© 2002 European Molecular Biology Organization EMBO reports vol. 3 | no. 8 | pp 767–773 | 2002 767
p21-activated kinase 1 interacts with and
phosphorylates histone H3 in breast cancer cells
Feng Li, Liana Adam, Ratna K. Vadlamudi, Hongyi Zhou, Subrata Sen, Jonathan Chernoff1,
Mahitosh Mandal & Rakesh Kumar+
The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 and 1The Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Received March 8, 2002; revised June 3, 2002; accepted June 18, 2002
Stimulation of p21-activated kinase-1 (Pak1) signaling pro-
motes motility, invasiveness, anchorage-independent growth
and abnormal mitotic assembly in human breast cancer
cells. Here, we provide new evidence that, before the onset of
mitosis, activated Pak1 is specifically localized with the
chromosomes during prophase and on the centrosomes in
metaphase and moves to the contraction ring during cyto-
kinesis. To identify mitosis-specific substrates of Pak1, we
screened a synchronized G2–M expression library by using a
glutathione transferase Pak1 solid-phase-based kinase reaction.
This analysis identified histone H3 as a substrate of Pak1 both
in vitro and in vivo, and it specifically interacted with Pak1 but
not Pak2 or Pak3. Site-directed mutagenesis indicated that
Pak1 phosphorylates histone H3 on Ser10. Expressions of the
wild-type, or catalytically active, Pak1 caused it to appear at
the poles corresponding to mitotic centrosomes in a variety of
mammalian cells. Together, these results suggest for the first
time that Pak1 interacts with and phosphorylates histone H3
and may thus influence the Pak1–histone H3 pathway, which
in turn may influence mitotic events in breast cancer cells.
The formation and functions of motile structures in mammalian
cells are regulated by the small GTPases, including Cdc42 and
Rac1 (Manser et al., 1997; Daniels and Bokoch, 1999). In
mammalian cells, p21-activated kinases (Paks) are one of the
targets of Cdc42 and Rac1, and binding of GTP-bound GTPases
to Pak stimulates its kinase activity via autophosphorylation
(Manser et al., 1997). Activation of Pak1 by diverse signals leads
to autophosphorylation at several sites, including Thr423 within
the activation loop of the kinase (Sells et al., 1997). Pak1
phosphorylation at Thr423 has been linked with its activation,
since substitution of the acidic residue glutamic acid at this site
produces a constitutively active T423E Pak1 mutant enzyme
(Sells et al., 2000). We recently generated a human breast
cancer cell line expressing T423E Pak1 and showed a close
linkage between the expression of T423E Pak1 and an abnormal
formation of mitotic spindles (Vadlamudi et al., 2000), which
suggests that Pak1 plays a role in mitosis and the formation of
Chromosomal and cytoskeleton-linked functions during
mitosis are regulated by a class of proteins termed chromosomal
passengers (Earnshaw and Bernat, 1991). These proteins, such as
the inner centromere protein (INCENP), the Aurora-B, the polo-
like kinases and survivin, associate with chromosomes along
their length during prophase and then concentrate at the inner
centromere and later by metaphase move to the centrosome. At
anaphase, the passengers abruptly move to the center of the
mitotic spindle and the cell cortex, where the contractile ring
originates (Adams et al., 2001).
The eukaryotic genome is compacted with histone and other
proteins to form chromatin, which consists of repeating units of
nucleosomes (Wolffe and Kurumizaka, 1998). The chromatin
structure plays an important role in gene regulation and is a
point where signaling pathways converge (Cheung et al., 2000).
The eukaryotic chromosomes contain five major classes of
histones: H1, H2A, H2B, H3 and H4. H3 histones are divided
into three subtypes: H3.1, H3.2 and H3.3. H3.3 is encoded by
two distinct genes: H3.3A and H3.3B, which code identical
proteins and are structurally similar to histones H3.1 and H3.2
except for the addition of the first initiation codon, methionine,
and three amino acid substitutions.
Cellular signaling pathways modify histones by a number of
methods, including phosphorylation of the histone tails, thereby
+Corresponding author. Fax: +1 713 745 3792; E-mail: email@example.com
F. Li, L. Adam and R.K. Vadlamudi contributed equally to this work
768 EMBO reports vol. 3 | no. 8 | 2002
F. Li et al.
controlling gene expression (Cheung et al., 2000; Jenuwein and
Allis, 2001). In addition, histone H3 Ser10 phosphorylation,
which has been linked with the functions of chromosomal
passengers, regulates mitotic chromosome assembly, congrega-
tion at the metaphase plate and segregation (Earnshaw and
Bernat, 1991; de la Barre et al., 2000; Adams et al., 2001).
Despite the well-known role of histones in chromatin structure
and function, very little is known about their role in the Pak1
pathway of mammalian cells. In the experiments described below,
we provide new evidence that Pak1, besides playing an important
role in the mitosis of eukaryotic cells, phosphorylates histone H3.
RESULTS AND DISCUSSION
Deregulation of Pak1 activation promotes the formation of
abnormal mitotic spindles in human breast cancer cells
(Vadlamudi et al., 2000). To understand the potential role of
activated Pak1 in mitotic spindle formation, we first determined
the location of activated Pak1 in mitotic MCF-7 breast cancer
cells transiently transfected with myc-tagged Pak1. About
40–50% of Pak1-transfected mitotic cells but <10% of the
mock-transfected cells had three or more spindle poles
(Figure 1A and B). myc-Pak1 was localized on and around the
spindles pole in the centrosomal region (Figure 1A’).
Endogenous Pak1 was localized inside the nucleus in 18–24%
of interphase cells (Figure 1C, panel 1), on the centrosomes and/or
metaphase plate in 100% of the metaphase-to-anaphase cells
(Figure 1C, panels 2 and 3) and on the midbody of 85% of the
telophase cells (Figure 1C, panel 4). This cell-cycle-related Pak1
translocation was observed in several eukaryotic cell lines, as
well as in NIH 3T3 murine fibroblasts (Figure 1C), in MCF-7
(Figure 1F and G), in MDA-MB231 (Figure 1D and E) and in
ZR-75R breast cancer cells (data not shown).
To examine the activation status of Pak1 in mitotic cells, we
next determined the distribution of endogenous activated Pak1
in mitotic cells using an anti-Pak1Phos antibody that recognizes
phosphorylated Thr423 Pak1 (Sells et al., 2000). Pak1Phos
staining had a strong, punctated pattern at the mitotic spindle
poles (Figure 1D, white arrows) and at the midbody (Figure 1E,
green arrow). Centrosomal Pak1 reactivity was reproduced by
another Pak1 antibody, Ab2 (from Cell Signaling), which was raised
against the phosphorylated Thr423 site (Figure 1F); it specifically
competed with the immunizing peptide (Figure 1G). Centro-
somal Pak1Phos was specific for Pak1, as neither Pak2 (Figure 1H)
nor Pak3 (Figure 1I) localized on the centrosome, as determined
by specific antibodies. Furthermore, Pak1 translocation to the
centrosomes depended on its kinase activity, as this localization
(Figure 1K) was blocked by the expression of a glutathione
S-transferase (GST)-tagged Pak1 inhibitor (amino acids 83–149)
(Figure 1L and J). Similar experiments using the dominant-
negative Cdc42 construct failed to block Pak1 centrosomal
localization (data not shown).
The newly observed localization of activated Pak1 raised the
possibility that Pak1 phosphorylates mitosis-specific substrates.
To identify such substrates, we screened a synchronized G2–M
expression library from HeLa cells using a GST–Pak1 solid-
phase-based kinase reaction (Figure 2A). After the third
screening, several clones were identified, and isolates from one
of the clones were 100% identical to histone H3.3A (DDBJ/
EMBL/GenBank accession No. BC001124), which is structurally
Fig. 1. Subcellular localization of Pak1 during mitosis. (A and A′) Merged
confocal image of α-tubulin (red) and myc-tagged wild-type (WT) Pak1 or
myc-tagged kinase-dead K299LL (KLL) Pak1 (green) overexpression in
MCF-7 cells. (B) Quantification of the multiple spindle poles induced by
myc-tagged WT or KLL Pak1 expression in MCF-7 cells. (C) Interphase (1),
metaphase (2), anaphase (3) and telophase (4) of NIH 3T3 cells showing
intranuclear, centrosomal and midbody (asterisk) localization of Pak1
(green). (D) Centrosome (white arrows) localization of phosphorylated Pak1 in
mitotic MDA-MB231 cells. (E) Midbody (green arrow) localization of
phosphorylated Pak during cytokinesis. (F and G) Pak1Phos at the spindle
poles (white arrows) of metaphase MDA-MB231 cells by another anti-
Pak1Phos antibody (Ab2) in the absence or presence of a competing peptide
used to generate this antibody. (H and I) Absence of Pak2 or Pak3 on
centrosomes in mitotic MDA-MB231 cells. (J) Centrosomal Pak1 localization
requires Pak1 activity. MCF-7 cells were transfected with GST–Pak1 inhibitor
(amino acids 83–149). Quantification of centrosomal Pak1 expression in the
GST-positive (GST+) and GST-negative (GST–) metaphase cells. (K) Example of
mitotic (GST–) cell with centrosomal Pak1. (L) Example of mitotic (GST+)
cell with no centrosomal Pak1. Scale bars = 4 µm.
EMBO reports vol. 3 | no. 8 | 2002 769
Pak1 regulation of H3 phosphorylation
similar to histone H3.1 and H3.2 except for the addition of the
first initiation codon, methionine, and three amino acids
substitutions. Histone H3.3 was strongly expressed in the human
breast cancer cells and in a number of murine tissues and was at
its highest level in mammary gland during pregnancy day 15
(Figure 2B). Since the N-terminal regions of histone H3 isoforms
are highly conserved and are similar to those of the histone H3.1
and histone H3.2 isoforms, H3.3 may not retain its first methio-
nine; therefore, we will refer to the H3.3 isoform of histone as
H3 in this report.
To determine whether histone H3 interacts with Pak1, we next
examined the ability of an in vitro-translated Pak1 protein to
Fig. 2. Identification of histone H3.3A as a Pak1 substrate. (A) G2–M expression library screening by Pak1. (B) mRNA expression of H3.3A in breast cancer cell
lines and murine tissues. (C) Direct interaction of Pak1 with histone H3 in a GST pull-down assay. In vitro-translated H3 bound to GST–Pak1 (left panel), and in
vitro-translated Pak1 bound to GST–H3 (right panel). (D) Non-interaction of H3 with Pak 2 and Pak3. cDNAs were translated in vitro, incubated with GST–H3
and analyzed by a GST pull-down assay. (E) Interaction of Pak1 domains with histone H3. Pak1 GST-fusion proteins containing full-length Pak1 amino acids (aa
1–546), the kinase domain (aa 270–546), the N-terminal domain (aa 1–132), the Nck-binding domain (aa 1–75), the Cdc42/Rac-interacting domain (aa 52–132)
and the autoinhibitory domain (aa 75–149) were all incubated with in vitro-translated H3, and binding was analyzed by a GST pull-down assay. (F) Pak1
specifically interacts with H3 but not H4 in TNT pull-down assays. GST pull-down assay using 35S-labeled Pak1 and GST fusion of H3 or H4. (G) Pak1 interacts
with H3 tail. GST pull-down assay using 35S-labeled Pak1 and GST fusions of H3 1–57 or H3 1–29.
770 EMBO reports vol. 3 | no. 8 | 2002
F. Li et al.
bind with the GST-tagged histone H3. As shown in Figure 2C,
Pak1 efficiently interacted with GST–H3 but not with GST alone
in GST pull-down assays. Conversely, an in vitro-translated
histone H3 protein also specifically interacted with GST–Pak1.
To evaluate the specificity of the histone H3–Pak1 interaction,
we examined whether histone H3 could interact with the Pak2
and Pak3 proteins. It did not interact with Pak2 or Pak3
(Figure 2D). To examine the H3 binding domain of Pak1, we
used a series of GST-fusion proteins expressing Pak1 domains
(Figure 2E). Histone H3 did not interact with the kinase domain,
Fig. 3. Histone H3 is a substrate of Pak1. (A) Pak1 phosphorylation of histone H3. Phosphorylation of GST–H3 by purified Pak1 (upper panel). Phosphorylated
GST–H3 was immunoblotted with an anti-H3 Ser10Phos antibody. Autophosphorylated Pak1 and GST–H3 are shown as controls (lower panels). (B) Ser10 of H3
is the Pak1 phosphorylation site. In vitro kinase assay of mutated histone H3 (H3.3 S10A and S95A) GST-fusion proteins by Pak1 enzyme. (C) Pak1
phosphorylation of native and purified histones in vitro. (D) Purified native histones H3 and H1 were phosphorylated in vitro using a Pak1 enzyme, blotted with an
anti-H3 Ser10Phos antibody. (E) Tet vector or Ha-T423E Pak1 vector stable MCF-7 cells were treated with doxycyclin (1 µg/ml) for 24 h to induce expression of
Pak1. Expression of Ha-T423EPak1 was analyzed by western blotting (upper panel) and its activity was analyzed by in vitro kinase assay (lower panel). (F) Ser10
phosphorylation of the endogenous histone H3 in T423E Pak1 (TA2) expressing cells as determined by in vivo labeling with [32P]orthophosphoric acid. Histones
were isolated and phosphorylation of H3 was analyzed by autoradiography. Histone H3 (upper band). NS, non-specific band present in both lanes. (G) Ser10
phosphorylation of H3 in T423E expressing TA2 cells as shown by blotting with an anti-H3 Ser10Phos antibody. (H) T423E Pak1 stimulates phosphorylation of
T7-H3. MCF-7 cells cotransfected with T7-H3 and T423E Pak1 or vectors were labeled with 32P-labeled orthophosphoric acid, and cell lysates were
immunoprecipitated with a T7 antibody. Phosphorylated histone H3 is shown. (I) The same blot was reprobed with anti-H3 Ser10Phos and T7 antibodies. (J) H3
fails to bind ∇1–82 Pak1 which lacks N-terminal 82 aa. T423E Pak1 or ∇1–82 Pak1 was translated in vitro and GST pull-down assays were performed with
GST–H3. (K) Pak1 mutant lacking H3 binding region fails to phosphorylate H3 on H3 Ser10Phos. MCF-7 cells were cotransfected with T423E Pak1 or ∇1–82 Pak1
along with T7-H3. The status of H3 Ser10Phos on T7-H3 was analyzed by western blotting.
EMBO reports vol. 3 | no. 8 | 2002 771
Pak1 regulation of H3 phosphorylation
the Cdc42/Rac1-interacting domain or the Pak1 autoinhibitory
domain. However, N-terminal region fusion proteins containing
amino acids 1–132 and 1–75 bound strongly to histone H3
(Figure 2E), indicating that a histone H3 binding site was located
in the N-terminal 75 amino acids, a region involved in Nck
binding to Pak1 (Lu et al., 1997). The noticed histone H3 inter-
action with Pak1 was specific, as Pak1 failed to interact with
histone H4 (Figure 2F). The Pak1 binding to histone H3 was
Fig. 4. Chromosomal localization of activated Pak1. (A) Metaphase plate localization of Pak1. Confocal micrographs of a single tangentially cut plane through a
metaphase cell. Pak1Phos alone (right panel) and merged image with α-tubulin (left panel). (B) Centromeric and extracentromeric patterns of chromosome-bound
activated Pak1, as revealed by three-color confocal microscopy of chromosome spreads obtained from colcemid-treated MDA-MB231 cells. Centromeric
colocalization of dynein (red) and activated Pak1 (green) (brown arrowheads); extracentromeric localization of Pak1Phos (yellow arrowheads). Dynein and Pak1
colocalization on centrosomes (white arrows) and in the centromeric region (brown arrowheads). (C) Centromeric and extracentromeric localization of activated
Pak1 on chromosome spreads from MDA-MB231 cells. Activated Pak1 associated with condensed chromosomes containing histone H3 Ser10Phos. Histone H3
Ser10Phos (red), activated Pak1 (green); Topro-3 DNA staining (blue). Representative spreads are shown in merged colors in the left panel and in two colors in the
middle and right panels. Note the activated Pak1 on both the centrosomes and chromosomes (white arrows and yellow arrowheads, respectively). Scale bar = 4 µm.
(D) Extracentromeric localization of activated Pak1 immunoreactivity without dynein labeling (yellow arrowheads); Pak1 and dynein colocalization in the
centromeric region (brown arrowheads). Unilateral Pak1 localization on the chromosome arm (purple arrowhead). (E) Representative examples of individual
chromosomes in which activated Pak1 colocalized with histone H3 Ser10Phos in the extracentromeric (yellow arrowheads) regions.
772 EMBO reports vol. 3 | no. 8 | 2002
F. Li et al.
localized within the histone H3 tail region amino acids 1–57
To determine whether histone H3 was a substrate of Pak1, we
performed in vitro kinase assays using a purified Pak1 enzyme
and GST–H3. As shown in Figure 3A (upper panel), recombinant
Pak1 efficiently phosphorylated histone H3. We next explored
whether a Pak1-phosphorylated histone H3 could be recognized
by a well-characterized anti-H3 Ser10Phos antibody (Zeitlin et al.,
2001). Interestingly, Pak1-mediated phosphorylation of histone
H3 was readily recognized by the anti-H3 Ser10Phos antibody
(Figure 3A, second panel), raising the possibility of a close
relationship between Pak1 and histone H3 phosphorylation.
Lack of phosphorylation in lane 1 was not due to lack of Pak1
activity, since Pak1 autophosphorylation status was similar in
both lanes (Figure 3A, third panel). The mutation of histone H3
Ser10 to alanine (S10A) completely blocked the ability of Pak1
to phosphorylate H3, whereas the mutation of histone H3 Ser95
to alanine (S95A) had no effect (Figure 3B), confirming that
Ser10 of H3 is the Pak1 phosphorylation site. Accordingly, the
recombinant Pak1 enzyme (Figure 3C) efficiently phosphory-
lated both native histone and highly purified histone H3 but
not histone H1. The anti-H3 Ser10Phos antibody also readily
recognized the Pak1-mediated phosphorylation of purified
histone H3 on Ser10 (Figure 3D).
To demonstrate that Pak1 phosphorylates histone H3 in vivo,
we examined the regulation of endogenous histone H3 phos-
phorylation in MCF-7 cells, which express the catalytically
active mutant Ha-tagged T423E Pak1 in the presence of doxy-
cyclin (TA2 clones; Vadlamudi et al., 2000) (Figure 3E). The
results of both in vivo labeling of cells with 32P-labeled ortho-
phosphoric acid (Figure 3F, upper band; NS, non-specific band
present in both lanes) and western blotting with the anti-H3
Ser10Phos antibody (Figure 3G) demonstrated that overexpression
of catalytically active Pak1 was accompanied by increased H3
phosphorylation on Ser10. To further confirm that H3 was
indeed phosphorylated in vivo by Pak1, we transfected MCF-7
cells with the T7-tagged histone H3 and myc-tagged T423E
Pak1. Immunoprecipitation of histone H3 with an anti-T7 mono-
clonal antibody showed that histone H3 was indeed phosphory-
lated in cells expressing T423E Pak1 but not in vector-transfected
cells (Figure 3H). The anti-H3 Ser10Phos antibody also recog-
nized this phosphorylation of T7-H3 (Figure 3I). Expression of a
Pak1 construct lacking the histone H3 binding region (∇1–82
Pak1) into MCF-7 cells failed to bind histone H3 (Figure 3J), as
well as failing to induce histone H3 phosphorylation (Figure 3K),
suggesting a close relationship between Pak1 interaction with
histone H3 and its phosphorylation by Pak1. Together, these
results suggest that Pak1 phosphorylates histone H3 and that
histone H3 is a substrate of Pak1 that interacts with Pak1 and
may influence its functioning in mitotic cells.
We observed a punctated pattern of Pak1 localization in the
nucleus (Figure 1C, panel 1), which suggested that activated
Pak1 might be associated with the kinetochore complex/centro-
meric region of mitotic chromosomes during prophase. To
support such a possibility, we show the presence of the Pak1
protein at the metaphase plates (20–30%), as shown in a
representative tangentially ‘cut’ confocal section of a metaphase
plate with a high Pak1 immunoreactivity (Figure 4A). A similar
pattern has been reported for another component of the kineto-
chore component dynein (Echeverri et al., 1996). To explore
whether the microtubules and Pak1 compete for the kineto-
chore in MDA-MB231 cells, we next eliminated the possible
contribution of microtubules using a tubulin-depolymerizing
agent (colcemid) and immunostaining the resulting chromo-
somal spread preparations for either histone H3 Ser10Phos or
dynein (Steuer et al., 1990) and activated Pak1. Representative
examples of these studies, shown in Figure 4B (brown arrow-
heads), indicated the presence of centromeric Pak1 colocalized
with the dynein intermediary chain. These findings suggested
the possibility that Pak1 leaves the kinetochores after the micro-
tubules attach. It is possible that under normal circumstances an
abundance of Pak1/dynein complex at the kinetochores may
help capture the microtubules and drive the chromosome
movement, after which Pak1 leaves the kinetochore as part of a
putative complex. Examples of individual chromosomes with
different patterns are shown in Figure 4D. Some displayed either
an occasionally extracentromeric parallel localization of the
activated Pak1 (Figure 4D, yellow arrowheads) or non-parallel
pattern (Figure 4D, purple arrowhead). The extracentromeric
Pak1 was colocalized with the abundantly phosphorylated
histone H3 Ser10Phos on the chromosome arms (Figure 4C,
yellow arrowheads). No colocalization of Pak1Phos and H3
Ser10Phos was detected on the two centrosomes (Figure 4C, white
arrows) or the centromeres (Figure 4E, brown arrowheads).
These results suggest that Pak1 may be a transitory component of
kinetochores in mammalian cells and that its association with
the kinetochore was sensitive to the presence of the micro-
tubules. Pak1 extracentromeric localization, as well as H3
binding and specific ability to phosphorylate histone H3 on
Ser10, may identify novel functions of Pak1 during mitosis.
In summary, our results show a putative chromosomal function
of Pak1. We have shown that, before the onset of mitosis, Pak1
becomes activated and translocates to the nucleus. From this
point on, Pak1 behaves like a chromosomal passenger protein.
Results presented here raise new questions about the functions
of Pak1 during mitosis, including its potential involvement in
the condensation, capture and/or movement of chromosomes
during mitosis, all of which are required for proper chromo-
somal segregation. Furthermore, in addition to Pak1’s potential
role in chromatin rearrangement at the chromosomal level, this
process may also be influenced by the potential substrates that
participate in microtubule dynamics. Because histone H3 phos-
phorylation has been shown to be required for the initiation of
chromosome condensation (Cheung et al., 2000) and cell-cycle
progression, our present findings suggest that Pak1, through histone
H3 phosphorylation, may play a role in regulating mitotic events.
Cell cultures and reagents. MCF-7 breast cancer cells were
maintained in DMEM-F12 (1:1, v/v) supplemented with 10%
fetal calf serum. Antibodies against Pak1, Pak2 and Pak3 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and
Zymed Laboratories (San Francisco, CA). Antibodies against T7
tag and histone H3 Ser10Phos were purchased from Novagen
(Madison, WI) and United Biochemical Inc. (Hauppage, NY).
Antibodies against phospho-T423E Pak1 (Sells et al., 2000) were
purchased from Cell Signaling (Beverly, MA).
Screening of G2–M expression library. HeLa cells were synchron-
ized with a double thymidine block and release protocol (Kumar
EMBO reports vol. 3 | no. 8 | 2002 773 Download full-text
Pak1 regulation of H3 phosphorylation
et al., 1994). The cDNA expression library was established using
the ZAP Express cDNA Synthesis Kit and the ZAP Express cDNA
Gigapack III Gold Cloning Kit (Stratagene, La Jolla, CA), as
described previously (Fukunaga and Hunter, 1997).
Plasmid construction. H3 expression vectors were constructed
by amplifying the coding region of H3.3 of the positive clone
obtained from expression library screening, by PCR amplifica-
tion and by subcloning into the EcoRI and XhoI sites of
pcDNA3.1 (T7-H3) and the PGEX5 vector (GST–H3). Mutations
of H3.3, S10A and S95A were created using the Stratagene
Quik Change kit and pcDNA 3.1-H3 in the vector as a template.
∇1–82 Pak1 was constructed by PCR amplification of Pak1
coding region containing amino acids 83–546 and subcloning
into pcDNA3.1 HIS vector (Invitrogen).
In vitro kinase and GST pull-down assays. In vitro kinase assays
using either purified histones or (GST)–H3 protein were
performed as described previously (Mazumdar et al., 2001).
Confocal studies and chromosome spreads. Confocal and
chromosome spreading studies were performed as described
previously (Saffery et al., 2000; Vadlamudi et al., 2000).
This work is supported by NIH grant CA80066, Cancer Center
core grant CA16672, M.D. Anderson Cancer Therapeutic
Discovery Program (to R.K.) and M.D. Anderson Intramural
Research Grant (to L.A.).
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