibody. The blot was then stripped and reprobed with
anti-HER3 antibody as a loading control. (B) BT474 cells were treated as
described above and total RNA was extracted for real-time RT-PCR analysis.
*p = 0.04.
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
dose-dependent manner. As expected, the level of p27Kip1gradually
increased with increasing concentrations of LY294002 (Fig. 6A).
LY294002 also inhibited the expression of NPAT protein in a
dose-dependent manner (Fig. 6B and C). The expression of NPAT
mRNA was not, however, significantly reduced by the treatment
with LY294002 as shown by real-time RT-PCR in Figure 6D.
Consequently, post-transcriptional regulation may be important.
Also similar to trastuzumab treatment, LY294002 inhibited the
serine phosphorylation at 780 and 795 sites and threonine
phosphorylation at 356 and 821 sites of the Rb protein (Fig. 6B).
Thus, the effect of LY294002 mimicked the effect of trastuzumab in
decreasing expression of CDK2, Rb phosphorylation, and NPAT.
The anti-HER2 antibody trastuzumab and the PI3K inhibitor
LY294002 reduce the expression of histone H4. CDK2/cyclin E
can regulate both histone synthesis and DNA replication during cell
cycle progression from G1to S phase.9-11NPAT has been shown to
regulate the expression of histone proteins.11,12Therefore, the effect
of anti-HER2 antibody on the expression of histone H4 was exam-
ined by Northern blot analysis. As shown in Figure 7, anti-HER2
antibody trastuzumab dramatically suppressed the mRNA expression
of histone H4 in a time-dependent manner. LY294002 at the
concentration of 10 µM/ml also dramatically reduced the level of his-
tone H4 mRNA (Fig. 7C). These results suggest that trastuzumab
may affect histone synthesis through the PI3K pathway.
In our previous reports, we have shown that anti-HER2 antibodies
including trastuzumab induce p27Kip1expression and arrest tumor
cells that overexpress HER2 in the G1phase of the cell cycle.17-19In
this report, we demonstrate that trastuzumab inhibits CDK2, Rb
phosphorylation, E2F activity, NPAT and histone H4. PI3K signaling
appears to be a critical pathway by which anti-HER2 antibodies
exert their inhibitory effect on growth of tumor cells.25Based on our
studies, we have proposed a model that illustrates the effect of
trastuzumab on the cell cycle. As shown in Figure 8, trastuzumab
inhibits the PI3K pathway, which leads to upregulation of p27Kip1
and downregulation of CDK2, CDK6, cyclin A, and cyclin D
through transcriptional and post-transcriptional mechanisms.
Downregulation of CDK2, CDK6, cyclin A, and cyclin D
decreases both the phosphorylation of Rb protein and the activity of
E2F. Downregulation of the CDK2-cyclin E complex also decreases
NPAT expression. Downregulation of NPAT decreases histone H4
expression. Inhibition of both E2F and histone synthesis blocks the
G1to S phase transition, which results in G1arrest and depletion of
cancer cells in S phase.
The dramatic effect of trastuzumab on CDK2 may present another
molecular mechanism, in addition to the upregulation of p27Kip1,
by which trastuzumab inhibits cell cycle progression and proliferation
Figure 6. Inhibition of the PI3K pathway by LY294002 mimics the effect of trastuzumab on Rb phosphorylation and expression of CDK2 and NPAT.
(A) BT474 cells were treated with different concentrations of LY294002, or vehicle (DMSO) as a control for 24 hrs and total protein was collected for
Western blot analysis with anti-CDK2 antibody. The blot was then stripped and reprobed with anti-p27Kip1antibody. (B) Western blot analysis with anti-NPAT
and Rb antibodies. BT474 cells were treated with LY294002 as described above. Western blots were probed with anti-NPAT. The blot was then stripped
and reprobed in sequential fashion with anti-P-T356-Rb, anti-P-S780-Rb, anti-P-T821-Rb, anti-P-S795-Rb, and anti-total Rb antibodies. (C) Desitometric
semi-quantification of NPAT protein expression based on the densities of the total Rb protein shown in (B). (D) NPAT expression by real-time RT-PCR. BT474
cells were treated with LY294002 as described above and total RNA was extracted for real-time RT-PCR analysis. *p = 0.06.
Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression
1660 Cell Cycle2006; Vol. 5 Issue 15
of tumor cells that overexpress HER2. CDK2, an upstream regulator
of p27Kip1, is reduced via transcriptional mechanism by trastuzumab
(Fig. 2), which contrast to the upregulation by trastuzumab of
p27Kip1through posttranslational mechanisms.17The differential
effects of trastuzumab on CDK2 and CDK4 may have resulted from
the differential effects of anti-HER2 antibodies on the inhibitors of
cyclin-dependent kinases (CDKI). We and others have shown that
the only CDKI that is upregulated by anti-HER2 antibodies is
p27Kip1,16,26,27which is mainly inhibitory factor for the CDK2-
cyclin E complex and is required to form and maintain the activity
of the CDK4-cyclin D complex.1The effects of trastuzumab on
CDK2 and CDK4 are similar to the effects of two CDK2 inhibitors:
olomoucine or roscovitine, which specifically suppress CDK2 but
not CDK4.28Since anti-HER2 antibodies have no significant effect
on CDK4, a CDK4 inhibitor such as fascaplysin29may be able to
enhance the effect of trastuzumab, especially in cancer cell lines that
have high levels of HER2 but that are insensitive to trastuzumab
treatment. If the combination of trastuzumab and CDK4 inhibitor
prove to further inhibit cancer growth, this approach may be translated
into clinical management of patients whose cancer overexpress
Cyclin E-CDK2 complexes have two major roles in promoting
the transition from G1to S. First, Cyclin E-CDK2 participates,
together with cyclin D-CDK4/6, in the control of E2F/DP
transcription factor via phosphorylation of Rb protein. E2F
transcription factors are key regulators of a number of genes
involved in the G1/S-phase transition and DNA replication in
mammalian cells.6Data in this report indicate that trastuzumab
blocks this process (Figs. 3 and 4). Cyclin E-CDK2 can function in
an E2F-independent manner to activate DNA replication through
NPAT. NPAT accelerates S-phase entry.30Data in this report suggest
that trastuzumab suppresses this process as well (Fig. 5).
We have observed that trastuzumab can downregulate a number
of genes involving in DNA synthesis and repair.25In this report, we
observed that trastuzumab can downregulate the synthesis of histone
H4 (Fig. 7). Production of both linker histone (H1) and the core
histones (H2A, H2B, H3, and H4) is required in S phase.9
Inhibition of both DNA synthesis and histone synthesis impedes the
G1to S phase progression and leads to G1arrest and S phase
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Figure 8. Proposed model for the effect of trastuzumab on the cell cycle. See
the Discussion for details.
Figure 7. Anti-HER2 antibody trastuzumab inhibits the expression of histone
H4 mRNA. (A) Histone H4 expression under trastuzumab treatment by
Northern blotting. BT474 cells were treated with trastuzumab at different
intervals and total RNA was extracted for Northern blot analysis. EtBr-staining
28S and 18S bands were used as the loading controls. (B) Desitometric
semi-quantification of histone H4 mRNA expression based on the densities
of the 18S bands shown in (A). (C) Histone H4 expression under LY294002
treatment by Northern blotting. BT474 cells were treated with LY294002 at
different concentrations for 24hrs and total RNA was extracted for Northern
blot analysis. EtBr-staining 28S and 18S bands were used as the loading
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Trastuzumab Inhibits CDK2, NPAT and Histone H4 Expression