ANTICANCER RESEARCH 26: 1909-1916 (2006)
DNA Damage Responses in Cell Cycle G2
Phase and Mitosis – Tracking and Targeting
ANNE HANSEN REE1,2, TROND STOKKE3, ÅSE BRATLAND1,2, SEBASTIAN PATZKE4,
RAGNHILD V. NOME1, SIGURD FOLKVORD1, LEONARDO A. MEZA-ZEPEDA1,
KJERSTI FLATMARK1,5, ØYSTEIN FODSTAD1 and YVONNE ANDERSSON1
Departments of 1Tumor Biology, 2Oncology, 3Radiation Biology, 4Immunology and
5Surgical Oncology, The Norwegian Radium Hospital, 0310 Oslo, Norway
Abstract. Background: In order to determine temporal
responses of cell cycle populations to DNA damage, a rational
combination of cell cycle analyses is critical. Moreover, the
targeting of cell cycle checkpoint responses may modify the
cytotoxic effect of DNA damage. Materials and Methods: The
characteristics of cell cycle populations (DNA content, cell
cycle transitioning of S phase cells and size of mitotic cell
fraction within the total G2/M phase population) in HeLa cells
exposed to ionizing radiation were analyzed using three
individual flow cytometry-based assays. The potential
radiosensitization from inhibiting DNA damage responses was
assessed by the colony formation assay. Results: Irradiation
resulted in an initial accumulation of S phase cells in G2
phase, from which the arrested cells were subsequently released
to enter mitosis. Upon drug inhibition of G2 checkpoint
signaling or mitotic progression, the cytotoxic effect of ionizing
radiation on the HeLa cells was amplified. Conclusion: DNA
damage-induced cell cycle responses, analyzed by selected
cytometry assays and modified by specific targeting, might
contribute to an understanding of how to improve radiotherapy
outcome.
In proliferating cells, DNA damage delays normal cell cycle
progression. A complex network of responses is activated as
soon as the damage is registered in the genome, and these
are rapidly manifested at cell cycle checkpoints. Massive
insults to DNA, such as double-strand DNA breaks
following cellular exposure to ionizing radiation, may induce
checkpoint responses in essentially any phase of the cell
cycle (1), ultimately leading to the outcome of cell survival
Correspondence to: Anne Hansen Ree, Department of Tumor
Biology, The Norwegian Radium Hospital, 0310 Oslo, Norway. Tel:
+47 22-935421, Fax: +47 22-522421, email: a.h.ree@medisin.uio.no
Key Words: Cell cycle, G2 checkpoint, mitosis, molecular targeting,
cervical carcinoma.
0250-7005/2006 $2.00+.40
if DNA is properly repaired or, if not, to cell death (2, 3).
Much effort has been directed to estimating the duration of
individual cell cycle phases and changes in their kinetics to
define strategies that might override checkpoint responses
and, thereby, enhance therapeutic tumor cell killing. In
order to determine such temporal responses, a rational
combination of cell cycle analyses is critical.
This study was aimed at presenting DNA damage-
induced cell cycle profiles deduced from the combination of
three individual cytometry assays. The relative distribution
of individual cell cycle populations was determined by
staining cell suspensions for DNA content. Cells in the
S phase were labeled with 5-bromo-2’-deoxyuridine (BrdU)
to allow tracking of their cell cycle transitioning, and the
size of the mitotic cell fraction within the total G2/M phase
population was specifically measured by simultaneous
analysis of DNA content and light scattering.
While the signaling pathway via the tumor-suppressor
protein p53, the primary regulator of the G1 checkpoint, is
often defective in human solid tumors, the DNA damage-
activated G2 checkpoint signaling, initiated by ATM,
communicates through mediators like Chk1 and Chk2 (1),
which are essential for the checkpoint response and might,
therefore, be candidates for therapeutic targeting (4). A
variety of pharmacological compounds, designed to target
cell cycle regulatory mechanisms, has been shown to
override the DNA damage defense response that prevents
mitotic entry (4). Such G2 checkpoint targeting agents may
have therapeutic potential as radio- or chemosensitizers by
facilitating cell death through mitotic catastrophe.
Moreover, taxanes, which disrupt chromosome segregation
in mitotis, are currently utilized clinically as radiosensitizers
in the treatment of non-small cell lung cancer and head and
neck cancer (5).
Cervical cancer, which is rated among the most common
malignancies globally, is etiologically linked to infection with
human papillomavirus types 16 and 18 (6). It has been
known, for more than a decade, that the cervical carcinoma
1909
 ANTICANCER RESEARCH 26: 1909-1916 (2006)
HeLa cell line harbors the human papillomavirus type 18
strain and therefore expresses the viral oncoprotein E6,
which stably complexes with the cellular p53 protein,
encoded by the wild-type TP53 gene, to facilitate ubiquitin-
mediated p53 degradation (7, 8). By this mechanism, E6
also disrupts cell cycle G1 arrest in response to DNA
damage (9). Given its lack of functional G1 checkpoint
along with its rapid proliferation rate (estimated cell cycling
time of ~18 h (data not shown)) and highly reproducible
colony formation capacity (for evaluation of the in vitro end-
point effect of DNA damage), the HeLa cell line appeared
to be a valid experimental model for tracking and targeting
cell cycle responses to DNA damage.
Materials and Methods
Cell cultures and experimental treatments. The HeLa cells were
routinely grown in RPMI 1640 medium supplemented with 10%
fetal bovine serum (FBS) and 2.0 mM glutamine. The breast
carcinoma MCF-7 cells were kept in Dulbecco’s Minimum
Essential Medium supplemented with 5% FBS in addition to
glutamine. High-energy radiation from a 60Co source was delivered
at a rate of approximately 0.75 Gy/min. The unirradiated control
cells were simultaneously placed at room temperature to obtain
comparable conditions. In experiments using the selective Chk1
kinase inhibitor UCN-01 (National Cancer Institute, Bethesda,
MD, USA), this compound was added at a final concentration of
100 nM to the cell medium, as recommended by the supplier, 15
min before irradiation.
Flow cytometry analyses.
a) Hoechst 33258 staining and analysis: Cells were pelleted in PBS
and fixed in 100% methanol. The cells were subsequently stained
with 1.5 Ìg/ml Hoechst 33258 in phosphate-buffered saline (PBS)
before the DNA content was analyzed by FACS flow cytometry
(Becton Dickinson, Franklin Lakes, NJ, USA), essentially as
previously described (10). The Modfit cell cycle program (Verity
Software House Inc., Topsham, ME, USA) was used to determine
the fractions of cells in the G1, S and G2/M phases from the cell
cycle distribution.
b) BrdU labeling, staining and analysis: Cells were pulse-labeled with
30 ÌM BrdU (Sigma-Aldrich Company Ltd., Dorset, UK) in the
culture medium for 30 min and the experimental treatment was then
applied. Harvested cells were fixed in 100% methanol, as described
above, and extracted in 2 M HCl and 0.5% Triton X-100. The cells
were subsequently stained for BrdU incorporation for 30 min at room
temperature with 20 Ìl anti-BrdU (Becton Dickinson) per one million
cells in 1 ml Tween 20 and 1% bovine serum albumin in 0.5% PBS,
followed by incubation for 30 min at room temperature with the
secondary FITC-labeled anti-mouse antibody (DakoCytomation A/S,
Glostrup, Denmark) diluted 1:25 in 0.5% Tween 20 and 1% bovine
serum albumin in PBS. Finally, the cells were stained for 15 min at
37ÆC with 5 Ìg/ml propidium iodide (Calbiochem/Merck Biosciences
Ltd., Nottingham, UK) and 100 Ìg/ml RNase A (Amersham
Biosciences Ltd., Buckinghamshire, UK) in PBS, and were analyzed
by flow cytometry.
c) Propidium iodide staining and analysis: Cells pelleted in PBS
were extracted in a 3/4 volume of 0.1% Nonidet P40, 150 mM
1910
NaCl, 0.5 mM EDTA, 0.1 mM phenylmethylsulphonyl fluoride and
10 nM phosphate buffer (pH7.4), before a 1/4 volume of 4%
paraformaldehyde was added. After fixation on ice for at least 1 h,
the cells were stained with 12.5 Ìg/ml propidium iodide in 0.1%
Nonidet P40, 150 mM NaCl, 0.5 mM EDTA and 10 mM phosphate
buffer (pH7.4) containing 100 Ìg/ml RNase A, and were analyzed
by flow cytometry. The simultaneous measurement of propidium
iodide fluorescence and light scattering allows for the
determination of mitotic cells since this population binds more dye
than G1- and S-phase cells and scatters less light than G2 phase
cells, respectively (11). Cells treated with 500 ng/ml nocodazole
(Sigma-Aldrich Company Ltd.), a mitotic spindle poison, and
sampled after 12 and 24 h, were analyzed as positive controls for
cell cycle arrest in mitosis.
All experiments for the flow cytometry analyses were repeated
at least three times independently.
Western blot analysis. Protein expression was measured by means of
the standard Western blot technique, essentially as previously
described (10). The primary antibodies were anti-p53 (SC-6243;
Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MDM2
(MAB4134; Chemicon International, Temecula, CA, USA), anti-
Phospho-Chk1 (#2341; Cell Signaling Technology Inc., Beverly,
MA, USA) and anti-·-tubulin (CP06; Calbiochem/Merck
Biosciences Ltd.). The MAB4134 recognizes an epitope, possibly
hypophosphorylated (12), within amino acid residues 154–167 of the
human MDM2 oncoprotein, and the anti-Phospho-Chk1 antibody
detects Chk1 only when activated by serine 345 phosphorylation.
Immune complexes were detected with appropriate horseradish
peroxidase-coupled secondary antibodies, and enzyme activity was
visualized with enzyme-linked chemiluminescence (Amersham
Biosciences Ltd.). All experiments for Western blot analyses were
repeated at least twice independently.
Assessment of clonogenic regrowth. The clonogenic regrowth
efficiency of the HeLa cells was determined by plating single cells
suspended in medium for 6 h prior to irradiation. The appropriate
plating density was aimed to produce 20 to 40 surviving colonies in
each well of 6-well culture plates. Medium containing paclitaxel
(Sigma-Aldrich Company Ltd.) was applied to the designated cell
cultures 12 h after irradiation and replaced by fresh medium after
a drug exposure period of 24 h. For all experimental conditions,
the cell colonies were fixed and stained with 0.1% crystal violet
after a total incubation time of 7 days following radiation exposure.
Colonies of ≥50 cells were counted for computation of the
surviving fraction, and at least four parallel samples were scored in
the thee independent repetitions performed for each treatment
condition.
Results
Cell cycle redistribution following irradiation. Exponentially-
proliferating HeLa cells were exposed to a radiation dose of
8.0 Gy, and the redistribution of the cells within the cell cycle
phases was followed for up to 48 h after the DNA-damaging
treatment. As seen from Figure 1, the radiation exposure
initially resulted in a progression of G1 and S phase cells into
the G2/M phase, which was ~50% of the total cell count
after 12 h. This G2/M phase accumulation seemed to persist
 Ree et al: Cell Cycle Responses to DNA Damage

Figure 1. Time-dependent distribution of HeLa cell cycle phases following exposure to ionizing radiation. The cells were irradiated with a dose of 8.0 Gy
and were further incubated for the indicated time-periods before cellular DNA content was determined by flow cytometry analysis gated for Hoechst
33258 fluorescence. Cells with DNA contents characteristic for G1 and G2/M phase cells were found in channel numbers ~200 and ~400 along the
x-axes, respectively. Scales indicating cell counts (y-axes) are provided, as are the percentages of G1, S and G2/M phase cells of each total cell count
(below the DNA histograms).

for a period of less than 24 h after the DNA damage, as an
almost equally large fraction of the cells (~45%) was also
detected as a G1 phase population at 24 h.
A more detailed profiling of the G2/M phase detention
showed that the DNA-damaged HeLa cells apparently
responded by initially delaying cell cycle progression throughout
the G2 phase, before the cells subsequently escaped G2 arrest
and were released into mitosis (Figures 2 and 3).
Indeed, those cells that had been BrdU-labeled shortly
before irradiation (S phase cells) were detected within the
G2/M phase population already 6 h later (Figure 2, lower left
histogram) and seemed to be consolidated therein 12 h after
DNA damage (Figure 2, upper right histogram). At this
time-point, the mitotic fraction of the G2/M phase
population was significantly smaller than in the unirradiated
HeLa cells (compare the left and middle histograms of
Figure 3), clearly indicating that cells that had arrested in G2
phase had failed to progress further into mitosis. But yet,
12 h after DNA damage, the HeLa cells that had been in G1
phase when irradiated seemed to retain the ability to make
cell cycle transition (observe the shift of cells negative for
BrdU labeling in the upper right histogram of Figure 2).
Furthermore, 24 h after DNA damage, when the majority
of the HeLa cells were detected in either the G1 or G2/M
phases of the cell cycle (as consistently demonstrated by all
sets of flow cytometry data), cells that had escaped G2 arrest
(BrdU-negative cells) initially after irradiation now seemed
to recur in the next G1 phase, as did some cells that had been
in the S phase (BrdU-positive cells) when DNA damage
occured (Figure 2, lower right histogram). This is in
accordance with the observed size of the mitotic fraction
(recovered to baseline; compare the right histogram of Figure
3 with the left and middle ones), consisting of cells that had
been released from the initial radiation-induced G2 arrest.
Lack of functional cell cycle G1 checkpoint in HeLa cells. The
HeLa cells displayed a lack of characteristics consistent with
a functional G1 checkpoint; e.g., absence of both radiation-
induced cell cycle G1 arrest (Figure 1) and mRNA
expression of CDKN1A, encoding the G1 cell cycle inhibitor
p21 (data not shown). All exons of the TP53 gene were
confirmed as wild-type upon analysis of DNA by constant
denaturant gel electrophoresis (data not shown), a method
that detects one-base substitutions, if any, in the DNA
fragments analyzed with high sensitivity (13).
Essentially, after DNA damage the protein encoded by
wild-type TP53 is rapidly stabilized by post-translational
modifications, leading to enhanced expression levels of p53
(14). However, as seen in Figure 4A, irradiation (8.0 Gy)
of the HeLa cells did not up-regulate the level of p53
protein expression. The transient radiation-induced p53
level in the wild-type TP53 MCF-7 cells was used as a
positive control.
p53 stabilization results primarily from disruption of the
interaction between p53 and the MDM2 oncoprotein, which
thereby protects p53 from ubiquitin-mediated degradation
(15). In contrast to the MCF-7 cells, in which the p53
induction 1 to 3 h after irradiation (8.0 Gy) was followed by
an up-regulation of MDM2 expression after 3 to 6 h, as
expected (16), the HeLa cells instead showed a rapidly
induced overexpression of an upper major MDM2 band
15 min after the DNA-damaging treatment had been

1911
 ANTICANCER RESEARCH 26: 1909-1916 (2006)

Figure 2. Time-dependent responses of HeLa S phase cells following exposure to ionizing radiation. The cells were pulse-labeled with BrdU before being
irradiated with a dose of 8.0 Gy and were further incubated for the indicated time-periods. The distribution of cells positive and negative for BrdU (x-axes)
was determined by flow cytometry analysis gated for anti-BrdU-FITC (R1), and emission values exceeding ~103 were considered positive for BrdU labeling.
The propidium iodide content was also analyzed, and G1 and G2/M phase cells were found in channel numbers ~50 and ~100 along the y-axes, respectively.

Figure 3. Time-dependent M phase effects in HeLa cells exposed to ionizing radiation. The cells were irradiated with a dose of 8.0 Gy and were further
incubated for the indicated time-periods before propidium iodide fluorescence (x-axes) and light scattering (y-axes) were determined by flow cytometry
analysis. The cells with DNA contents characteristic for G1 and G2/M phase cells were found in channel numbers ~200 and ~400 along the x-axes,
respectively. Along the y-axes, however, G1 and M phase cells were detected in channel numbers 200-400, whereas S and G2 phase cells were registered
in channel numbers 400-500 and 500-700, respectively. Mitotic cell populations are indicated along with the percentages of M phase cells of each total
cell count. The percentages of M phase cells upon treatment with 500 ng/ml nocodazole for 12 and 24 h were 8.6 and 11.4, respectively (data not shown).

1912
 Ree et al: Cell Cycle Responses to DNA Damage

Figure 4. Checkpoint regulatory proteins in HeLa cells after exposure to ionizing radiation (IR). The cells were treated (+) with IR (8.0 Gy) or were left
untreated (–), and the expression of p53, MDM2 and phosphorylated Chk1 (Chk1-P) was analyzed by Western blot hybridization after the indicated
time- periods. In both sets of analyses, the expression of ·-tubulin was measured as the loading control. (A) The levels of p53 and MDM2 in the HeLa
cells were compared with those found in the breast carcinoma MCF-7 cell line, harboring intact p53 function. (B) The IR-dependent HeLa cell expression
of Chk1 phosphorylated on serine residue 345 was analyzed in the absence (–) or presence (+) of UCN-01 (100 nM).

initiated (Figure 4A). Human tumors with MDM2 gene

amplification, with or without concomitant oncogenic
MDM2 activity, may often show phenotypic inactivation of
their wild-type TP53 gene (17). In the HeLa cells, however,
MDM2 displayed as a normal copy number gene by Southern
blot analysis (18) (data not shown).

Clonogenic regrowth upon ionizing radiation – the impact of

cell cycle G2 and M phase responses. Based on the
observations that the HeLa cells were arrested in the G2
phase 12 h after irradiation but had entered mitosis 24 h
after DNA damage, the possibility of radiosensitization upon
inhibitory targeting of G2 checkpoint signaling or mitotic
progression, essentially by amplifying the cytotoxic effect of
ionizing radiation on clonogenic regrowth, was evaluated.
The HeLa cells were exposed to increasing doses of
ionizing radiation (2.0 to 8.0 Gy) to determine clonogenic
survival (Figure 5). This treatment caused a dose-dependent
loss of colony formation, with a surviving fraction of ~0.12
with the highest radiation dose applied. Figure 5. Inhibition of Chk1 or mitosis modulates the outcome of HeLa
clonogenic regrowth after exposure to ionizing radiation (IR). The cells
The regulatory mechanism of ionizing radiation on G2 phase
were treated with increasing IR doses in the absence (●) or presence of
responses signals through Chk1, as demonstrated previously either UCN-01 (100 nM ● ) or paclitaxel in increasing concentrations (1
(10, 19, 20). Consistently, as seen in Figure 4B, Chk1 nM ■ , 5 nM ■ and 10 nM ▼), to determine relative colony formation
phosphorylation was strongly induced in HeLa cells 1 h after compared to the unirradiated control (mean±SEM, n=3).

1913
 ANTICANCER RESEARCH 26: 1909-1916 (2006)
radiation exposure (8.0 Gy). This induction was attenuated after
12 h. Furthermore, treatment with the Chk1 inhibitor UCN-01
(100 nM) counteracted the Chk1 phosphorylation response
almost completely after 1 h, but apparently less efficiently
throughout the observation period of 12 h.
As a consequence of this inhibitory effect on DNA
damage-induced G2 phase responses mediated by Chk1, the
possible radiosensitizing effect of UCN-01 (100 nM) on the
HeLa cell clonogenicity was measured (Figure 5). Whereas
the ability of clonogenic regrowth after irradiation with the
lower doses (2.0 and 5.0 Gy) was modestly reduced when
UCN-01 was present compared to when absent, the
cytotoxic effect of 8.0 Gy of ionizing radiation was ~3-fold
amplified by UCN-01.
Since G2 phase-arrested HeLa cells had entered mitosis
24 h after radiation exposure, we chose to evaluate the
possible radiosensitizing effect of a mitotic inhibitor during
the period of cell cycle redistribution when the cells were
regenerating the ability of G2/M transitioning. Hence,
paclitaxel was added to the cells 12 h after irradiation and
was subsequently removed after 24 h of drug exposure, and
HeLa cell clonogenicity was compared with that of cells
treated with ionizing radiation alone. In the cells treated
with paclitaxel (1-10 nM), the ability for clonogenic
regrowth after irradiation was reduced by a factor of 2-10,
depending on which paclitaxel concentrations and radiation
doses applied (Figure 5).
Discussion
In this report, we aimed to distinguish temporal cell cycle
responses to DNA damage by combining flow cytometry-
based assays that characterize the distribution of individual
cell cycle phase populations and the kinetics of their
transitioning. We chose to treat the relatively radioresistant
HeLa cell line with rather high radiation doses in the hope
of obtaining clearly defined effects on cell cycle phenotypes
and colony formation capacity. Irradiation resulted in an
initial accumulation of S phase cells in the G2 phase, from
which the arrested cells were subsequently released to enter
mitosis. Upon drug inhibition of the G2 checkpoint signaling
or mitotic progression, the cytotoxic effect of ionizing
radiation on HeLa cells was amplified, consistent with the
concept of an increased probability of tumor cell death
when cellular DNA damage-responses are targeted.
Double-strand DNA breaks may induce checkpoint
responses in essentially any phase of the cell cycle (1). In
the present study, we could follow the responses of BrdU-
labeled cells (S phase cells) as well as the size of the mitotic
fraction within the total G2/M phase population. Whereas
the DNA-damaged S phase cells initially arrested in G2
phase before they progressed into mitosis, their irradiated
counterparts from G1 phase retained the immediate ability
1914
of cell cycle phase transitioning. Twenty-four hours after
irradiation, the cell cycle G1 population did not exclusively
consist of cells that had escaped G2 arrest, but also of cells
positive for BrdU labeling (irradiated S phase cells). Thus,
the G2 checkpoint detention of DNA-damaged S phase cells
did not seem to be absolute, and a portion of the irradiated
S phase population seemed capable of escaping cell death,
which might be directly associated with the relative
resistance of HeLa cells to irradiation (observed clonogenic
regrowth fraction of ~0.12 at 8.0 Gy).
The HeLa cells displayed a typical phenotype of tumor
cells lacking a functional G1 checkpoint as the G1 phase cells
rapidly disappeared while the G2/M phase population
expanded following DNA damage. The HeLa TP53 genotype
was confirmed to be wild-type but was inconsistent with any
radiation-induced p53 protein expression. It has been known,
for more than a decade, that the viral oncoprotein E6
expressed by HeLa cells facilitates ubiquitin-mediated p53
degradation (7) by a mechanism that overrides endogenous
MDM2 activity (8, 21). Yet, the HeLa p53 protein has been
found to be functionally reactivated by therapeutics that
interfere with the p53-MDM2 interaction (22, 23).
In the MCF-7 cells, irradiation led to a rapid decrease in
the expression level of the two major MDM2 polypeptides
(possibly hypophosphorylated (12)) detected with an
antibody against amino acid residues 154-167, clearly
preceding the accumulation of the p53 protein (12, 24). In
the HeLa cells, however, an apparent overexpression of the
upper MDM2 band, rather than its down-regulation, was
seen 15 min after the DNA-damaging treatment was
initiated. This might suggest that the mechanism involving
MDM2 phosphorylation (12, 24) in the signaling pathway
from DNA damage to p53 activation is defective and may,
in addition to the oncogenic E6 activity, partially account
for the lack of p53 stabilization.
The DNA damage response communicated through the
G2 checkpoint signaling pathway was evaluated as a
therapeutic target to possibly enhance HeLa cell
radiosensitivity. Even though UCN-01 counteracted
radiation-induced Chk1 phosphorylation on serine residue
345, consistent with data demonstrated by others (25), the
radiosensitizing effect of this targeting drug seemed to be
modest compared to the enhanced cytotoxic effect
previously reported in irradiated HeLa cells with a defective
Chk1 phenotype (26). This difference might be explained by
a transient nature of the pharmacological inhibition
indicated by the partial recovery of radiation-induced Chk1
phosphorylation after 3 to 12 h of UCN-01 incubation.
Moreover, the signal transduction cascade downstream of
the damaged DNA is very complex and involves a variety of
G2 checkpoint effector mechanisms (1), inviting alternative
effector pathways to compensate when one particular
effector molecule is inhibited.
 Ree et al: Cell Cycle Responses to DNA Damage
The possible radiosensitizing effect of the mitotic
inhibitor paclitaxel was evaluated using a concentration
range of the compound within which the fraction of mitotic
HeLa cells has been found to increase sharply from lack of
mitotic arrest (at 1 nM) to 50-70% arrested cells (at 10 nM)
(27, 28). Yet, the clonogenic survival data indicated that
paclitaxel sensitized the HeLa cells to the cytotoxic effect of
ionizing radiation within the entire concentration range.
The temporal aspect of using a mitotic inhibitor to obtain
tumor cell radiosensitization, i.e., timing of paclitaxel
treatment to the period of cell cycle redistribution when the
cells regenerate the ability of G2/M transitioning, is
probably critical (29).
There is strong scientific evidence that concomitant
cisplatin-based chemotherapy improves disease-free and
overall survival compared to radiotherapy alone for high-
risk early stage cervical cancer. Concurrent use of
cisplatin is also recommended in radiation treatment of
locally advanced disease stages, even though randomized
studies have failed to prove that the addition of cisplatin
to irradiation is clearly advantageous for outcome.
Antimetabolites (hydroxyurea and 5-fluorouracil) have
also been evaluated as radiosensitizing compounds in
cervical cancer, but have not been found to be superior
to cisplatin (30, 31). Whether cell cycle response data
obtained from cell line models like ours might translate
into strategies to improve the radiotherapy outcome in
cervical carcinoma requires further experimental
approaches, but hints, if anything, at an appealing
concept.
Acknowledgements
This work was supported by the Norwegian Cancer Society grant
C-04083 (to Anne Hansen Ree). We thank Kyowa Hakko Kogyo
Co., Ltd. (Tokyo, Japan) and the Division of Cancer Treatment
and Diagnosis, National Cancer Institute (Bethesda, MD, USA) for
the gift of UCN-01. We greatly appreciate the technical support
from Mali Strand Ellefsen and Kirsti Solberg Landsverk
(Department of Radiation Biology, The Norwegian Radium
Hospital, Oslo, Norway).
References
1 Kastan MB and Bartek J: Cell-cycle checkpoints and cancer.
Nature 432: 316-323, 2004.
2 Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R
and Kroemer G: Cell death by mitotic catastrophe: a molecular
definition. Oncogene 23: 2825-2837, 2004.
3 Brown JM and Attardi LD: The role of apoptosis in cancer
development and treatment response. Nat Rev Cancer 5: 231-
237, 2005.
4 Zhou BBS and Bartek J: Targeting the checkpoint kinases:
chemosensitization versus chemoprotection. Nat Rev Cancer 4:
1-10, 2004.
5 Choy H: Taxanes in combined modality therapy for solid
tumors. Crit Rev Oncol Hematol 37: 237-247, 2001.
6 zur Hausen H: Papillomavirus infections – a major cause of
human cancers. Biochim Biophys Acta 1288: F55-F78, 1996.
7 Scheffner M, Werness BA, Huibregtse JM, Levine AJ and
Howley PM: The E6 oncoprotein encoded by human
papillomavirus types 16 and 18 promotes the degradation of
p53. Cell 63: 1129-1136, 1990.
8 Hoppe-Seyler F and Butz K: Repression of endogenous p53
transactivation function in HeLa cervical carcinoma cells by
human papillomavirus type 16 E6, human mdm-2, and mutant
p53. J Virol 67: 3111-3117, 1993.
9 Kessis TD, Slebos RJ, Nelson WG, Kastan MB, Plunkett BS,
Han SM, Lorincz AT, Hedrick L and Cho KR: Human
papillomavirus 16 E6 expression disrupts the p53-mediated
cellular response to DNA damage. Proc Natl Acad Sci USA 90:
3988-3992, 1993.
10 Ree AH, Bratland Å, Nome RV, Stokke T and Fodstad Ø:
Repression of mRNA for the PLK cell cycle gene after DNA
damage requires BRCA1. Oncogene 22: 8952-8955, 2003.
11 Larsen JK, Munch-Petersen B, Christiansen J and Jørgensen K:
Flow cytometric discrimination of mitotic cells: resolution of M,
as well as G1, S, and G2 phase nuclei with mithramycin,
propidium iodide, and ethidium bromide after fixation with
formaldehyde. Cytometry 7: 54-63, 1986.
12 de Toledo SM, Azzam EI, Dahlberg WK, Gooding TB and
Little JB: ATM complexes with HDM2 and promotes its rapid
phosphorylation in a p53-independent manner in normal and
tumor human cells exposed to ionizing radiation. Oncogene 19:
6185-6193, 2000.
13 Hovig E, Smith-Sørensen B, Brøgger A and Børresen AL:
Constant denaturant gel electrophoresis, a modification of
denaturing gradient gel electrophoresis in mutation detection.
Mutat Res 262: 63-67, 1991.
14 Fei P and El-Deiry WS: p53 and radiation responses. Oncogene
22: 5774-5783, 2003.
15 Meek DW and Knippschild U: Posttranslational modification
of MDM2. Mol Cancer Res 1: 1017-1026, 2003.
16 Xia L, Paik A and Li JJ: p53 activation in chronic radiation-
treated breast cancer cells: regulation of MDM2/p14ARF.
Cancer Res 64: 221-228, 2004.
17 Zhang R and Wang H: MDM2 oncogene as a novel target for
human cancer therapy. Curr Pharm Des 6: 393-416, 2000.
18 Forus A, Flørenes VA, Maelandsmo GM, Meltzer PS, Fodstad
Ø and Myklebost O: Mapping of amplification units in the
q13-14 region of chromosome 12 in human sarcomas: some
amplica do not include MDM2. Cell Growth Diff 4: 1065-
1070, 1993.
19 Ree AH, Bratland Å, Nome RV, Stokke T, Fodstad Ø and
Andersson Y: Inhibitory targeting of checkpoint kinase
signaling impairs radiation-induced cell cycle gene regulation:
a therapeutic strategy in tumor cell radiosensitization?
Radiother Oncol 72: 305-310, 2004.
20 Nome RV, Bratland Å, Harman G, Fodstad Ø, Andersson Y
and Ree AH: Cell cycle checkpoint signaling involved in histone
deacetylase inhibition and radiation-induced cell death. Mol
Cancer Ther 4: 1231-1238, 2005.
21 Chen J, Marechal V and Levine AJ: Mapping of the p53 and
mdm-2 interaction domains. Mol Cell Biol 13: 4107-4114,
1993.
1915
 ANTICANCER RESEARCH 26: 1909-1916 (2006)
22 Hietanen S, Lain S, Krausz E, Blattner C and Lane DP:
Activation of p53 in cervical carcinoma cells by small molecules.
Proc Natl Acad Sci USA 97: 8501-8506, 2000.
23 Wesierska-Gadek J, Schloffer D, Kotala V and Horky M:
Escape of p53 protein from E6-mediated degradation in HeLa
cells after cisplatin therapy. Int J Cancer 101: 128-136, 2002.
24 Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y and Shkedy
D: Rapid ATM-dependent phosphorylation of MDM2 precedes
p53 accumulation in response to DNA damage. Proc Natl Acad
Sci USA 96: 14973-14977, 1999.
25 Tian H, Faje AT, Lee SL and Jorgensen TJ: Radiation-induced
phosphorylation of Chk1 at S345 is associated with p53-
dependent cell cycle arrest pathways. Neoplasia 4: 171-180, 2002.
26 Koniaras K, Cuddihy AR, Christopoulos H, Hogg A and
O’Connell MJ: Inhibition of Chk1-dependent G2 DNA damage
checkpoint radiosensitizes p53 mutant human cells. Oncogene
20: 7453-7463, 2001.
27 Jordan MA, Toso RJ, Thrower D and Wilson L: Mechanism of
mitotic block and inhibition of cell proliferation by taxol at low
concentrations. Proc Natl Acad Sci USA 90: 9552-9556, 1993.
1916
28 Jordan MA, Ojima I, Rosas F, Distefano M, Wilson L, Scambia
G and Ferlini C: Effects of novel taxanes SB-T-1213 and
IDN5109 on tubulin polymerization and mitosis. Chem Biol 9:
93-101, 2002.
29 Yildiz F, Perez R and Redpath JL: Paclitaxel exposure time
determines the nature of the interaction with radiation in HeLa
cells: the role of apoptosis. Eur J Cancer 36: 1426-1432, 2000.
30 Einhorn N, Trope C, Ridderheim M, Boman K, Sorbe B and
Cavallin-Stahl E: A systematic overview of radiation therapy
effects in cervical cancer (cervix uteri). Acta Oncol 42: 546-
556, 2003.
31 Loizzi V, Cormio G, Loverro G, Selvaggi L, Disaia PJ and
Cappuccini F: Chemoradiation: a new approach for the
treatment of cervical cancer. Int J Gynecol Cancer 13: 580-
586, 2003.
Received January 11, 2006
Accepted March 16, 2006