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Analysis of Cell-Cycle Checkpoint Pathways in Head and Neck Cancer Cell Lines
Implications for Therapeutic Strategies
Sean C. Coleman, MD;
Zoe A. Stewart, PhD;
Terry A. Day, MD;
James L. Netterville, MD;
Brian B. Burkey, MD;
Jennifer A. Pietenpol, PhD
Arch Otolaryngol Head Neck Surg. 2002;128:167-176.
ABSTRACT
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Objective To determine the mechanism of action of paclitaxel (Taxol) and carboplatin
in cell lines of head and neck squamous cell carcinoma (HNSCC).
Design Four HNSCC cell lines were treated with paclitaxel and carboplatin,
alone or in combination, and evaluated for cell-cycle position by means of
flow cytometry, for molecular determinants of cell cycle by means of Western
blotting and kinase analysis, and for anchorage-independent growth by means
of soft-agar assays.
Results Paclitaxel was more effective at inducing apoptosis and inhibiting anchorage-independent
cell growth, compared with carboplatin. The activity of paclitaxel was correlated
with an elevation of cyclin B1/CDC2 activity, prolonged mitotic arrest, and
Bcl-2 phosphorylation. In contrast, carboplatin arrested cells before mitosis.
Combination treatment with both agents, simultaneously or sequentially, was
more effective at inhibiting cell growth than either single agent. Cellular
outcome was the same regardless of which drug was used first. The order of
addition of these 2 drugs differentially affected cell-cycle position. Paclitaxel
pretreatment arrested cells in mitosis, whereas carboplatin pretreatment or
cotreatment resulted in premitotic arrest.
Conclusions To our knowledge, this study is the first to explore how paclitaxel
and carboplatin, alone or in combination, differentially affect cell-cycle
checkpoint response and HNSCC cell growth. These results provide molecular
validation for the current clinical use of both drugs in combination and set
the stage for analyses of patient tumor specimens.
INTRODUCTION
STRATEGIES FOR the treatment of squamous cell carcinoma of the head
and neck (HNSCC) have traditionally involved surgery and radiotherapy. For
stages I and II cancer, this technique yields a 5-year survival rate of 70%
to 95%. Unfortunately, more than 60% of HNSCC presents as locally advanced,
stages III and IV cancer. Despite aggressive surgery and radiotherapy, disease-free
survival is less than 30% at 3 years in this latter population.1
In patients with stages III and IV cancer, efforts have been made to provide
better functional and oncological outcomes with various chemotherapeutic agents.
Two agents used increasingly in the treatment of HNSCC are paclitaxel (Taxol)
and carboplatin.
Paclitaxel is a natural product from the bark of the western yew tree
and one of a new class of agents known as taxanes.
The effects of paclitaxel are correlated with tubulin polymerization and stabilization
and subsequent arrest of the cells in mitosis.2-6
Phase 2 studies have indicated a response rate to paclitaxel of 20% to 43%
when it is used as a single agent in HNSCC, with a median survival of more
than 9 months and a 1-year survival of 33%.7-9
Carboplatin intercalates into DNA to form a bifunctional covalent link that
interferes with DNA synthesis in the S phase of the cell cycle.10
Used as a single agent, carboplatin has yielded a response rate of 26% in
initial phase 1 trials in HNSCC.11
When combined in the treatment of HNSCC, paclitaxel and carboplatin
have better overall clinical response rates ranging from 32% to 54%.12-13 In a study of advanced stages III
and IV tumors, the combination of paclitaxel and carboplatin resulted in complete
histological response in 28% of patients.13
Clinically, some of the most encouraging results have come from the use of
radiotherapy administered concurrently with paclitaxel and carboplatin. In
phase 2 trials in patients with advanced HNSCC, response rates of 90% to 100%
have been reported with concomitant administration of paclitaxel, carboplatin,
and radiotherapy, with histological response in as many as 66%.14
In patients with metastatic cervical adenopathy at presentation, this regimen
has resulted in histologically proven negative findings in neck specimens
in 63% to 71%.14-16
Although numerous clinical trials have evaluated the efficacy of chemotherapeutic
agents used to treat HNSCC, little attention has been focused on drug mechanism,
specifically as it relates to cell-cycle checkpoint signaling.
Normal eukaryotic cells progress through the cell cycle in a regulated
manner owing to a cascade of biochemical events that coordinates the transition
of cells from one phase to another. During a normal cell cycle, the completion
of mitosis is followed by the G1 phase, in which a regulated series
of events must take place before entry into the S phase. These events include
elevations in D- and E-type cyclin levels, activation of cyclin-dependent
kinases (CDKs), phosphorylation of the retinoblastoma protein (pRb), and subsequent
activation of the E2F transcription factor family.17
Once activated, E2F stimulates the transcription of genes whose protein products
are required for S-phase entry and transition. After S phase, cells enter
the G2 phase, in which additional cyclin/CDK complexes are activated,
in particular, the cyclin B1/CDC2 complex that stimulates mitotic entry.18
Cell-cycle transitions are regulated by checkpoint signaling pathways.
These checkpoint pathways monitor cellular integrity and ensure the completion
of one phase of the cell cycle before initiation of the next phase. When activated
by various forms of cellular or genotoxic stress, checkpoint signaling can
halt cell-cycle progression if abnormalities such as DNA damage, aneuploidy,
or mitotic spindle anomalies exist. At the G1/S-phase transition,
p53, pRb, and a host of CDK inhibitors (p21Waf1/Cip1, p27Kip1, p57Kip2, and p16INK4A) are necessary for
checkpoint function.19 Arrest of the G1 phase mediated by p53 depends on p21Waf1/Cip1 (p21) transactivation;
embryonic fibroblasts from mice null for p21 (p21-/-) demonstrate
a defective G1 checkpoint after genotoxic stress.20
In eukaryotic cells, progression from the G2 phase into mitosis
depends on the activity of cyclin B1/CDC2,18
and is negatively regulated by CDC2 phosphorylation on threonine 14 and tyrosine
15 residues.21-22 Phosphorylation
of CDC2 is regulated by the opposing effects of the activating CDC25C phosphatase
and inhibitory protein kinases, Wee1 and Myt1.23
Phosphorylation of CDC2 appears to play a critical role in enforcing the G2-phase cell-cycle checkpoint after DNA damage. During mitosis, the
spindle checkpoint monitors spindle microtubule structure and chromosome alignment.24-25
In the current study, we explored how the chemotherapeutic agents paclitaxel
and carboplatin modulate cell-cycle events in HNSCC cell lines. A panel of
HNSCC cell lines were treated with various combinations of paclitaxel and
carboplatin, and the effects on cell-cycle progression, checkpoint signaling
pathways, and cell growth were examined. Our primary objective was to gain
insight into the mechanisms of drug action as they pertain to cell-cycle checkpoints
in HNSCC. The ultimate goal is to use these preclinical molecular findings
to devise improved clinical trials of paclitaxel and carboplatin and to provide
clues to additional molecular mechanisms that can be targeted for the discovery
of new drugs for the treatment of HNSCC.
MATERIALS AND METHODS
CELL LINES, GROWTH CONDITIONS, AND TREATMENT
All HNSCC lines used were maintained at 37°C under 5% carbon dioxide
in a monolayer culture in Dulbecco Modified Eagle Hi-glucose Medium (GIBCO
BRL, Grand Island, NY) supplemented with 10% fetal bovine serum; 1% penicillin
(100 U/mL); streptomycin (100 U/mL); a combination of insulin, transferrin,
and selenium (50 µg/mL) (Roche Molecular Biochemicals, Indianapolis,
Ind); and 0.1mM nonessential amino acids (GIBCO BRL). When indicated, cells
were treated with 100 nM of paclitaxel (Taxol; Sigma-Aldrich Corp, St Louis,
Mo) resuspended in dimethyl sulfoxide or 100 µM of carboplatin (Bristol-Myers
Squibb, Princeton, NJ) added directly to the cell media. Dimethyl sulfoxide
was used alone as a vehicle control. The human colorectal carcinoma cell line,
HCT116, and the isogenic derivative lines, HCT116 p53-/- and HCT116
p21-/-, were cultured in McCoys 5A medium (GIBCO BRL) supplemented
with 10% fetal bovine serum and a combination of 1% penicillin and streptomycin
(100 U/mL). Cells were treated with doxorubicin hydrochloride (Adriamycin)
as indicated.
FLOW CYTOMETRY
Control and treated cells were trypsinized, the trypsin was inactivated
with serum, and 1 x 106 cells were divided into aliquots
for flow cytometry. The remaining cells were processed for protein analysis.
Cells were incubated in propidium iodide (20 µg/mL) (Sigma-Aldrich Corp),
and the DNA content was measured for 15 000 events for each sample (FACSCaliber
instrument; Becton, Dickinson & Co, Franklin Lakes, NJ). Data were plotted
using CellQuest software (Becton, Dickinson & Co).
WESTERN BLOTTING
Cellular proteins were prepared for Western blotting as previously described.26 The membranes were incubated with mouse monoclonal
antibodies against p53 (PAb 1801), p21 (EA10) (Calbiochem; Oncogene Research
Products, San Diego, Calif), cyclin B1 (GNS 1), Bcl-2 (clone 100), CDC2 (clone
17) (Santa Cruz Biotechnology, Santa Cruz, Calif), MPM-2 (Upstate Biotechnology,
Lake Placid, NY), and goat polyclonal antibodies against actin (Santa Cruz
Biotechnology). Primary antibodies were detected using goat anti–mouse
and rabbit anti–goat horseradish peroxidase–conjugated secondary
antibodies (Pierce, Rockford, Ill) and subjected to enhanced chemiluminescence
detection.
KINASE ASSAY
For each condition, protein extract was prepared. We immunoprecipitated
150 µg of cellular protein with a cyclin B1–specific antibody
(GNS1; Santa Cruz Biotechnology) and performed in vitro kinase assays as previously
described.26
SOFT-AGAR ASSAY
The UNC-7, UNC-10, UM-14C, and UM-38 cells were plated at 1 x
104 per 35-mm dish in supplemented growth media with 0.4% agar
and the indicated concentrations of carboplatin and paclitaxel. Colonies were
counted after 14 days (Omnicon 3800 Image Analysis System; BioLogics Inc,
Gainesville, Va) and Tumor Colony Analysis Software (Version 2.11a; BioLogics
Inc). Values represent 2 independent experiments performed in quadruplicate.
RESULTS
EFFECTS OF PACLITAXEL AND CARBOPLATIN ON CELL-CYCLE PROGRESSION
To determine the effects of paclitaxel and carboplatin on cell-cycle
progression, we initially treated 2 HNSCC cell lines, UNC-7 and UM-38, with
each drug and observed the effects for 72 hours by means of flow cytometry
(Figure 1). Treatment of the cells
with paclitaxel (100 nmol/L) caused a significant increase in cells at the
G2/M phase, with most cells arrested in mitosis by 24 hours. By
48 to 72 hours, a significant fraction of the UNC-7 cells had a subdiploid
DNA content indicative of apoptosis (Figure
1). The increase in the percentage of the population with a subdiploid
DNA content was accompanied by the appearance of cells with nuclear blebbing
and chromatin condensation as visualized by staining with 4',6-diamidino-2-phenylindole
(data not shown). The UM-38 line underwent a similar arrest of the G2/M phase by 24 hours after paclitaxel treatment; however, most of the
UM-38 cells maintained this arrest through 72 hours (Figure 1). Carboplatin (100 µmol/L) treatment of both cell
lines caused an increase in the S-phase fraction between 18 and 24 hours and
an eventual arrest of the UM-38 cells at the G2/M phase.
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Figure 1. Paclitaxel (Taxol) and carboplatin
induce differential cell-cycle profiles in cell lines of head and neck squamous
cell carcinoma. Results of flow cytometric analysis of asynchronous cultures
of UNC-7 and UM-38 cell lines treated with paclitaxel (100 nmol/L) and/or
carboplatin (100 µmol/L) are seen. Cells were harvested at the indicated
times; 15 000 events were analyzed for each sample. Content of DNA is
represented on the x-axis; number of cells counted is represented on the y-axis.
Data represent 3 independent experiments.
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Since these 2 chemotherapeutics are administered concurrently in the
clinical setting, we next examined the effect of simultaneous addition of
paclitaxel (100 nmol/L) and carboplatin (100 µmol/L) to both cell lines
in culture. The concurrent treatment of UNC-7 cells with both drugs resulted
in a flow cytometric profile that was similar to that observed after treatment
of the cells with paclitaxel alone; however, arrest of the G2/M
phase was attenuated, with more cells remaining in the S-phase fraction (Figure 1). Simultaneous treatment of the
UM-38 cells with both drugs also resulted in a similar cell-cycle profile
to that observed with paclitaxel alone (Figure
1).
EFFECTS OF PACLITAXEL AND CARBOPLATIN ON CHECKPOINT SIGNALING PATHWAYS
To gain insight into the molecular mechanisms behind the differential
cell-cycle responses and outcomes observed after paclitaxel and carboplatin
treatment, alone or in combination, we analyzed the drug-induced modulation
of cell cycle and cell viability relative to the levels and activity of proteins
involved in cell-cycle checkpoint signaling. For all subsequent experiments,
we analyzed the following 4 HNSCC cell lines: UNC-7, UNC-10, UM-14C, and UM-38.
All 4 cell lines were treated with paclitaxel (100 nmol/L), carboplatin (100
µmol/L), or a combination of both drugs. Cell-cycle and molecular analyses
were performed at 6, 12, 24, and 36 hours after treatment. Paclitaxel induced
an accumulation of cells in the G2/M phase by 24 hours in all cell
lines. Carboplatin treatment stimulated an increase in the fraction of S-phase
cells in all cell lines when used as a single agent. When given in combination
with paclitaxel, carboplatin uniformly attenuated arrest of the G2/M
phase. Thus, regardless of the differing genetic alterations present in the
4 tumor cell lines under examination, modulation of cell-cycle position was
relatively similar after a given drug treatment. These observations prompted
an examination of the levels, phosphorylation status, and activity of select
cell-cycle regulatory proteins.
Cellular response to genotoxic agents and microtubule inhibitors has
been linked to p53-mediated signaling. Thus, we examined p53 protein levels
and activity by means of Western blotting. We assessed p53 activity by looking
for increased levels of the p53 downstream target gene product, p21. We observed
a modest increase in p53 levels after 36 hours of carboplatin treatment, alone
or in combination with paclitaxel, in the UNC-7 cells only (Figure 2A). However, the increase in p53 level was not sufficient
to induce expression of the downstream target p21, suggesting that the p53
signaling pathway is altered in this cell line. Levels of p53 were significantly
elevated and not altered when compared with controls in the remaining lines,
consistent with the presence of a mutant p53 protein (Figure 2B-D).
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Figure 2. Molecular analyses of cell-cycle
regulatory proteins in head and neck squamous cell carcinoma (HNSCC) after
paclitaxel (Taxol) and carboplatin treatment. Asynchronous cultures of UNC-7
(A), UNC-10 (B), UM-14C (C), and UM-38 (D) cells were treated with paclitaxel
(100 nmol/L) or carboplatin (100 µmol/L) for the indicated times, and
protein was harvested. Western blotting of actin, cyclin B1, p53, p21, CDC2,
MPM-2, and Bcl-2 was performed. Cyclin B1/CDC2 kinase activity was also evaluated.
The indicated HNSCC lines and HCT116 cells positive for p53 and HCT116 cells
null for p53 were treated with doxorubicin hydrochloride (Adriamycin) (350
nmol/L) for 24 hours, and protein was harvested (E). Western blotting of p53
and p21 was performed. Results represent 2 independent experiments. IP indicates
immunoprecipitation.
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To further analyze the p53 status of the 4 cell lines, we compared,
side by side, the basal p53 and p21 protein levels in all 4 HNSCC cell lines
under control conditions (Figure 2E).
Levels of p53 protein were elevated in the UNC-10, UM-14C, and UM-38 cells
compared with the UNC-7 cells, consistent with the presence of mutant p53
in the first 3 lines. In parallel, UNC-7 cells were treated with doxorubicin,
an anticancer agent that induces stabilization of only wild-type p53 protein
and elevation of p21 protein levels in cells containing functional, wild-type
p53. After doxorubicin treatment, we performed Western blotting to compare
the relative levels of p53 and p21 proteins under control and treated conditions
with those observed in a well-characterized isogenic set of colon carcinoma
epithelial cell lines that contains wild-type p53 (HCT116) or is null for
p53 (HCT116 p53-/-) owing to homologous recombination at the p53
locus.27 As seen in Figure 2E, relative to the robust elevation in p53 and p21 protein
levels in the HCT116 cells, the elevation in p53 and p21 protein levels in
the UNC-7 cells was minimal. Furthermore, the very low level p53-independent
elevation of p21 observed in the HCT116 p53-/- cells after doxorubicin
treatment was similar to that observed in the UM-14C and the UM-38 cells after
paclitaxel and carboplatin treatment (Figure
2C-D). The doxorubicin-induced, p53-independent elevation of p21
levels was not sufficient to inhibit CDK activity in the HCT116 p53-/-
cells as previously shown by Flatt et al,28
and it was not sufficient to induce cell-cycle arrest in the UNC-7 and UNC-10
cells.
Several studies indicate that the mitotic arrest mediated by microtubule
inhibitors such as paclitaxel may result in cytotoxicity through alteration
of normal mitotic signal transduction pathways, including prolonged CDC2 activity.29-30 Numerous studies suggest a link between
CDC2 activation and apoptosis.31-34
In the current study, all of the HNSCC cells arrested with a 4N DNA content
by 24 hours after paclitaxel treatment. The cell-cycle arrest was accompanied
by elevated levels of cyclin B1 protein and cyclin B1–immunoprecipitable
kinase activity (Figure 2A-D). The
presence of a faster migrating or hypophosphorylated form of CDC2 after paclitaxel
treatment was consistent with transition into mitosis and elevated levels
of cyclin B1/CDC2 activity. We verified that paclitaxel induced mitotic arrest
in all 4 HNSCC cell lines when we detected significant increases in MPM-2
epitope positivity on results of Western blotting (Figure 2A-D). A previous study by Davis et al35
has shown that the MPM-2 antibody recognizes phosphorylated protein epitopes
found only in mitotic cells. Thus, the MPM-2 antibody can distinguish 4N DNA–containing
mitotic cells from those in G2 phase. The length of the paclitaxel-mediated
elevation in CDC2 activity varied among the different cell lines; however,
a peak in activity preceded the accumulation of subdiploid cells that was
observed at 24 hours after treatment.
Previously, sensitivity to anticancer agents has been shown to be influenced
by alterations in posttranslational modifications of Bcl-2 family members,
including the antiapoptotic protein Bcl-2.36-40
After paclitaxel treatment, Bcl-2 phosphorylation was evident in all the HNSCC
cell lines by 12 hours, when cells first began to accumulate in mitosis and
to acquire cyclin B1/CDC2 activity (Figure
2A-D). When peak levels of cyclin B1/CDC2 activity were apparent,
there was a significant conversion of Bcl-2 to the phosphorylated forms in
all the HNSCC cell lines. In fact, we observed a correlation between the relative
levels of cyclin B1/CDC2 activity and Bcl-2 phosphorylation.
Significant differences in the regulation of select cell-cycle signaling
pathways accompanied the differential cell-cycle arrest induced by carboplatin
in all the HNSCC cell lines (Figure 2A-D).
Carboplatin treatment resulted in an almost complete inhibition of cyclin
B1/CDC2 activity, which was likely integral for the observed S-phase arrest.
Between 24 and 36 hours, we saw a significant conversion of CDC2 from the
hypophosphorylated, active form in the control cells to the hyperphosphorylated,
inactive form of CDC2. Consistent with these changes in the phosphorylation
status of CDC2 was an almost complete loss of cyclin B1–immunoprecipitable
kinase activity and the absence of MPM-2 positivity on results of Western
blotting. When carboplatin was given simultaneously with paclitaxel, attenuation
of the paclitaxel-induced elevation in cyclin B1/CDC2 activity, mitotic entry,
and Bcl-2 phosphorylation were seen in all 4 cell lines examined (Figure 2A-D).
ALTERATION OF CELL-CYCLE SIGNALING AFTER DIFFERENTIAL ORDER OF DRUG
ADDITION
Because simultaneous treatment of the HNSCC cells with paclitaxel and
carboplatin led to an attenuation of mitotic arrest, we explored whether pretreatment
of the cells with one drug before addition of the second would provide clues
to the molecular changes that may be consistent with apoptosis. To accomplish
this, parallel experiments were designed in which HNSCC cell lines were subjected
to one drug (paclitaxel or carboplatin) for 8 hours, followed by cotreatment
with the second drug for 6, 12, 24, and 36 hours (Figure 3).
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Figure 3. Effect of paclitaxel (Taxol) or
carboplatin pretreatment on the cell-cycle kinetics of head and neck squamous
cell carcinoma. Results of flow cytometric analysis of asynchronous cultures
of UNC-7 (A), UNC-10 (B), UM-14C (C), and UM-38 (D) cells treated with paclitaxel
(100 nmol/L) or carboplatin (100 µmol/L) are seen. Prepaclitaxel or
precarboplatin indicates that the cells were pretreated with the drug for
8 hours before simultaneous treatment with both drugs. Cells were harvested
at the indicated times; 15 000 events were analyzed for each sample.
Content of DNA is represented on the x-axis; number of cells counted is represented
on the y-axis. Data represent 3 independent experiments.
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In the first set of experiments, cells were treated with paclitaxel
for 8 hours, then treated with carboplatin. After this treatment regimen,
an arrest of the G2/M phase occurred at 6 to 12 hours and the subdiploid
population increased at 24 to 36 hours in the UNC-7, UNC-10, and UM-14C cells
(Figure 3). The increase in S-phase
fraction seen in the previous carboplatin treatments was reduced with this
treatment regimen. As seen in the experiment, the UM-38 line maintained the
arrest of the G2/M phase (Figure
3D). The same lines were exposed to carboplatin for 8 hours before
paclitaxel treatment. The accumulation of a G2/M-phase population
was delayed in all lines except the UM-38 cells (Figure 3). In the UM-38 cells, there was a transient elevation in
S-phase cell levels at 6 to 12 hours, followed by an arrest of cells with
a 4N DNA content by 24 hours (Figure 3).
The remaining lines, UNC-7, UNC-10, and UM-14C, accumulated cells in the S
phase without the appearance of a G2/M-phase arrest, similar to
carboplatin treatment (Figure 3).
To determine what effect order of addition of paclitaxel and carboplatin
had on molecular cell-cycle determinants, we examined the G1/S-phase
checkpoint regulators, p53 and p21, and the mitotic markers examined in previous
treatments (Figure 4). Modulation
of p53 was not significantly different from that shown in the previous section.
Levels of mutant p53 remained unchanged in the UM-14C, UM-38, and UNC-10 cells
(Figure 4B-D). In the UNC-7 cells,
p53 levels increased with the carboplatin pretreatment schedule, but not with
paclitaxel pretreatment (Figure 4A).
Again, p21 was regulated in a p53-independent manner in the UM-14C and UM-38
cells (Figure 4C-D). In the UM-38
cells, a decrease in p21 protein was seen by 24 to 36 hours after pretreatment
with paclitaxel or carboplatin. This latter change is likely the result of
altered downstream p53 signaling. Again, as in the previous section, these
differences did not significantly affect the cell-cycle kinetics.
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Figure 4. Molecular analyses of cell-cycle
regulatory proteins in head and neck squamous cell carcinoma after paclitaxel
(Taxol) and carboplatin pretreatment. Asynchronous cultures of UNC-7 (A),
UNC-10 (B), UM-14C (C), and UM-38 (D) cells were treated with paclitaxel (100
nmol/L) and carboplatin (100 µmol/L). Pretreatment consisted of 8 hours'
exposure to the first drug followed by the second drug as indicated, and protein
was harvested. Western blotting of actin, cyclin B1, p53, and p21, CDC2, MPM-2,
and Bcl-2 was performed. Cyclin B1/CDC2 kinase activity was also evaluated.
Results represent 2 independent experiments. IP indicates immunoprecipitation.
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Perhaps the most accurate molecular indicators of mitosis in paclitaxel-treated
cells were the levels of MPM-2, Bcl-2, and cyclin B1 and the activity of CDC2
(Figure 4). In the UNC-7, UNC-10,
and UM-38 cells pretreated with paclitaxel for 8 hours before the addition
of carboplatin, the mitotic arrest was prolonged compared with the simultaneous
treatment regimen (Figure 4A, B,
D). Coadministration resulted in the mitotic markers reverting from the mitotic
form to the premitotic form between 12 and 24 hours. In contrast, after paclitaxel
pretreatment, the mitotic indices were still present at 24 hours, consistent
with a prolonged mitotic arrest. In the UM-14C cells, the patterns for simultaneous
administration were similar to those for the paclitaxel pretreatment arm,
which likely indicates cell-to-cell variability (Figure 4C). In the carboplatin pretreatment schedule, there was
a conversion of CDC2 to the slower migrating, inactive form, a loss of cyclin
B1 activity and MPM-2 positivity, and maintenance of Bcl-2 in the premitotic,
dephosphorylated form (Figure 4).
These data correlate with results of the flow cytometric analyses (Figure 3) and suggest that these cells never
entered mitosis.
EFFECTS OF PACLITAXEL AND CARBOPLATIN ON ANCHORAGE-INDEPENDENT GROWTH
To extend the studies performed in monolayer and to further examine
the chemosensitivity of the HNSCC cell lines, the effect of paclitaxel and
carboplatin on anchorage-independent growth was assayed by means of growth
in soft agar (Figure 5). In 1 arm
of the experiment, cells were treated in soft agar with paclitaxel or carboplatin
alone or simultaneously. Alternatively, cells were pretreated with paclitaxel
or carboplatin for 8 hours in monolayer culture, followed by anchorage-independent
growth in the presence of both agents. All 4 cell lines displayed the least
sensitivity to carboplatin as a single agent. In the UNC-7 and UM-38 cells,
paclitaxel treatment alone or combined with carboplatin reduced colony formation
by approximately 50%, compared with controls, regardless of whether the cells
were treated with both drugs simultaneously or sequentially. In the UNC-10
and UM-14C cells, the combination of both agents resulted in a more significant
reduction in colony number than use of either agent alone. Overall, order
of addition did not significantly affect outcome relative to simultaneous
addition of both drugs in this assay.
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Figure 5. Effect of paclitaxel (Taxol) and
carboplatin on the anchorage-independent growth of head and neck squamous
cell carcinoma. UNC-7 (A), UNC-10 (B), UM-14C (C), and UM-38 (D) cells were
treated with paclitaxel (100 nmol/L) and/or carboplatin (100 µmol/L).
Cells were treated in soft agar with paclitaxel alone, carboplatin alone,
or simultaneously, or pretreated with carboplatin or paclitaxel for 8 hours
in monolayer culture followed by soft-agar growth in the presence of both
agents. Colonies were grown for 14 days in soft agar before quantification.
Results represent 8 independent determinations. Error bars represent SDs.
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COMMENT
In an effort to increase effectiveness and decrease the toxicity of
chemotherapeutic agents in the treatment of HNSCC, combination therapy has
evolved. Until recently, these treatments have been reserved for inoperable
or recurrent tumors. In the treatment of HNSCC, chemoradiotherapy may have
similar efficacy to surgery in select cases. By avoiding surgery, organ preservation
and improved quality of life may be enjoyed by the patient. Several groups
have demonstrated significant increases in response rates of head and neck
tumors when treated with concomitant paclitaxel, carboplatin, and radiation
therapies14-16,41;
however, improvement in survival has not yet been shown. The optimistic results
of these phase 2 trials were the impetus for determining mechanism of action
of these drugs in HNSCC cells.
We undertook the current study to delineate the response of 4 HNSCC
cell lines to treatment with paclitaxel or carboplatin alone or in combination.
Specifically, we examined the cell-cycle progression of these lines and determined
the effect of these treatments on molecular mechanisms of cell-cycle control.
There was a defect in the G1/S-phase checkpoint in all of the lines
studied as determined by the lack of G1-phase arrest after treatment
with the genotoxic agent carboplatin. However, regardless of the genetic alteration(s)
that led to the loss of G1-phase checkpoint control, paclitaxel
was more effective at inhibiting anchorage-independent cell growth compared
with carboplatin, in all of the cell lines. The activity of paclitaxel was
correlated with an elevation of cyclin B1/CDC2 activity, a prolonged mitotic
arrest, and Bcl-2 phosphorylation. In contrast, carboplatin induced predominantly
an S-phase arrest, except in the UM-38 cells. Combination treatment, simultaneously
or sequentially, was more effective than the use of either agent alone in
the inhibition of HNSCC growth. Furthermore, when used in combination, the
order of drug administration differentially affected cell-cycle position.
However, overall cell outcome was the same, regardless of which drug was used
first.
The mechanism of action of the agents under study is proposed to be
cell-cycle dependent. Paclitaxel exerts its activity by polymerizing tubulin,
causing its stabilization and the subsequent arrest of cells in mitosis.2-6
Carboplatin intercalates into DNA and interferes with DNA synthesis, arresting
cells in the S phase.10 Since both drugs cause
differential cell-cycle arrest with respect to one another, the activity of
one drug may be affected by the second drug in the treatment of HNSCC. We
hypothesized that the order of addition of these drugs would alter the effectiveness
of the combination treatment against HNSCC. However, this was not the case.
Although combination treatment was more effective than use of a single agent,
the order of addition did not significantly alter outcome. Pretreatment or
simultaneous cotreatment with carboplatin abrogated the mitotic response of
paclitaxel as determined by molecular markers (eg, elevation of cyclin B1/CDC2
activity, MPM-2 positivity, and Bcl-2 phosphorylation). Other studies have
shown that paclitaxel can act as a radiosensitizer by arresting cells in mitosis,
the most radioresponsive phase of the cell cycle.42
If mitotic arrest is required for radiosensitization, then we hypothesize
that the premitotic cell-cycle arrest we observed in HNSCC cells after simultaneous
carboplatin and paclitaxel treatment would abrogate the radiosensitizing effects
of paclitaxel. Additional preclinical studies will be required to test this
hypothesis, the results of which may have significant clinical relevance.
Several studies have shown that tumor cells with defective checkpoint
function are more vulnerable to anticancer agents.23
If one considers the frequency of alterations in p53 and pRb and their upstream
and downstream regulatory pathways in HNSCC, most have defective checkpoint
function. For example, more than 80% of primary HNSCCs examined by Reed et
al43 contain inactivated p16INK4a, which would
result in defective G1/S-phase checkpoint function. Likewise, p5344-45 and pRb46
mutations have been found in many HNSCCs examined. Approximately 50% of patients
with HNSCC had a mutation within the p53 gene.47
Overexpression of MDM-2, which can inactivate p53, was found in 78% of oral
squamous cell carcinomas and 52% of dysplastic, premalignant lesions, suggesting
a role for MDM-2 overexpression as in the genesis of HNSCC.48
Elevated CDK6 levels, which can lead to hyperphosphorylation of pRb and deregulated
S-phase entry, were found in all the HNSCC specimens examined in a recent
study by Timmermann et al.49 However, it remains
to be determined whether these genetic alterations have prognostic significance
in HNSCC.
In patients with advanced inoperable HNSCC treated with platinum-based
radiochemotherapy, mutant p53 was associated with improved local progression-free
survival.50 One possible explanation for this
observation is that defective DNA repair in tumor cells may lead to increased
tumor cell death. Chomchai et al51 have shown
improvement in overall survival and a trend toward improved disease-free survival
in patients with p53 mutation, regardless of the treatment method. However,
in a recent survey of patients undergoing primary radiochemotherapy with carboplatin,
Haas et al52 showed no correlation of p53,
p21, pRb, p16INK4A, or Bcl-2 status with remission rate, locoregional recurrence
rate, or survival. Thus, the latter results refute the notion that these cell-cycle
regulators may provide prognostic significance. However, Haas et al demonstrated
that cyclin D1 overexpression was correlated with a significantly shortened
overall survival. In other studies, the apoptotic family of proteins has been
examined with regard to prognosis. In 1 study, ectopic expression of the antiapoptotic
protein, Bax, in HNSCC cells was found to increase sensitivity of the cells
to a variety of anticancer agents, including paclitaxel.53
Although an ever-increasing number of tumor specimens are undergoing molecular
analyses, the prognostic significance of the findings is not well defined,
and in some cases is controversial. Indeed, this area of research requires
further investigation.
CONCLUSIONS
To our knowledge, this study is the first to explore how paclitaxel
and carboplatin, used alone or in combination, differentially affect cell-cycle
checkpoint response and HNSCC cell growth. Paclitaxel was a more effective
single agent than carboplatin; however, combination therapy was the most effective
at inhibiting tumor cell growth. Sequential combination of both drugs was
equally effective at inhibiting cell growth as simultaneous cotreatment. Carboplatin
pretreatment or cotreatment resulted in a prevention of the mitotic arrest
seen with paclitaxel. This premitotic arrest may have implications for the
radiosensitizing ability of this combination. These results provide molecular
validation for the current clinical use of these 2 drugs in combination and
set the stage for analyses of patient tumor specimens.
Many laboratories are now searching for compounds that interfere with
cell-cycle checkpoints, in the hope that such agents will be more effective
in anticancer therapy. As our knowledge of cell-cycle checkpoint regulation
and the mechanism of action of currently used anticancer agents in the treatment
of HNSCC increases, so will the number of signaling molecules and pathways
that can be used as targets for rational drug design. We hope that a detailed
understanding of these processes will lead to evolution of more incisive approaches
to HNSCC treatment that exploit the molecular defects in HNSCC cell-cycle
control.
AUTHOR INFORMATION
Accepted for publication September 10, 2001.
This study was supported by grants CA70856 (Dr Pietenpol) and ES00267
and CA68485 (Core services) from the National Institutes of Health, Bethesda,
Md; and a Burroughs Wellcome Fund Grant, Research Triangle Park, NC (Dr Pietenpol).
The UM-14C and UM-38 HNSCC cell lines were kindly provided by Thomas
Carey, PhD, Kresge Research Institute, University of Michigan, Ann Arbor;
UNC-7 and UNC-10, by Wendell Yarbrough, MD, University of North Carolina,
Chapel Hill; and HCT116, HCT116 p53-/-, and HCT116 p21-/-,
by Bert Vogelstein, MD, Johns Hopkins Oncology Center, Baltimore, Md.
Corresponding author: Jennifer A. Pietenpol, PhD, Department of Biochemistry,
Vanderbilt University School of Medicine, 652 The Preston Building, 2220 Pierce
Ave, Nashville, TN 37232-6305 (e-mail: pietenpol{at}toxicology.mc.vanderbilt.edu).
From the Vanderbilt Bill Wilkerson Center for Otolaryngology and Communication
Sciences, Department of Otolaryngology (Drs Coleman, Day, Netterville, and
Burkey), the Department of Biochemistry (Drs Stewart and Pietenpol), the Center
in Molecular Toxicology (Dr Pietenpol), and the Vanderbilt-Ingram Cancer Center
(Dr Pietenpol), Vanderbilt University School of Medicine, Nashville, Tenn.
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