From the Departments of Oncology and Biochemistry and
Molecular Biology, Drug Discovery Program, H. Lee Moffitt Cancer Center
and Research Institute, University of South Florida, Tampa, Florida
33612, the § Department of Chemistry, Yale University, New
Haven, Connecticut 06520-8107, and the ¶ Cell and Developmental
Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania
19111
Received for publication, July 13, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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Even though farnesyltransferase inhibitors
(FTIs), a novel class of therapeutic agents presently in clinical
trials, have preclinically outstanding anticancer activity and
impressive lack of toxicity, their mechanism of action is not well
understood. To enhance our understanding of how FTIs inhibit the growth
of tumors, we have investigated their effects on cell cycle progression of two human lung cancer cell lines, A-549 and Calu-1. In this report,
we show in synchronized A-549 and Calu-1 cells that FTI-2153 treatment
resulted in a large accumulation of cells in the mitosis phase of the
cell division cycle, with some cells in the
G0/G1 phase. Furthermore, microtubule
immunostaining and 4,6-diamidino-2-phenylindole DNA staining
demonstrated that the FTI-2153-induced accumulation in mitosis is due
to the inability of these cells to progress from prophase to metaphase.
FTI-2153 inhibited the ability of A-549 and Calu-1 cells to form
bipolar spindles and caused formation of monoasteral spindles.
Furthermore, FTI-2153 induced a ring-shaped chromosome morphology and
inhibited chromosome alignment. Time-lapse videomicroscopy confirmed
this result by showing that FTI-2153-treated cells are unable to align
their chromosomes at the metaphase plate. FTI-2153 did not affect
the localization to the kinetochores of two farnesylated centromeric
proteins, CENP-E and CENP-F. Thus, a mechanism by which FTIs inhibit
progression through mitosis and tumor growth is by blocking bipolar
spindle formation and chromosome alignment.
Protein farnesyltransferase catalyzes the covalent transfer of the
lipid farnesyl from farnesyl pyrophosphate
(FPP)1 to the cysteine thiol
of proteins that end with a CAAX motif at their
carboxyl-terminal (C = cysteine, A = aliphatic
amino acid, and X = any amino acid but preferably
methionine and serine but not leucine) (1, 2). Protein farnesylation is
required for the localization and function of several proteins pivotal to signal transduction pathways (1, 2). Among these is a family of low
molecular weight GTPases called Ras (K-, N-, and H-Ras). Ras proteins
transduce biological signals from cell surface receptors to the nucleus
regulating important processes, including the cell division cycle,
programmed cell death, and differentiation (3, 4). Furthermore, Ras
proteins are found constitutively activated (due to point mutations) in
about 30% of all human cancers, resulting in uncontrolled
proliferation and tumor cell survival (5). The interest in
farnesyltransferase was heightened when it was discovered that Ras
requires farnesylation for its cancer-causing activity. This prompted
many researchers to design farnesyltransferase inhibitors (FTIs) as
potential anticancer drugs (6-8).
Intense research efforts over the last decade using several approaches
have resulted in potent and selective FTIs (6-8). The approaches
included rational design of CAAX peptidomimetics, FPP
analogs, and FPP/CAAX bisubstrate transition state analogs as well as screening of natural product and chemical libraries. Some of
the FTIs were shown to inhibit potently farnesyltransferase in
vitro (IC50 in the picomolar range) and in whole cells
(nanomolar range), to antagonize oncogenic H-Ras signaling, and to
induce apoptosis in Ras-transformed fibroblasts when deprived of either serum or substratum attachment. More recently, FTIs were shown to
induce apoptosis of attached human cancer cells in the presence of
serum by a mechanism involving, at least in part, the
phosphatidylinositol 3-kinase/Akt-2 survival pathway (9). Finally, FTIs
have been shown to be potent inhibitors of tumor growth in several
animal models. Their impressive antitumor activity and lack of toxicity to normal cells have led to ongoing human clinical trials with several
FTIs (10).
Despite these major advances, the mechanism by which FTIs manifest
their outstanding antitumor activity remains largely unknown. Although
initially FTIs were hypothesized to inhibit tumor growth by inhibiting
Ras farnesylation, several key observations suggested that farnesylated
proteins other than Ras may be involved. First, the Ras mutation status
does not predict sensitivity of human tumors to FTIs in soft agar
assays (11). Second, K-Ras, the most prevalent mutated form of Ras in
human tumors, becomes geranylgeranylated in the presence of FTIs (12,
13). Third, although both FTIs and geranylgeranyltransferase
inhibitors are necessary for inhibition of K-Ras prenylation,
FTIs alone are sufficient for inhibition of the growth in nude mice of
human tumors with K-Ras mutations (14). Finally, the kinetics of
FTI-induced reversal of transformation do not correlate with that of
inhibition of Ras farnesylation (15). One candidate, RhoB, another
low molecular weight GTPase that is both farnesylated and
geranylgeranylated, has been suggested as a target for FTI antitumor
activity in fibroblasts (16). However, recent data in human cancer
cells of epithelial origin argue against RhoB as a target, because both
farnesylated and geranylgeranylated RhoB were shown to antagonize
transformation and potently suppress human tumor growth in nude mice
(17). Thus, to date, the critical target for FTIs has not been identified.
To further investigate the mechanism of action of FTIs we have studied
the effects of FTIs on cell cycle progression of human cancer cells.
Our goal is to identify the precise step of the cell cycle where a
farnesylated protein or proteins is(are) required. Ultimately, we hope
to identify such a protein using this strategy. Previously, we had
shown that in NIH 3T3 murine fibroblasts protein farnesylation is not
required for G1 to S phase transition (18). In contrast, in
human cancer cells we had shown that FTIs can induce a G1
block, accumulate cells in G2/M or have no effect on cell
cycle distribution, depending on the cell line (19). Here we show by
flow cytometry, immunostaining, and time-lapse videomicroscopy, using
synchronized human lung cancer cells, that FTIs induce a G1
and G2/M block and that the latter occurs through inhibition of bipolar spindle formation and chromosome alignment and
causes a significant accumulation of the cells in prometaphase during mitosis.
Cell Culture--
A-549 cells (ATCC, CCL-185) were maintained in
Kaighn's F-12 medium, and Calu-1 cells (ATCC, HTB-54) were maintained
in McCoy's medium. All media were adjusted to contain 1.5 g/liter
sodium bicarbonate and were supplemented with 10% fetal bovine serum. All cells were maintained in a humidified incubator at 37 °C and 10% CO2.
Flow Cytometry--
Cells were plated to subconfluency in
100-mm2 plates to obtain 5 × 105 to
1 × 106 cells for DNA analysis. After 24 h, the
cells were treated with 30 µM lovastatin for 48 h
and released with 2 mM mevalonic acid in the presence of
vehicle (Me2SO) or 15 µM FTI-2153. Cells were harvested at indicated time points (0 h indicates time of release from
lovastatin block) with trypsin (0.05%)/EDTA (0.53 mM),
washed two times with PBS, resuspended in 500 µl of PBS, and fixed in 4.5 ml of 70% ethanol. Cells were stored in ethanol at Immunocytochemistry--
Cells are grown on 2-well Lab-Tek II
chamber slides (Nunc, Inc., Naperville, IL) to subconfluency and
treated as described above. The cells are fixed in 50% methanol/4%
paraformaldehyde (diluted with PBS from 16% stock (Electron Microscopy
Sciences, Fort Washington, PA)) in a humidified chamber for 20 min at
4 °C and permeabilized with 0.5% Triton X-100 in PBS for 1 h.
For microtubule immunocytochemistry, the fixed cells were stained with
Hematoxylin and Eosin Staining--
The cells were fixed in B5
fixative for 2 h at room temperature. Cells were rinsed with water
and incubated with Lugol's iodine. After several rinses with reagent
alcohol, cells were then stained with hematoxylin and eosin.
Time-lapse Videomicroscopy--
Cells were plated at a density
of 100,000 per 60-mm2 plate and treated with lovastatin as
for flow cytometry. Upon release of the cells from the lovastatin
block, they were viewed using an Olympus K70 phase contrast microscope
for up to 96 h. Still images were captured using Sony SV05800 SVHS
VTR and Vidcap software. Images were manipulated using Adobe Premier
and Adobe Photoshop.
FTI-2153 accumulates synchronized human lung cancer cells in
G0/G1 and G2/M. To determine the
effects of inhibition of protein farnesylation on cell cycle
progression, we have used a standard method to synchronize cells (18).
This was accomplished by blocking cells in G1 by treatment
with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor
lovastatin for 48 h and releasing the block by supplying mevalonic
acid (MVA), the product of the reaction catalyzed by
3-hydroxy-3-methylglutaryl-coenzyme A reductase. The human cancer cells
were either released in the presence or absence of the
farnesyltransferase inhibitor FTI-2153 (21), as described under
"Experimental Procedures." Fig.
1A shows that, in the absence
of FTI-2153, G1-blocked A-549 cells exited G1
16-20 h after MVA release, entered and exited S phase between 20 and 32 h, entered and exited G2/M between 24 and 36 h, re-entered a second cycle reaching G1, S, and
G2/M peaks at 36, 40, and 44 h. In contrast, A-549
cells released in the presence of FTI-2153 were delayed in their
release from the G1 block by about 6 h, entered and
exited the first S phase, and then entered G2/M (Fig. 1A). Some cells accumulated in G2/M but some
exited and entered G1. Although control cells had 59%,
26%, and 11% in G1, S, and G2/M phases,
respectively (96 h after MVA release), FTI-2153-treated cells had 42%,
9%, and 45% (Fig. 1C). A small proportion (2.5% and 1%)
of cells contained 6N and 8N DNA peaks, but FTI-2153 treatment did not
affect these proportions (Fig. 1C). Thus, FTI-2153 induced a
delayed exit from G1 and accumulated A-549 cells in both a
G1 and G2/M phases of the cell division cycle.
Similar results were obtained with another human lung adenocarcinoma,
Calu-1 (Fig. 1B). Here again, FTI-2153 induced a delay in
the first cell cycle and resulted in accumulation of cells in
G1 and G2/M. Calu-1 cells were able to exit
G1, enter and exit S, and enter G2/M. However, a large proportion accumulated in G2/M (Fig.
1B). In contrast to A-549 cells, FTI-2153-treated Calu-1
cells had cells not only with 2N (G0/G1), 3N
(S), and 4N (G2/M) DNA content but also accumulated cells
with 6N and 8N DNA content (96 h after MVA release) (Fig. 1C). However, control cells had 54%, 28%, 12%, 4%, and
2% in G1, S, G2/M, 6N, and 8N peaks,
respectively, FTI-2153-treated cells had 11%, 10%, 31%, 20%, and
28% 96 h after MVA release (Fig. 1C).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. When ready to stain with propidium iodide, cells were centrifuged to
remove the ethanol and washed once in PBS. The cell pellet was then
resuspended in 1 ml of PI/Triton X-100 staining solution (0.1% (v/v)
Triton X-100 in PBS, 0.2 mg/ml RNase A, and 20 µg/ml propidium
iodide) and incubated at room temperature for at least 30 min. DNA
analysis was done using a FACScan flow cytometer (Becton Dickinson, San
Jose, CA) and ModFit LT 2.0 (Verity Software House, Topsham, ME).
-tubulin ((clone B-5-1-2), Sigma T5168) diluted in 0.1% Tween 20, 1% bovine serum albumin in PBS for 1 h and fluorescein isothiocyanate-conjugated goat anti-mouse (Sigma F9006) diluted in
0.1% Tween 20, 1% bovine serum albumin in PBS for 25 min in the dark.
For CENP-E and CENP-F immunostaining, a similar procedure was used
except anti-CENP-E or anti-CENP-F antibodies (20) were used as primary
antibodies and Alexa Fluor 594 goat anti-rabbit antibody (Molecular
Probes, A-11012) was used for the secondary antibody. After final
washes, slides were mounted with Vectashield mounting medium with DAPI
(Vector Laboratories, Inc., Burlingame, CA) and viewed at
magnifications of × 400 and × 1000 with an Orthoplan 2 fluorescence microscope (Leitz) and SmartCapture VP software (Digital Scientific).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FTI-2153 treatment causes an accumulation of
A-549 and Calu-1 cells in G2/M. A-549 (A)
or Calu-1 (B) cells blocked in G1 with
lovastatin were released with mevalonic acid in the absence
(closed diamonds) or presence (open circles) of
FTI-2153 (15 µM), as described under "Experimental
Procedures." Samples were collected every 4 h from 16 to 48 h (A and B) and at 72 h (A), then
analyzed for DNA content by flow cytometry. C, histographic
representation 96 h after mevalonic acid release. a and
b, control and FTI-2153-treated A-549 cells; c
and d, control and FTI-2153-treated Calu-1 cells. Data are
representative of three independent experiments.
FTI-2153 Induces Accumulation of A-549 and Calu-1 Cells in
Prometaphase during Mitosis--
The demonstration that FTI-2153
resulted in accumulation of cells in G2/M suggested that
protein farnesylation is critical for either entering G2,
exiting G2, entering mitosis, or exiting mitosis. To
determine the precise step at which inhibition of protein farnesylation
results in disruption of the G2/M transition, we used
immunostaining techniques under the synchronization conditions of Fig.
1. Synchronized A-549 and Calu-1 cells were grown in the presence or
absence of FTI-2153 and stained for tubulin and DAPI at different
phases of the cell cycle. FTI-2153 treatment had no detectable effect
on the distribution of chromatin and microtubules at interphase when
compared with untreated cells (Fig. 2).
In the absence of FTI-2153 treatment, Fig. 2 (A and
B) shows that Calu-1 and A-549 cells traverse all phases of
mitosis. In prophase the nuclear membrane is disassembled, DNA is
condensed, the spindle poles have begun to separate, and mitotic
bipolar spindle assembly has begun. At this point, asters radiating
from bipolar centrosomes have formed. In metaphase, the chromosomes are
aligned at the metaphase plate, and the mitotic bipolar spindles are
fully formed. At anaphase the spindles begin to depolymerize, pulling
the chromosomes toward each pole. By telophase the chromosomes have
reached their respective poles, and the cell begins to undergo
cytokinesis. In contrast, FTI-2153-treated cells accumulated in mitosis
with chromosomes organized into a rosette configuration with
microtubules radiating from the center (Fig. 2, C and
D). The proportion of FTI-2153-treated cells with a mature
bipolar spindle and chromosomes aligned at the spindle equator are
dramatically reduced (see below).
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We next compared the number of A-549 and Calu-1 cells in the different mitotic phases with and without drug treatment. At least 100 mitotic figures were counted per sample. Fig. 2F shows that, in the absence of FTI-2153, most A-549 cells in mitosis were in telophase, whereas in FTI-2153-treated cells most cells in mitosis were in prometaphase. For example, 48 h after MVA release, 14%, 26%, 2%, and 58% of control cells and 69%, 16%, 0%, and 15% of the FTI-2153-treated A-549 cells were in prometaphase, metaphase, anaphase, and telophase/cytokinesis, respectively. Similarly, in Calu-1 cells, 48 h after MVA release, control cells in mitosis had about 5%, 37%, 3%, and 55%, whereas FTI-2153-treated cells had 84%, 8%, 1%, and 7% in prometaphase, metaphase, anaphase, and telophase/cytokinesis, respectively (Fig. 2E). Thus, treatment of FTI-2153 resulted in a significant accumulation of both A-549 and Calu-1 cells in prometaphase during mitosis.
FTI-2153 Inhibits Chromosome Alignment-- The data described above suggest that a farnesylated protein is critical to a step between prophase and metaphase involving chromosome and/or spindle organization. We first determined the effects of FTI-2153 on organization of chromosomes by three methods, H & E staining, DAPI staining, and time-lapse videomicroscopy.
H & E Staining--
For H & E staining, cells were treated as
described under experiments of Figs. 1 and 2, were harvested at 48 h, fixed with B5 fixative, and stained with H & E to visualize DNA.
Fig. 3A shows that in
vehicle-treated Calu-1 cells, chromatin condenses at prophase, aligns
at metaphase, begins to separate at anaphase, and reaches the cellular
poles at telophase/cytokinesis. In contrast, in FTI-2153 cells, these
chromatin figures are rarely seen. Instead, chromosomes in these cells
are organized into a ring-like shape (Fig. 3A). We counted
the ring shape mitotic figures in control and treated cells. In Calu-1
cells, out of 53 control prometaphase cells, 30% and out of 56 FTI-2153-treated prometaphase cells, 86% had their chromosomes in a
ring-like shape, respectively. Similarly, in A-549 cells (39 control
and 59 FTI-2153-treated prometaphase cells) 5% and 68% of control and
FTI-2153-treated prometaphase cells had this shape, respectively.
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DAPI Staining-- To further characterize these effects of FTI-2153 on chromatin structure, we stained the DNA with DAPI as described under "Experimental Procedures." For each sample at least 38 and as many as 139 mitotic figures at prometaphase were counted. Fig. 3B shows that, in control A-549 cells, 79.0% and 21.0% had normal and ring shapes, respectively, whereas FTI-2153-treated A-549 cells had 26.0% and 74.0% normal and ring-shaped chromosomes, respectively. Similar results were obtained with Calu-1 cells where FTI-2153 increased the ring-shaped chromosome morphology from 27.9% to 71.6% (Fig. 3B).
Time-lapse Videomicroscopy--
The above studies clearly
demonstrate that, in both Calu-1 and A-549 cells, FTI-2153 interferes
with a step leading to prometaphase involving chromosome alignment.
However, a drawback of these studies is that they only reflect pictures
of what happened every 4 h. Yet the whole process of mitosis
occurs around 1-2 h for most cells. To follow in a continuous fashion
the effects of FTI-2153 on the progression through mitosis, we
performed time-lapse videomicroscopy. To this end, A-549 and Calu-1
cells, which had been synchronized and released in the presence or
absence of FTI-2153, were recorded. Several cells were observed for up
to 70 h after MVA release. Figs. 4
and 5 show the cell cycle progression for one cell per sample. In the
absence of FTI-2153, Calu-1 cells
progressed normally through mitosis (Fig.
4A). For the cell shown (marked with the white
star) mitosis started about 35.5 h after MVA release. At this
time, the cell rounded and its DNA (black arrow) condensed initiating prophase. The chromosome then lined up along the metaphase plate, separated during anaphase, and migrated toward the two opposing
poles (see two black arrows). The cells then pinched off in
the middle during telophase/cytokinesis. After completion of mitosis,
the resulting two daughter cells (two white stars) flattened
out again. The average length of mitosis for the 12 cells (from two
independent experiments) analyzed was 56.8 ± 12.6 min. In
contrast, FTI-2153-treated cells behaved quite differently. The cell
followed (white star) was able to round up 45 h after MVA release (Fig. 4B). However, progression through mitosis
did not occur, but the cells eventually flattened back out. No apparent metaphase, anaphase, or telophase was observed. The chromosomes did not
align on the metaphase plate. In the 10 cells (two independent experiments) observed, the cells were able to flatten out again after
an average of 286.3 ± 110.4 min. Therefore, FTI-2153-treated Calu-1 cells appear to synthesize DNA, but they are not able to actually divide. These results are consistent with those of Fig. 1B where FTI-2153 accumulated Calu-1 cells in mitosis.
Furthermore, because these cells are not able to divide, cells with
more than 4N DNA should be more prevalent in FTI-2153-treated cells. We have confirmed this by flow cytometry where 20% and 28% of Calu-1 cells had 6N and 8N DNA, respectively, in FTI-2153-treated samples but
only 4% and 2% in control sample (Fig. 1C).
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As was the case for Calu-1 cells, A-549 cells that were not treated with FTI-2153 were able to traverse all phases of mitosis. Fig. 5A shows that one of these A-549 cells (white star) rounded up to initiate prophase at 68 h after MVA release, aligned its chromosomes (black arrow) at metaphase, and separated them at anaphase. The cell then pinched off into two daughter cells during telophase/cytokinesis (Fig. 5A). The two daughter cells then flattened out. The average time it took 18 cells (from two independent experiments) to go through mitosis was 58.7 ± 15.1 min. A-549 cells released in the presence of FTI-2153 did round up but never went through metaphase, anaphase, or telophase (Fig. 5B). However, out of the 12 cells (from two independent experiments) observed, 11 actually divided and the two daughter cells flattened (Fig. 5B). The average time for the 11 cells to divide is 83.0 ± 25.6 min.
FTI-2153 Inhibits Bipolar Spindle Formation but Has No Effect on
CENP-E and -F Localization--
The data described so far demonstrate
that inhibition of protein farnesylation results in accumulation of
cells in a prometaphase-like state and suggest disruption of
interactions between the spindle and chromosomes or perturbation of
spindle assembly. CENP-E, a kinetochore-associated microtubule
motor that is important for chromosome alignment (20, 22), contains a
potential farnesylation site at its carboxyl terminus. Because the
kinetochore-targeting domain of CENP-E is also located at its carboxyl
terminus (23), farnesylation might be important for kinetochore
binding. We therefore examined the effect of FTI-2153 treatment on the
distribution of CENP-E in Calu-1 and A-549 cells that were in mitosis.
Immunofluorescence staining shows that CENP-E accumulates at the
kinetochores of chromosomes in both control prometaphase cells and
drug-treated cells that were arrested in a prometaphase-like state
(Fig. 6). Thus, FTI-2153 does not affect
the ability of CENP-E to bind to kinetochores. Similar results were
obtained for CENP-F (data not shown), another kinetochore protein that
has a potential farnesylation site at its carboxyl terminus (24).
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We next determine whether FTI-2153 affected bipolar spindle formation.
We treated A-549 and Calu-1 cells as described for the experiments of
Fig. 2, immunostained with an antibody against -tubulin and counted
mono and diaster figures during prometaphase for both vehicle- and
FTI-2153-treated cells. For every sample, at least 33 and as many as 75 mitotic figures were counted. Fig. 3C shows that, in the
absence of FTI-2153, 67.1 ± 0.7% (average of three experiments)
of the Calu-1 mitotic figures counted had diasters. In contrast, in
FTI-2153-treated Calu-1 cells only 2.3 ± 2.7% of the mitotic
figures counted had diasters. Similarly, Fig. 3C shows that,
in A-549 cells treated with vehicle, 76.4% and 87.0% (two
experiments) whereas, in those treated with FTI-2153, only 18.2% and
18.9% of the mitotic figures at prometaphase had diasters. FTI-2153
treatment increased the percentage of mitotic figures with monoasters
from 32.9 ± 0.7% to 97.7 ± 2.7% in Calu-1 cells (Fig.
3C). Similarly, FTI-2153 increased monoaster formation from
13% and 23.6% to 81.8% and 81.1% in A-549 cells. Thus, FTI-2153 inhibited bipolar spindle formation in both A-549 and Calu-1 cells.
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DISCUSSION |
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Our previous results demonstrated that FTIs induce a G2/M accumulation in some human cancer cells, but the precise stage of the cell cycle where cells are arrested was not known (19). In this manuscript we present data demonstrating that inhibition of protein farnesylation blocks cells in prometaphase. FTI-2153-treated Calu-1 and A-549 cells were able to enter mitosis as evident from cell rounding, chromosome condensation, and nuclear membrane disassembly. However, these cells were not able to form bipolar spindles and their chromosomes failed to form a metaphase plate. Thus, although FTIs disrupt mitosis, they are distinct from other anti-cancer drugs, such as Taxol, that stabilize tubulin polymerization and block mitosis at the latter stage of metaphase to anaphase transition (25). FTI treatment outcome appears to be more similar to tubulin polymerization-destabilizing agents, such as vinblastine, which disturb events leading to metaphase (26).
FTI-2153-treated Calu-1 cells accumulated in prometaphase and were not able to divide (Fig. 4). However, these cells were not permanently arrested in mitosis, because they eventually exit mitosis without dividing to produce 6N- and 8N-containing cells. This was confirmed by the flow cytometry data of Fig. 1C where, after prolonged FTI-2153 treatment (96 h), 20% and 28% of the Calu-1 cells contained 6N and 8N DNA content, respectively, as compared with 4% and 2% with untreated cells. In A-549, where cells were able to divide even in the presence of FTI-2153, the percentage of cells containing 6N and 8N DNA is only 2.6% and 0.9%, respectively. Thus, even though in human lung adenocarcinoma cells protein farnesylation appears to be pivotal for the proper progression through mitosis, A-549 but not Calu-1 cells are able to adapt and divide despite the fact that they are not able to form bipolar spindles. It is possible, however, that at higher concentrations of FTI-2153, A-549 would not be able to divide.
Our results suggest that a farnesylated protein(s) is (are) involved in a step critical to bipolar spindle formation and chromosome alignment. We examined the possibility that FTI-2153 arrested cells in mitosis by interfering with the localization of the kinetochore proteins CENP-E and CENP-F, because both proteins contained potential farnesylation sites. However, FTI-2153 treatment did not affect the ability of these proteins to bind to kinetochores during mitosis. This is consistent with a recent study (reported while this work was being reviewed) that shows that another FTI, SCH66336, did not affect the localization of CENP proteins (27). Although SCH66336 did not affect CENP-E localization, the authors suggested that CENP-E might be involved based on the ability of SCH66336 to inhibit the association of CENP-E with microtubules (27). It is possible that FTIs could interfere with the functions of these proteins when they are bound to kinetochores. In the case of CENP-E, its carboxyl terminus has been shown to bind microtubules in vitro (22), and, thus, inhibition of farnesylation might affect its ability to interact with microtubules in vivo. Despite this possibility, we do not believe that the mitotic arrest induced by FTI-2153 is due to disruption of CENP-E function at kinetochores. It has been shown that the primary defect in cells that are deficient for CENP-E function is the inability to align chromosomes properly at the metaphase plate. These cells arrest in mitosis for extended periods of time with their chromosomes distributed in a characteristic pattern with some positioned near one of the two separated poles while others lie in the center of the spindle (20, 23, 28, 29). This pattern is distinctly different from the rosette configuration that is seen in FTI-treated cells. Furthermore, both in vitro and in vivo experiments show that CENP-E is not essential for spindle pole separation, because cells that lack CENP-E function are still able to establish a bipolar spindle (20, 23, 28-30). This outcome is clearly also distinct from that seen for FTI-treated cells where spindle poles failed to separate. Therefore, if CENP-E is the target, the FTI-treated cells should have arrested with a bipolar spindle but unaligned chromosomes.
Based on the large number of cells that accumulated in mitosis with unseparated spindle poles, we believe that the primary targets of FTI-2153 are components that are essential for separation of the spindle poles. It is noteworthy that disruptions of cytoplasmic dynein as well as the Eg5 kinesin-related protein all arrested cells in mitosis with unseparated spindle poles that were surrounded by a ring of chromosomes (31, 32). Indeed, Eg5 has recently been shown to be the target for Monastrol, a compound that was isolated based on its ability to arrest cells in mitosis and that was subsequently shown to inhibit bipolar spindle formation (33). However, neither cytoplasmic dynein nor Eg5 contain the consensus CAAX motif that specifies isoprenylation. Thus, proteins that interact with dynein or Eg5 or those involved in their regulation in cancer cells should be prominent candidates for FTIs. A potential candidate is H-Ras, which is exclusively farnesylated and is known to activate the cyclin-dependent kinase, cdc2, which in turn phosphorylates Eg5 to promote bipolar spindle formation.
Thus, the results presented in this report enhance our understanding of
how inhibition of protein farnesylation results in the accumulation of
cells in the mitotic phase of the cell division cycle. This appears to
involve a critical step for bipolar spindle formation and chromosome
alignment resulting in accumulation of cells in prometaphase during
mitosis. The results implicate protein farnesylation in the regulation
of pathways pivotal to the prophase/metaphase transition.
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ACKNOWLEDGEMENTS |
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We thank Santo Nicosia, Beatrice Saunders, and Debbie Bir from the University of South Florida for their help with time-lapse videomicroscopy and H & E staining. We also thank Jodi Kroeger for her help with flow cytometry analysis. We also thank Laura Francis for typing the manuscript.
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FOOTNOTES |
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* This work was supported by Grant CA67771 from NCI, National Institutes of Health (to S. M. S. and A. D. H.) and by the Flow Cytometry, Pathology and Molecular Imaging cores at the H. Lee Moffitt Cancer Center and Research Institute.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: Drug Discovery
Program, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Dr., MRC-DRDIS, Tampa, FL 33612. Tel.: 813-979-6734; Fax: 813-979-6748; E-mail: sebti@moffitt.usf.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M006213200
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ABBREVIATIONS |
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The abbreviations used are: FPP, farnesyl pyrophosphate; FTI, farnesyltransferase inhibitor; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; MVA, mevalonic acid; H & E, hematoxylin & eosin.
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