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INTRODUCTION |
Protein prenylation is an important posttranslational modification
that is required for cellular localization and biological function of
many proteins (1). These covalent attachments of farnesyl
(C15) or geranylgeranyl (C20) to cysteines at
the carboxyl-terminal of some proteins are catalyzed by three different
enzymes. Farnesyltransferase (FTase)1 and
geranylgeranyltransferase I (GGTase I) modify cysteines of proteins
that end in CAAX (C = Cys, A = aliphatic amino acid, X = any amino acid) at their
carboxyl-terminal with GGTase I preferring leucine or isoleucine and
FTase preferring Met or Ser in the X position (1). GGTase II, on the
other hand, geranylgeranylates on two cysteines in proteins that end in
XXCC, CCXX, or CXC sequences. Many
prenylated proteins are small G-proteins that are integral components
of proliferative signal transduction pathways (1). For example, Ras
farnesylation is required for its ability to transduce growth signals
from receptor tyrosine kinases to transcription factors and the cell
cycle machinery that regulates cell division (2). Furthermore, mutated
Ras is found in about 30% of all human cancers and is believed to
cause malignant transformation by constitutive activation of abnormal
growth (3). Farnesylation of oncogenic Ras is required for its
cancer-causing activity (4). Similarly, the Rho family of small
G-proteins such as RhoA and Rac1 require geranylgeranylation for their
biological function. One of their key biological roles is to allow
cells to traverse the G1 phase of the cell cycle and begin
DNA synthesis in S phase (5). Recently, RhoA and Rac1 have been shown
to be required for malignant transformation by Ras (6, 7). Furthermore, constitutively activated RhoA and Rac1 can also lead to oncogenic transformation (6, 7). The overwhelming evidence implicating the Ras
and Rho family of proteins in aberrant proliferative pathological conditions such as cancer and cardiovascular diseases prompted us and
others (reviewed in Ref. 8) to design and synthesize FTase and GGTase I inhibitors.
FTI-277 and GGTI-298 are CAAX peptidomimetics that potently
and selectively inhibit FTase and GGTase I, respectively (9, 10). We
have found FTI-276 and its methyl ester FTI-277 to be potent inhibitors
of oncogenic Ras processing and signaling (9). FTI-276 also potently
inhibited the growth in nude mice of human tumors with multiple genetic
alterations such as K-Ras mutation and p53 deletion (11, 12). FTIs from
several other groups have also shown potent antitumor efficacy without
toxicity in several animal models (8). Furthermore, several FTIs are
presently in phase I clinical trials (13).
Selective inhibition of protein geranylgeranylation with GGTI-298 has
major consequences on several biological pathways. Pretreatment of
fibroblasts with GGTI-298, blocks PDGF- and epidermal growth factor-dependent tyrosine phosphorylation of their
corresponding tyrosine kinase receptors (14). In contrast, selective
inhibition of protein farnesylation has no effect on receptor tyrosine
kinase phosphorylation (14). Furthermore, GGTI-298 inhibits the
growth in nude mice of human tumors by a mechanism that is not yet
known (12). One possible mechanism may involve GGTI-298-mediated
G1 phase block and apoptosis in cultured human tumor cells
(15). The ability of GGTI-298 to inhibit proliferation is not limited to human tumor cells that are of epithelial and fibroblast origin. GGTI-298 also has a major effect on the proliferative pathways of
smooth muscle cells (16). For example, GGTI-298 is very effective at
inducing G1 arrest and apoptosis in rat pulmonary artery
smooth muscle cells. Furthermore, GGTI-298 enhances the ability of
interleukin-1
to induce nitric oxide synthase-2 in the same cells
(17). This induction of nitric oxide synthase-2 results in a large
increase in the production of the nitric oxide radical, which is known to be inhibitory to smooth muscle cell proliferation. GGTI-298 also
inhibited the ability of PDGF, interleukin-1
, and activated Ras to
induce superoxide production in smooth muscle cells (18). Taken
together, the data indicate that GGTI-298 has antiproliferative effects
on fibroblasts, epithelial, and smooth muscle cells, and this cell
growth inhibition appears to be mediated through a G1 phase
arrest. Recently, we have shown that in human tumors this G1 arrest correlated with an induction of a
cyclin-dependent kinase inhibitor p21WAF1 (19).
However, whether this induction of p21WAF1 is responsible
for the G1 arrest is not yet known.
The G1 to S phase transition of the mammalian cell division
cycle is a highly regulated step. The key regulators of
G1/S progression are a series of kinases that depend on
cyclins for activation (for review, see Refs. 20-22). For example, the
D-type cyclins are made in early G1, bind to
and activate the G1 cyclin-dependent kinases
CDK4 and CDK6. Activation of these kinases also requires phosphorylation by CDK kinases such as CAK or cyclin H-CDK7 and dephosphorylation by phosphatases such as cdc25 (20). At this stage,
the cell has reached a critical checkpoint of its cell cycle called the
restriction point (R) at which time the cell checks that all is ready
for DNA synthesis. Cyclin D·CDK4 and cyclin D·CDK6 complexes are
now active and phosphorylate the retinoblastoma gene product pRb.
Phosphorylated pRb disassociates from the transcription factor E2F,
which, once freed from pRb, is able to induce the expression of several
genes that prepare the cell for DNA synthesis (20). Among these is
cyclin E, which activates CDK2, a kinase required for late
G1 to early S phase transition. The cyclin E·CDK2 complex
hyperphosphorylates pRb, and the cell proceeds into S phase. At this
time cyclin A expression is high, whereas cyclins D and E have been
degraded (20). The cyclin A·CDK2 complex maintains the
phosphorylation of pRb to sustain DNA replication. Finally CDK kinase
activities are highly regulated by two families of CDK inhibitors, CKIs
such as p21 and p27 and the INKs such as p15 and p16 (20-22). These
inhibitors play a key role in making sure the cells stop at the R point
if any DNA damage is detected. This allows the cells to repair the
damage before replicating their DNA. Growth factors such as PDGF and
epidermal growth factor activate several pathways, some of which have
been shown to directly regulate the cell cycle (22). For example,
activation of the Ras/Raf/MEK/ERK kinase cascade, which results in
increased expression of cyclin D1 that in turn will activate CDK4/CDK6
allowing cells to traverse the R point. PDGF activation of the Ras/RhoA
pathway results in the degradation of p27, which also will have the
same overall effect of allowing cells to traverse the R point and enter S phase of the cell cycle (23).
The G1 arrest brought about by GGTI-298 inhibition of
protein geranylgeranylation could be due to effects on several
important steps in G1/S such as the inhibition of
expression of cyclins D and/or E, CDK2, CDK4 and/or CDK6, or increased
expression of CKIs and/or INKs. The work described in this study
suggests a possible mechanism for GGTI-298 growth arrest involving
increased expression and partner switching of CDK inhibitors, resulting in inhibition of CDK2 and CDK4, and pRb phosphorylation.
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EXPERIMENTAL PROCEDURES |
Synthesis of CAAX Peptidomimetic--
The GGTase I-specific
peptidomimetic GGTI-298 was synthesized as described previously (10,
14).
Cells and Culture--
Human tumor cell lines Calu-1 and A-549
(lung carcinomas), T-24 (bladder carcinoma), and A-253 (head and neck
squamous cell carcinoma) were purchased from ATCC (Manassas, VA) and
grown in McCoy's 5A (Calu-1, T-24, and A-253) and F12K (A-549) media
at 37 °C in a humidified incubator containing 10%
CO2.
Western Blotting--
Cells were treated with GGTI-298 (15 µM) for 48 h, harvested, and lysed in HEPES lysis
buffer as described previously (11, 14). Proteins were then resolved by
12.5% or 7% SDS-PAGE gel and immunoblotted with antibodies against
Rap1A/Krev-1(121), p21WAF1 (C-190), cyclin E (C-19), cyclin
D1 (72-13G), CDK2 (M2), CDK4 (H-22), CDK6 (C-21), p16INK4A
(C-20), p15INK4B (C-20), cyclin A (H-432) (all from Santa
Cruz Biotechnology, Santa Cruz, CA), p27KIP1 (G173-524),
and pRb (G3-245) (from Pharmingen, San Diego, CA). The ECL blotting
system (NEN Life Science Products) was used for detection of positive
antibody reactions (14).
Flow Cytometry Analysis--
Cells were treated and harvested as
described for Western blotting, and nuclei were stained with propidium
iodide. DNA content was analyzed by fluorescence-activated cell sorter
as described previously (19).
Immunoprecipitations--
Cells were treated and harvested as
described above for Western blotting. Lysates (500 µg) were then
immunoprecipitated with polyclonal antibodies to CDK2 (M2), CDK4
(H-22), and CDK6 (C-21). The immunoprecipitates were then
electrophoresed on a 12.5% SDS-PAGE, transferred to nitrocellulose,
and immunoblotted with p15, p16, p21, and p27 as described above.
Cyclin-dependent Kinase Assay--
To measure the
activity of CDK2, histone H1 was used as the substrate; for CDK4 and
CDK6, GST-Rb (C-terminal fragment of pRb) was used as a substrate. The
CDK immunoprecipitates were resuspended in 10 µl of 50 mM
Hepes (pH 7.4) containing 10 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 10 µCi [
-32P]ATP and 100 µg/ml histone H1 (BM) or 20 µg/ml GST-Rb, and then incubated for 30 min at 30 °C with
occasional mixing. The reaction was terminated with an equal volume of
2× loading buffer (93.75 mM Tris, pH 6.8, 15% glycerol,
3% SDS, 7.5%
-mercaptoethanol). The sample was fractionated by
SDS-PAGE, and phosphorylated proteins were visualized by autoradiography.
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RESULTS |
GGTI-298 Induces Accumulation of Hypophosphorylated pRb in the
Human Lung Carcinoma Calu-1--
GGTI-298 was previously shown to
inhibit the growth in nude mice and to induce G1 block of
human tumor cells (12, 15). We sought to understand how GGTI-298
prevents Calu-1 cells from traversing G1 and entering S
phase of the cell cycle. We first determined the ability of GGTI-298 to
affect phosphorylation of pRb, one of the key events required for
G1/S transition. Calu-1 cells were treated for 48 h
with GGTI-298 (15 µM), and the cell lysates were
immunoblotted with an anti-pRb antibody that recognizes both hypo- and
hyperphosphorylated forms of pRb as described under "Experimental
Procedures." Other cell lysate aliquots were immunoblotted with
antibodies to either Rap1A or RhoA, small G-proteins that are
exclusively geranylgeranylated. Finally, a set of cells were analyzed
by flow cytometry to determine the proportion of cells at different
phases of the cell cycle. Fig.
1A shows that Calu-1 cells
treated with the vehicle contained predominantly hyperphosphorylated pRb. Treatment with GGTI-298 resulted in hypophosphorylation of pRb
(Fig. 1A). Hypophosphorylation of pRb correlated with
inhibition of the geranylgeranylation of the GGTase I substrates, Rap1A
and RhoA, and increased the proportion of Calu-1 cells in the
G1 phase of the cell cycle (Fig. 1A). Because
processed and unprocessed RhoA migrated closely (Fig. 1A),
we confirmed the effects of GGTI-298 on the processing of RhoA by
isolating membranes and cytosolic fractions and showing that GGTI-298
decreases the membrane levels of RhoA while inducing accumulation of
RhoA in the cytosol (Fig. 1B).

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Fig. 1.
GGTI-298 treatment induces pRb
hypophosphorylation. A, Calu-1 cells were treated for
48 h with GGTI-298 (15 µM), cell lysates were
prepared, separated by SDS-PAGE, and immunoblotted with either an
anti-pRb1, anti-RhoA, or anti-Rap1A antibody as described under
"Experimental Procedures." U and P designate
unprocessed and processed forms of Rap1A. The proportion of Calu-1
cells in G1 was determined by flow cytometry as described
under "Experimental Procedures." B, Calu-1 cells were
treated as described in A, except that membrane
(Mem) and cytosolic (cyto) fractions were
isolated before SDS-PAGE immunoblotting with anti-RhoA antibody. Data
are representative of five independent experiments except for RhoA (two
independent experiments).
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GGTI-298 Inhibits the Kinase Activities of CDK2 and CDK4--
We
next evaluated the ability of GGTI-298 to inhibit G1 phase
cyclin-dependent kinases that phosphorylate pRb. Calu-1
cells were treated with GGTI-298 for 48 h, and the lysates
immunoprecipitated with anti-CDK2, anti-CDK4, or anti-CDK6 antibody as
described under "Experimental Procedures." Fig.
2 shows that CDK2, CDK4, and CDK6 from
control Calu-1 cells were active and phosphorylated histone H1 (CDK2)
and GST-Rb (CDK4 and CDK6) in vitro. Treatment with GGTI-298
blocked the activity of CDK2, inhibited CDK4 and CDK6 activities by
75% and 30%, respectively(Fig. 2).

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Fig. 2.
GGTI-298 treatment results in inhibition of
the kinase activities of CDK2 and CDK4. Calu-1 cells were treated
with GGTI-298, cell lysates were prepared and immunoprecipitated with
CDK2, CDK4, and CDK6 antibodies, and kinase assays were carried out as
described under "Experimental Procedures." Data are representative
of at least three independent experiments.
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Effects of GGTI-298 on the Expression of Various Cell Cycle
Components--
The mechanism by which GGTI-298 results in inhibition
of the activities of CDKs could involve inhibition of the expression of
the G1 kinases themselves or their cyclins A, D, and E, or because of an increase in the levels of cyclin-dependent
kinase inhibitors. To further investigate these possibilities, Calu-1 cells were treated with GGTI-298 for 48 h, and the lysates
immunoblotted with antibodies to CDK2, CDK4, CDK6, cyclins D1, E, and
A, p21, p27, p15, and p16 as described under "Experimental
Procedures." Fig. 3 shows that
control-dividing Calu-1 cells expressed all cell cycle components
evaluated except p21, which was barely detectable. Treatment of Calu-1
cells with GGTI-298 did not alter the levels of CDK2 and CDK4 or those
of cyclins E and D1, indicating that the inhibition of CDK2 and CDK4
activities is not because of a decrease in the levels of cyclins E and
D1 or the kinases (Fig. 3). However, the amount of cyclin A was
decreased by 20%, and this could contribute to the block of CDK2
kinase activity. The levels of CDK6 decreased by 15%, and this could
account for the decrease in its kinase activity (Fig. 2). Furthermore
GGTI-298 greatly increased (7-fold) the levels of p21, and to a lesser extent (2-fold) increased the levels of p15 and had little effect on
the levels of p16 and p27 (Fig. 3).

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Fig. 3.
Effects of GGTI-298 on the protein
levels of CKIs, cyclins, and CDKs. Calu-1 cells were treated
and processed as described in the legend for Fig. 1, except lysates
were immunoblotted with antibodies to the indicated proteins as
described under "Experimental Procedures." Data are representative
of at least six independent experiments.
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GGTI-298 Induces Partner Switching of CDK Inhibitors--
The
above data suggest that the inhibition of CDK2 and CDK4 and the
subsequent hypophosphorylation of pRb are caused by increased protein
levels of some of the CDK inhibitors. Therefore we investigated whether
association of the CDKs with their inhibitors was also affected. Calu-1
cells were treated with vehicle (control) or GGTI-298 for 48 h,
the lysates were immunoprecipitated with various CDKs, the
immunoprecipitates were separated by SDS-PAGE and immunoblotted by
various CKIs. Fig. 4 shows that in
control cells, p21 and p15 were associated with primarily CDK6, whereas
p27 was primarily associated with CDK4 and CDK6. Treatment with
GGTI-298 increased (7-fold) association of p21 with CDK2 while
decreasing (70%) its association with CDK6. Similarly, GGTI-298
treatment decreased binding of p27 to CDK6 (85%) and CDK4 (22%) and
increased binding to CDK2 (2-fold) (Fig. 4). Furthermore, GGTI-298
treatment increased (2-fold) association of p15 to CDK4 with no effects
on association with CDK6 (Fig. 4). Finally, GGTI-298 had no effect on
the association of p16 with CDK4 or CDK6 (data not shown). Thus,
GGTI-298 treatment resulted in a partner switch for p21 and p27 from
CDK6 to CDK2. Furthermore, GGTI-298 treatment also induced an increased
association of CDK4 with p15 while decreasing its association with
p27.

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Fig. 4.
GGTI-298 treatment induces partner switching
of CKIs. Calu-1 cells were treated and processed as described in
the legend to Fig. 2, except the immunoprecipitates were separated by
SDS-PAGE and immunoblotted with antibodies for p21, p27, and p15. Data
are representative of at least four independent experiments.
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We next determined whether the GGTI-298-induced partner switching is
unique to Calu-1 cells. To this end, we treated three other human tumor
cell lines; A-549 lung carcinoma, T-24 bladder carcinoma, and A-253
head and neck squamous cell carcinoma with GGTI-298, immunoprecipitated
the lysates with CDK2 and CDK6 antibodies, and blotted with p27
antibody as described under "Experimental Procedures." Fig.
5 shows that in all three cell lines
treatment with GGTI-298 increased the levels of p27 associated with
CDK2 while it decreased the levels associated with CDK6.

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Fig. 5.
GGTI-298 induces partner switching in several
human tumor cell lines. A-549, T-24, and A-253 cells were treated
and processed as described in the legend to Fig. 4. Data are
representative of two independent experiments.
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Removal of GGTI-298 Results in Reversal of Partner
Switching--
To determine whether removal of GGTI-298 would result
in reversal of G1 block and partner switching, we treated
Calu-1 cells with GGTI-298 for 48 h, fresh medium lacking GGTI-298
was then added, and the cells collected after 0, 4, 24, and 48 h
post-GGTI-298 removal. The cells were then harvested and processed for
p21waf induction, immunoprecipitation/blotting and flow
cytometry as described under "Experimental Procedures." Fig.
6 shows that, consistent with Fig. 4,
GGTI-298 induced p21waf, partner switching of p27 from CDK6 to
CDK2 and G1 block (compare control versus
GGTI-298 lanes of Fig. 6). GGTI-298 had little effect on the
association of p27 with CDK4. Removal of GGTI-298 resulted in a
time-dependent reversal of p21waf induction, partner switching, and G1 block. Little change was observed
4 h after removal. However, significant reversal occurred at
48 h (Fig. 6).

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Fig. 6.
Time course reversal of GGTI-298
effects. Calu-1 cells were treated with either vehicle
(control) or with GGTI-298 for 48 h at which time fresh
medium lacking GGTI-298 was added, and the cells were collected at 0, 4, 24, and 48 h. The cells were then processed for p21waf
immunoblotting, flow cytometry, and immunoprecipitation with CDK2,
CDK4, and CDK6 antibodies, followed by immunoblotting with p27 antibody
as described under "Experimental Procedures." Data are
representative of two independent experiments.
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DISCUSSION |
Although there are more geranylgeranylated proteins than
farnesylated proteins, more efforts have been spent on designing, synthesizing, and biologically characterizing FTase rather than GGTase
I inhibitors (8). The intense search for FTase inhibitors was prompted
some years ago by the realization that farnesylation is required for
the cancer-causing activity of the important oncoprotein Ras (8).
Recently, however, more attention has been directed toward
understanding the effects of inhibiting (by GGTase I inhibitors) the
function of geranylgeranylated proteins. This is primarily due to the
discovery that some geranylgeranylated proteins such as those from the
Rho family are essential for normal and aberrant proliferation in
several cell types (1, 5). In addition to their ability to inhibit
human tumor growth, GGTase I inhibitors may also have great therapeutic
potential in cardiovascular diseases. For example, GGTI-298 blocks the
ability of smooth muscle cells to proliferate by inducing a
G1 block and apoptosis (16). This may be related to the
inhibition by GGTI-298 of PDGF and Ras induction of superoxide
formation (18) suggesting that a geranylgeranylated protein downstream
of Ras is critical to events regulating cell division. This is
consistent with a recent report, which shows that the
geranylgeranylated protein Rac1 mediates superoxide formation and
transformation by Ras (24). In this study, we demonstrate that GGTI-298
treatment of the human lung carcinoma Calu-1 cells results in a large
increase of the CDK inhibitor p21 and a modest increase of p15 with
little effect on p27 and p16. In nontreated dividing cells, CDK2 and
CDK4 bound mainly p27 and their kinases were active, whereas CDK6 bound
all inhibitors and was also active. Upon treatment with GGTI-298, p21
and p27 switched partners from CDK6 to CDK2, whereas p15 became bound
to CDK4. The effects of GGTI-298 on the observed partner switching was
reversible. We found that removal of GGTI-298 resulted in reversal of
the G1 phase block, which was paralleled by a reversal of
partner switching of p27 from CDK2 to CDK6.
The fact that in dividing cells, CDKs are bound to some inhibitors is
not surprising, because low levels of such inhibitors may be required
to stabilize cyclins with the corresponding CDKs (25). This is also
consistent with the suggestion that some CDKs may serve as cellular
reservoirs for low levels of inhibitors (26). Furthermore, although
there was little increase in total cellular levels of p27 after
GGTI-298 treatment, the amount of p27 bound to CDK2 was increased. This
suggests that p27 that was released from CDK6, and to a lesser degree
from CDK4, bound to CDK2. Taken altogether, the data suggests that
GGTI-298 induced partner switching for p21 and p27 from CDK6 to CDK2.
This partner switching was not unique to Calu-1 cells and was found to
occur in three other human tumor cell lines, A-549, T-24, and A-253. These results are similar to those obtained from cells that were arrested in late G1 following treatment with transforming
growth factor
(27). In these cells, transforming growth factor
induced the expression of p15, which bound CDK4 and CDK6 and displaced p27 from cyclin D·CDK4 and cyclin D·CDK6 complexes (27). The free
p27 bound the cyclin E·CDK2 complex and inhibited its activity, which
resulted in preventing cells from entering S phase (27).
The fact that inhibition of protein geranylgeranylation results in
G1 arrest suggests that some geranylgeranylated proteins are required for cell progression from G1 to S. The most
likely candidates are members of the Rho family of proteins such as
RhoA and Rac1. In this study, we showed that GGTI-298 inhibits the processing of RhoA, and recently we have demonstrated that RhoA represses p21waf transcription and suggested that GGTI-298
induction of p21waf is mediated by inhibition of RhoA
geranylgeranylation (29). Furthermore, it has been shown that both RhoA
and Rac1, when microinjected into G1-arrested cells,
promote progression to S phase (5). Furthermore, dominant-negative Rac1
and RhoA reverse oncogenic Ras transformation (6, 7). Rac1 may regulate
transcription by activating the Jun kinase pathway that in turn will
affect genes that are regulated by AP-1. Furthermore, very recently
Tapon et al. (28) discovered a novel effector of Rac1, POSH,
a kinase that appears to be required for activation of Jun kinase by
Rac1. It would be of great interest to determine whether activated Jun kinase or POSH would rescue the cells from the GGTI-298 mediated G1 block. These studies, which are in progress, will
further enhance our understanding of how inhibition of protein
geranylgeranylation results in G1 arrest.