Int. J. Radiation Oncology Biol. Phys., Vol. 59, No. 4, pp. 928 –942, 2004
Copyright © 2004 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/04/$–see front matter
doi:10.1016/j.ijrobp.2004.03.005
CRITICAL REVIEW
ROLE OF CELL CYCLE IN MEDIATING SENSITIVITY TO RADIOTHERAPY
TIMOTHY M. PAWLIK, M.D., M.P.H.,*
AND
KHANDAN KEYOMARSI, PH.D.†
Departments of *Surgical Oncology and †Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer
Center, Houston, TX
Multiple pathways are involved in maintaining the genetic integrity of a cell after its exposure to ionizing
radiation. Although repair mechanisms such as homologous recombination and nonhomologous end-joining are
important mammalian responses to double-strand DNA damage, cell cycle regulation is perhaps the most
important determinant of ionizing radiation sensitivity. A common cellular response to DNA-damaging agents is
the activation of cell cycle checkpoints. The DNA damage induced by ionizing radiation initiates signals that can
ultimately activate either temporary checkpoints that permit time for genetic repair or irreversible growth arrest
that results in cell death (necrosis or apoptosis). Such checkpoint activation constitutes an integrated response
that involves sensor (RAD, BRCA, NBS1), transducer (ATM, CHK), and effector (p53, p21, CDK) genes. One of
the key proteins in the checkpoint pathways is the tumor suppressor gene p53, which coordinates DNA repair
with cell cycle progression and apoptosis. Specifically, in addition to other mediators of the checkpoint response
(CHK kinases, p21), p53 mediates the two major DNA damage-dependent cellular checkpoints, one at the G1–S
transition and the other at the G2–M transition, although the influence on the former process is more direct and
significant. The cell cycle phase also determines a cell’s relative radiosensitivity, with cells being most radiosensitive in the G2-M phase, less sensitive in the G1 phase, and least sensitive during the latter part of the S phase.
This understanding has, therefore, led to the realization that one way in which chemotherapy and fractionated
radiotherapy may work better is by partial synchronization of cells in the most radiosensitive phase of the cell
cycle. We describe how cell cycle and DNA damage checkpoint control relates to exposure to ionizing radiation.
© 2004 Elsevier Inc.
Radiotherapy, Cell cycle, p53, p21, Synchronization.
In 2003, an estimated 1.3 million new cases of cancer were
diagnosed in the United States (1). Many of these patients
received some form of radiotherapy (RT) as part of their
treatment. After surgery, RT is arguably the most important
treatment for cancer, especially for localized disease that
has not spread. Ionizing radiation is used to treat virtually all
types of solid malignancies, but to varying degrees of success. That is, some tumors are highly responsive to low
doses of radiation (e.g., lymphomas, seminomas), and other
tumors are typically very radioresistant and tend to progress
even after high radiation doses (e.g., melanoma, glioblastoma) (2). A further difficulty is that treatment fails in a
considerable number of patients treated with ionizing radiation with curative intent not only because of distant metastatic spread, but also because of local treatment site failure. The reasons for such local RT failure are multiple and
varied. Tumor factors, such as location, size, and inadequate
vascular supply (hypoxia), can all play a role in the lack of
responsiveness of neoplasms to ionizing radiation. Perhaps
most important, however, are the cellular and genetic factors, such as differential tissue-specific gene expression
(e.g., p53, ataxia telangiectasia mutated [ATM] status), that
may result in radiation-resistant cellular phenotypes (3, 4).
Support for the role of differential gene expression in
determining radiation sensitivity comes in part from observations that cells from the same tissue of origin, but from
different patients, can show varying radiation sensitivities.
That is, tumors from different patients with the same histologic diagnosis can show varied responses to ionizing radiation (5). Such differential radiosensitivity can also be
present within a single tumor. This was the observation of
Weichselbaum et al. (6) who reported that four cell lines
clonally derived from the same tumor source showed different radiation sensitivities.
Recently, studies have focused more specifically on how
cell cycle checkpoints, including mutations in p53 and p21,
as well as the cell cycle phase, determine radioresponsiveness. More needs to be known about this, however, before
Reprint requests to: Khandan Keyomarsi, Ph.D., Department of
Experimental Radiation Oncology, The University of Texas M. D.
Anderson Cancer Center, Box 66, 1515 Holcombe Blvd., Houston,
TX 77030. Tel: (713) 792-4845; Fax: (713) 794-5369; E-mail:
[email protected]
T. M. Pawlik is supported by NIH training Grant CA09599.
Acknowledgments—The authors thank Dr. Ray Meyn for his critical reading of the manuscript and helpful suggestions.
Received Nov 6, 2003, and in revised form Mar 1, 2004.
Accepted for publication Mar 8, 2004.
INTRODUCTION
928
RT and the cell cycle
the knowledge can be applied and significant improvements
made in the clinical outcome of patients treated with ionizing radiation. The aim of this article is to review the major
aspects of this knowledge, which include information on (1)
radiation-induced cell death, (2) radiation damage to DNA
and subsequent cell cycle checkpoint alterations, and (3)
cell cycle phase-dependent radiosensitivity and kinetically
based use of chemoradiotherapy.
AN OVERVIEW: RADIATION-INDUCED
CELL DEATH
There is no doubt that DNA is the critical target in RT.
Irradiation induces both single- and double-strand DNA
breaks, with the double-strand breaks generally considered
the lethal event. Evidence that DNA is the effective target
for achieving cell death after RT comes from numerous
studies spanning several decades. In 1977, Warters and
Hofer (7) showed that ⬎19,600 disintegrations of 125I were
needed at the membrane level compared with only 60 disintegrations of 125I at the nucleus to generate similar cell
survival. More recently, Radford et al. (8) showed in several
cell lines that a high rate of apoptosis was induced by the
incorporation of 125I into the cellular DNA. Additional
evidence that radiation’s effects on DNA are the main cause
of cell death has come from studies examining DNA incorporation of 5-bromedeoxyuridine (9). This research has
shown that substituting 5-bromedeoxyuridine for the normal
DNA precursor thymidine amplifies the effects of radiation
on cells. More specifically, the combination of radiation and
5-bromedeoxyuridine incorporation significantly enhanced
apoptosis in the cell lines examined (9). Experiments using
severe combined immunodeficient mice have also provided
insight into the mechanism of radiation-induced cell death.
Severe combined immunodeficient mice are deficient in
DNA-dependent protein kinase, which functions in a complex at the site of DNA double-strand breaks to promote
repair (10). Normally, DNA double-strand breaks activate
Ku proteins, which bind to the area of DNA breakage (10).
The DNA-dependent protein kinase catalytic subunit then
forms a complex with the Ku proteins to facilitate repair.
Because of the defect in the DNA repair pathway, severe
combined immunodeficient mice are acutely sensitive to
radiation (11). From this, it is clear that the type of nuclear
damage and the nature of DNA repair processes together
determine the response of cells to ionizing radiation.
Mammalian cells have evolved a number of repair systems to deal with the DNA double-strand breaks induced by
exposure to ionizing radiation (12). The two major types of
double-strand break repair are homologous recombination
and nonhomologous end-joining. The relative contribution
of each of these types of repair is controversial (13–15).
Homologous recombination repair may play a more prominent role during the late S and G2 stages of the cell cycle
(14, 16), and nonhomologous end-joining may be more
important during the G1 and early S phases (17, 18). Although nonhomologous end-joining is the predominant
● T. M. PAWLIK et al.
929
mechanism of double-strand break repair, homologous recombination is known to be critical in cell signaling and is
also regulated by the cell cycle (12).
Radiation can produce cell death by one of two mechanisms: apoptosis or necrosis. Necrosis is a passive process
in which cells pass through mitosis with unrepaired DNA
strand breaks, leading to lethal chromosomal aberrations
(micronuclei) in nonclonogenic daughter cells (19). Necrotic cells are characterized by a loss of membrane integrity, cell swelling, dilation of cytoplasmic vesicles, and the
subsequent random degradation of DNA (20). In contrast,
apoptosis is an active process characterized by programmed
cell death in which a cascade of events is triggered in
response to cellular stress (e.g., radiation) (21–23). Apoptosis is characterized by nuclear DNA fragmentation, condensed chromatin, a fragmented nucleus, and an eosinophilic or poorly staining cytoplasm.
The relation of apoptosis and radiosensitivity is, however,
controversial. Although some investigators have reported
that apoptosis is an important mechanism by which RT kills
cells (24 –26), others have argued that apoptosis is not the
predominant form of cell death after exposure to ionizing
radiation (27). A basic principle in the radiobiology of
tumors is that an essential difference exists between cell
death and the loss of reproductive integrity of tumor cells,
and that for the outcome of curative RT, it is the latter that
matters most (27, 28). Use of apoptotic assays concentrates
on the first 90% of cell killing, but the outcome of treatment
depends on multi-log cell kill. The clonogenic cell survival
assay is, therefore, a more appropriate method to assess
radiation sensitivity. Clonogenic cells are defined as those
neoplastic cells within the tumor that have the capacity to
produce an expanding colony of descendants, and, therefore, the capacity to regrow the tumor if left intact at the end
of treatment (27). Clonogenic cells can be scored by means
of a clonogenic assay, of which many types have been used
(28). Loss of colony-forming ability is likely to be the key
event in radiation-treated tumor cells, and the appearance of
morphologic and molecular evidence of apoptosis is probably downstream from this event (27). Because the cell
cycle is strongly affected by irradiation, and radiosensitivity
depends on cell cycle position and cell cycle progression, it
is not surprising, however, that some association between
apoptosis and radiosensitivity has been observed.
The degree of radiation-induced apoptosis has been
shown to correlate with the p53 wild-type status (29). In
addition, apoptosis is induced when wild-type p53 is transfected into certain cell lines lacking p53 (30, 31). This
indicates that p53 not only plays a role in regulating the
progression through the cell cycle, it can also induce apoptosis in cells. p53 is not an essential component of the
machinery that carries out apoptosis; however, it appears to
be an activator of apoptosis. Although the exact means by
which p53 activates apoptosis is unclear, evidence has
shown that p53 mediates apoptosis by way of transcriptionindependent and transcription-dependent mechanisms (32–
36). p53 is known to regulate the expression of several
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Volume 59, Number 4, 2004
Fig. 1. DNA damage induced by ionizing radiation activates p53 pathway through ATM. p53 activates pro-apoptotic
BAX protein, which initiates permeability transition of mitochondrial membrane allowing release of several factors,
including cytochrome C. A key protein in electron transport, cytochrome C activates caspase-9 and the caspase cascade,
leading to cell death.
proteins involved in the apoptotic pathway, including
CD95, PIDD, PIGs, PERP, and KILLER/DR5 (35, 37– 41).
p53 also interacts with BAX, BCL-XL, and BCL-2 to exert
a direct apoptotic effect at the level of the mitochondria
(Fig. 1) (42, 43). Additionally, Fas, a cell-surface protein
that triggers apoptosis when it binds to its ligand, is encoded
by a target gene transcriptionally activated by p53 (37).
Despite p53’s known interaction with all of these antiapoptotic genes, none of them, however, appears to be the
principal mediator of the p53 apoptotic signal. This leaves
open the possibility that a uniqueness exists among p53
targets, and a tissue/cell-type specificity in their regulation
in response to ionizing radiation (43).
Although not as important as radiation-induced DNA
damage, damage to the cell membrane also initiates signaling events involved in the apoptotic response. For example,
radiation can induce the cleavage of the membrane-bound
protein sphingomyelin, resulting in the formation of ceramide, a lipid second messenger (44). Conversely, ceramide
production can be inhibited by BCL-2 (45), an anti-apoptotic membrane-protein; in turn, ceramide itself has been
implicated in the downregulation of BCL-2 (46). The ceramide produced in response to ionizing radiation, therefore,
appears to act as a positive regulator of apoptosis. This has
been further borne out by the finding that cells deficient in
sphingomyelinases, the enzymes needed to produce ceramide from sphingomyelin, are more resistant to apoptosis
after irradiation (46). Similarly, experiments using mem-
brane preparations from cells devoid of nuclei have shown
increased ceramide production after ionizing radiation, also
suggesting that ionizing radiation acts directly on the cell
membrane to induce ceramide production (47). Although
membrane events appear to contribute to radiation-induced
apoptosis, it remains unclear whether they are sufficient to
initiate cellular apoptosis.
RADIATION AND CELL CYCLE SIGNALING
The phosphatidyl-inositol kinase-related protein ATM is
the most proximal signal transducer initiating cell cycle
changes after the DNA damage induced by ionizing radiation (48). Likewise, the rapid induction of ATM serine/
threonine protein kinase activity after ionizing radiation has
also suggested that ATM acts at an early stage of signal
transduction in mammalian cells (49, 50). Mammalian
ATM is a member of a family of protein kinases that include
ATM-Rad3-related (ATR), DNA-dependent protein kinase,
and FRAP, which are related because they have a similar
carboxy-terminal kinase domain (51, 52). Until recently, the
mechanism by which ATM kinase activity increases after
radiation exposure was poorly understood. It was initially
believed that double-strand breaks in the DNA induced by
ionizing radiation either directly or indirectly signaled to
ATM. However, the rapidity with which ATM is phosphorylated after ionizing radiation suggested that ATM was not
activated by direct binding to the DNA strand breaks. Re-
RT and the cell cycle
● T. M. PAWLIK et al.
931
Fig. 2. Ionizing radiation rapidly induces the protein kinase activity of the ATM gene, which in turn interacts with a
broad network of proteins to block progression through the cell cycle, allowing time for DNA repair. ATM activates
both p53 and CHK2, leading to either a G1/S or G2/M cell cycle block, depending on interactions with downstream
target genes. Adapted from Samuel T, Weber HO, Funk JO. Linking DNA damage to cell cycle checkpoints. Cell Cycle
2002;1:162–168, with permission from Landes Bioscience (48).
cently, Bakkenist and Kastan (53) showed that, instead,
ATM activation may result from changes in the structure of
chromatin brought about by intermolecular autophosphorylation and ATM dimer dissociation. Once dissociated, ATM
can then potentially phosphorylate numerous downstream
targets, including p53, MDM2, CHK2, NBS1, RAD9, and
BRCA1.
ATM’s essential role in DNA damage and repair is highlighted by the extreme sensitivity to ionizing radiation of
cells with defective ATM and/or lacking ATM (54 –57).
This is also the case in patients with ataxia telangiectasia
who have a mutated ATM gene. These patients have a
characteristic phenotype consisting of a heightened cancer
predisposition, extreme sensitivity to radiation, and cell
cycle abnormalities (58, 59). In particular, cells from ataxia
telangiectasia patients show defective G1, S, and G2 arrest
after ionizing radiation (60, 61).
ATM has various targets. After cells are exposed to
ionizing radiation, ATM phosphorylates p53, stabilizing the
protein and prolonging its half-life (62). Ionizing radiation
also leads to the phosphorylation of serines 15/20 on p53,
which negatively influences the binding of p53 to the oncoprotein MDM2 (63, 64). MDM2 normally binds to p53,
thereby targeting it for degradation in the ubiquitin-dependent proteosome pathway (65– 67). By disrupting p53–
MDM2 binding, ATM inhibits the degradation process, thus
prolonging the half-life of p53. This prolongation of p53’s
otherwise short half-life after a DNA-damaging event has
been extensively studied (68 –70) and has been found to
correlate with cellular responses such as cell cycle arrest
and apoptosis (71, 72). ATM also activates human checkpoint kinase 2 (CHK2) in cells after exposure to ionizing
radiation. CHK2 in turn phosphorylates p53, further stabilizing p53 (73–75). CHK2 activity also is necessary for the
phosphorylation of the dual-specificity phosphatases
Cdc25A/C, which inactivates the enzymes, blocking CDK1
activation and causing a G2 arrest (Fig. 2) (76). Other
targets of ATM include BRCA1, NBS1, and RAD9 (77–
85). The ATM-mediated phosphorylation of NBS1 is required for the proper execution of the intra-S phase checkpoint, that of BRCA1 is associated with both the S phase
checkpoint and the G2/M transition, and RAD9 is linked to
the G1/S checkpoint activation (77– 85).
Cells with mutations in the NBS1 gene share a variety of
phenotypic similarities with ATM deficient cells such as
chromosomal instability, increased radiation sensitivity, and
defects in cell cycle checkpoints in response to ionizing
radiation (86 – 89). Although NBS1 is not required for the
activation of ATM and its downstream targets after ionizing
radiation, cells mutated at the ATM phosphorylation site of
NBS1 do display an abrogated S phase checkpoint after
exposure to ionizing radiation (81). ATM appears to control
S phase arrest after ionizing radiation by phosphorylating
NBS1 on Ser 343; however, the mechanism by which phos-
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● Biology ● Physics
phorylation of Ser 343 affects DNA replication after ionizing radiation remains unknown (81). Similar to NBS1,
ATM is required for phosphorylation of BRCA1 in response
to ionizing radiation. ATM resides in a complex with
BRCA1 and phosphorylates BRCA1 in a region that contains clusters of serine-glutamine residues (77). Phosphorylation of this domain appears to be functionally important
because a mutated BRCA1 protein that lacks these key
phosphorylation sites is unable to rescue the radiation hypersensitivity of BRCA1-deficient cell lines (77). Cells deficient in BRCA1 show genetic instability, defective G2/M
checkpoint control, and reduced homologous recombination
(90, 91). Additionally, BRCA1 regulates expression of both
the p21 and GADD45 proteins (92–94).
RAD9 is a 1309 amino acid protein, with a C-terminal
region that shows localized sequence identity with BRCA1
(95). RAD9 is required for DNA damage checkpoint in all
phases of the cell cycle (96 –98), and loss of RAD9 impairs
checkpoint-induced cell cycle arrest and increases genomic
instability (98, 99). The RAD9 protein is constitutively
phosphorylated in undamaged cells and undergoes hyperphosphorylation on exposure to ionizing radiation (85).
Hyperphosphorylation of RAD9 is induced by ionizing radiation through ATM phosphorylation of Ser 272. Cells
mutated at the RAD9 Ser 272 residue are defective in the
G1/S checkpoint after exposure to ionizing radiation (85).
DNA damaged-induced hyperphosphorylation of RAD9 appears to be normal in NBS1-deficient cells. This may be
because RAD9 operates upstream of NBS1, or alternatively,
that RAD9 functions separately from NBS1 (85).
p53 is a key DNA damage checkpoint protein that is
indispensable for the mounting of a complete DNA damage
response. However, whether p53 induces apoptosis or cell
cycle arrest is a complex matter and depends in part on the
abundance of the p53 protein (in general, low protein levels
lead to cell cycle arrest and high protein levels lead to
apoptosis), specific posttranslational modifications, and
p53’s interaction with such downstream activators as
GADD45 as opposed to p21 (48, 100). Thus, although p53’s
upregulation of GADD45 may play a role in apoptosis by
activating the JNK and/or p38 MAPK signaling pathways
(94, 101), p53’s activation of p21 after exposure to ionizing
radiation leads to cell cycle arrest (102). p21 belongs to the
Cip/Kip family of CDK inhibitors, which also includes p27
and p57 (103). Although members of the Cip/Kip family
share broad specificity in their binding to, and inhibition of,
most CDK/cyclin complexes (104 –106), only p21 is directly involved in DNA damage-induced cell cycle arrest
(48). Specifically, the p21 protein binds to, and inactivates,
cyclin-E-CDK2 complexes, which results in hypophosphorylation and cell cycle arrest at the G1/S transition (102,
107). p53 can also upregulate the transcription of 14-3-3␴
which inhibits G2 progression by sequestering CDK1 in the
cytoplasm. In this way, ATM and p53 play important roles
in both G1/S and G2 checkpoint regulation after exposure to
ionizing radiation.
Volume 59, Number 4, 2004
IRRADIATION AND CELL CYCLE ARREST
Multiple pathways are involved in the maintenance of
genetic integrity after exposure to ionizing radiation, most
of which are related to the cell cycle (108). Cells commonly
respond to DNA-damaging agents by activating cell cycle
checkpoints. These checkpoints provide for a controlled
temporary arrest at a specific stage of the cell cycle to allow
the cell to correct possible defects (108, 109). Ionizing
radiation induces arrests in the G1, S, and G2 phases of the
cell cycle. The G1 checkpoint prevents the replication of
damaged DNA before the cell’s entry into S phase, and the
G2 checkpoint prevents the segregation of aberrant chromosomes during M phase (110). Two molecularly distinct
G2/M checkpoints can be identified (111). The first of these
G2/M checkpoints occurs early after exposure to ionizing
radiation, is very transient, is ATM dependent, and is dose
independent (111). This first G2/M block represents the
failure of cells that had been in G2 at the time of irradiation
to progress into mitosis. This “early” G2/M checkpoint may
be the mechanism by which low-dose hyperradiosensitivity
is converted to resistance (112). In contrast, the “late” G2/M
accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after ionizing radiation, is ATM independent, is dose dependent, and
represents the accumulation of cells that had been in earlier
phases of the cell cycle at the time of radiation exposure
(111). G2/M accumulation after exposure to ionizing radiation is not affected by the early G2/M checkpoint and is
enhanced in cells lacking the radiation-induced S phase
checkpoint, such as those lacking NBS1 or BRCA1 function
(111).
Most cells with wild-type p53 exhibit only a transient
delay in the G1 and G2 phases of the cell cycle after RT.
Although it is widely accepted that p53 mediates G1 arrest
through the mechanisms outlined above, some departures
from this have been reported, in that wild-type p53 cells do
not always display G1 arrest after exposure to radiation
(113–115). It also appears that when irradiated cells undergo wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. However, if wild-type p53 cells are
irradiated after the G1 checkpoint, the cells do arrest in G2
but do not show a delay in the subsequent G1 phase (116).
p53’s role in the G2/M checkpoint is not as clear. Numerous studies have shown that p53 and p21 mutant cells
are capable of G2 arrest in response to DNA-damaging
agents, including ionizing radiation (117, 118). In many of
these studies, however, high doses of radiation were applied
to cells that were growing asynchronously or synchronized
in the S phase. Under these conditions, the data suggest that
neither p53 nor p21 is involved in the G2/M checkpoint,
because cells deficient in p53 or p21 were still able to arrest
in G2 after exposure to ionizing radiation (117, 119). Although p53 appears to be dispensable for the initiation of the
delay at the G2/M checkpoint after exposure to ionizing
radiation, p53- or p21-deficient cells do show a shorter
G2/M delay. Thus, it appears that although p53 and p21 are
RT and the cell cycle
not needed for the initiation of G2/M arrest, they are required for the sustaining of G2/M arrest after DNA damage
(120). This has also been borne out by data showing that a
given dose of radiation induces a longer G2/M delay in
radiosensitive cell lines than in matched normal or resistant
cells (121). A G2 delay in tumor cells may provide time for
repair processes to operate that are critical for ensuring cell
survival after sublethal DNA damage (122). In contrast,
numerous studies have shown that the disruption or abrogation of the G2/M checkpoint leads to the radiosensitization of p53-mutated cells (123). Likewise, tumor cells
treated with either caffeine or pentoxifylline, compounds
that disrupt the G2 checkpoint, are sensitized to ionizing
radiation (122, 124, 125).
Although p53 is dispensable for the initiation of the delay
at the G2 checkpoint, the ATM-CHK protein kinase pathway appears to be essential, because the inhibition of CHK1
in p53-deficient cells greatly sensitized them to radiation
(126). This validates the use of CHK inhibitors as an anticancer strategy. The CHK inhibitor UCN-01 (7-hydroxystaurosporine) represents one such attempt (127, 128).
UCN-01, which has significant in vivo activity (unlike its
parent compound staurosporine) was originally developed
as a selective protein kinase C inhibitor (129, 130). However, recent studies have suggested that UCN-01 has multiple divergent effects on cell cycle dynamics. In particular,
UCN-01 functions not only as a CDK inhibitor causing G1
arrest, it can also inhibit CHK1, and in so doing, abrogates
the G2 checkpoint (127, 131). Additionally, numerous
DNA-damaging agents, including radiation (132), 5-fluorouracil (133), camptothecin (134), and temozolomide
(135), appear to act supra-additively with UCN-01 in terms
of cytotoxicity (136). For example, the inhibition of CHK1
in p53-deficient cells greatly sensitized the cells to radiation
(126, 137). Although preclinical testing showed therapeutic
efficacy for UCN-01, clinical trials of UCN-01 have yielded
mixed results (136, 138 –143). In addition, in Phase I clinical trials, UCN-01 was found to bind avidly to human
plasma proteins, resulting in a long half-life that required
adjustment of the administration schedule (136). In many
patients, the subsequent dose of UCN-01 therapy after the
first course was reduced by 50%. The dose-limiting toxicities included hyperglycemia, acidosis, and adverse pulmonary events. One partial response occurred in a patient with
melanoma, and a protracted (⬎4 year) period of stabilization of minimal residual disease was observed in a patient
with anaplastic large-cell lymphoma (136).
CELL CYCLE: EFFECT OF CELL SYNCHRONY
ON RADIOSENSITIVITY
The importance of the p53 and p21 status in determining
radiosensitivity is somewhat complex. In general, loss of
p53 is associated with a more radioresistant phenotype (114,
144 –148), but in some instances, loss of p53 either has no
effect on radiation sensitivity or, conversely, is associated
with a more sensitive phenotype (149, 150). As an expla-
● T. M. PAWLIK et al.
933
Fig. 3. Synchronized human kidney cells show a differential survival depending on cell cycle phase in which they are irradiated.
Cells are most sensitive to irradiation during mitosis and in G2, less
sensitive in G1, and least sensitive during the latter part of S phase.
From Sinclair and Morton (160), with permission.
nation for such discrepancies, it has been suggested that
p53-mediated radioresistance is more important in cells that
depend on apoptosis, instead of necrosis, for cell death. In
the case of p21 mutation, when examined in vitro using a
clonogenic assay that assessed cell survival, the loss of p21
appeared to affect more the mode of cell death (i.e., apoptosis vs. necrosis) than the overall level of cell killing
(151–153). However, when using tumor regrowth delay or
in vivo clonogenic assays to assess for differences, a p21
mutation did appear to sensitize tumors to radiation. Furthermore, the loss of p21 in ATM knockout mice caused
increased radiosensitivity (154). Such contrasting results
emphasize the importance of considering the cellular context when dissecting the role of p53 and p21 in radiosensitivity.
Beginning in the late 1960s, researchers started to examine the dependence of the radiation response on the age or
phase of the cell in the growth cycle (155–159). Initial
studies in synchronized Chinese hamster cells showed a
differential response of the cells to radiation depending on
the phase of the cell cycle they were in at irradiation (155,
156). In general, cell survival data showed that cells were
most sensitive to irradiation during mitosis and in G2, less
sensitive in G1, and least sensitive during the latter part of
the S phase (Fig. 3) (160, 161). In the 1960s and 1970s, the
effects of the cell cycle phase or age were examined in
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synchronization studies in a variety of cell lines (e.g., HeLa
cells, Yoshida sarcoma cells, mouse fibroblasts, and mouse
L cells) (157, 158, 162–168). In most of these early experiments, synchronization was achieved using excess thymidine (thymidine block), serum starvation, mitotic “shakeoff,” or hydroxyurea (155, 160, 163, 169 –171). More
recently, lovastatin, centrifugal elutriation, and fluorescence
activated cell sorting have been used as methods to isolate
phase-specific populations of cells (172–175). The method
of synchronization determines the phase of the cell cycle
that cells are arrested in. For example, excess thymidine
blocks cells in the S phase, and lovastatin arrests cells in the
early G1 phase. Regardless of the method of synchronization, however, maximal radiosensitivity has been universally found to occur during mitosis, with resistance rising
during the S phase and reaching a maximum in the latter
part of the S phase (161). Given these initial findings, the
concept of synchronizing tumor cells in a phase of the cell
cycle that is sensitive to radiation was recognized as a
potentially important way to enhance the clinical efficacy of
RT (176 –181).
KINETICALLY BASED ADMINISTRATION OF
CHEMOTHERAPY AND RT
Because varying types of chemotherapeutic agents are
able to arrest cells at specific cell cycle checkpoints, researchers have explored the use of different chemotherapeutic agents to synchronize and arrest cells in the radiosensitive phases of the cell cycle (i.e., G1 and G2/M) (182–185).
For example, agents such as vinblastine and vincristine tend
to arrest cells in the radioresistant S phase, and other drugs
such as paclitaxel (Taxol) block cells at the radiosensitive
G2/M phase of the cell cycle (186, 187). CDK inhibitors
such as flavopiridol are also known to accumulate cells in
the G1 and G2 phases.
In vitro work examining the effects of Taxol in human
lymphoblasts and glioma cells has shown that the degree of
radiation cytotoxicity depends on both the duration of Taxol
treatment and the location of the cells in the cell cycle
(186 –189). Taxol cytotoxicity, however, cannot be completely attributed to increasing numbers of cells in G2/M
and is more likely to be determined by multiple factors,
including both the direct cytotoxic effects of the agent and
the cell cycle effects (186, 187). Experimental conditions,
such as the Taxol concentration, incubation time, radiation
fractionation, radiation schedule, and the sequence of Taxol
and RT have also been found to influence the effectiveness
of combined treatment, implying the involvement of other
mechanisms, in addition to G2/M accumulation, in Taxolinduced radiosensitization (190, 191).
The role of the cell cycle phase is believed to be critical
to the mechanism of action of other chemotherapeutic
agents as well. For example, one way in which 5-fluorouracil is thought to increase radiation sensitivity at the cellular level is by killing cells in the S phase, which are not as
radiosensitive (192, 193). Similarly, maximal sensitization
Volume 59, Number 4, 2004
with gemcitabine appears to require the redistribution of
cells into S phase, along with deoxyadenosine triphosphate
pool depletion (192, 194). It has also been proposed that the
radiosensitization of log phase cells using gemcitabine occurs through the selective sensitization of radioresistant S
phase cells (192, 195). In vitro work using flavopiridol has
corroborated the importance of cell cycle phase redistribution with regard to chemosensitivity. In particular, flow
cytometry has shown that flavopiridol accumulates cell in
the G1 and G2 phases, with a significant reduction in the S
phase component, leading to increased radiosensitivity
(196). Cell synchronization has also been found to have
positive therapeutic benefits in in vivo animal tumor systems
(197, 198). Given this, some investigators have advocated a
“kinetic” approach to chemoradiotherapy, emphasizing the
need for the appropriate timing of chemotherapy in relation
to RT (182, 199, 200). Although kinetic approaches may be
conceptually appealing on the basis of in vitro data, in
practice, kinetic treatment regimens have largely been unsuccessful. The major reason for the clinical failure of this
approach is undoubtedly because tumors, unlike cells in
controlled culture conditions, are wildly heterogeneous with
regard to their cell cycle characteristics.
As already noted, ionizing radiation can retard the rate of
progression of proliferating cell populations through various
phases of the cell cycle (201–203), causing cells to accumulate in the G2 phase (204) and keeping cells from undergoing mitotic division (208). In general, the effects of G2
blockade increase with radiation dose, but even low doses of
radiation can result in cell cycle phase redistribution and,
with time, may lead to partial synchronization (201, 206).
Given this, split or fractionated doses of radiation may be
more efficacious, in part, by inducing a transient cell cycle
arrest, after which a secondary RT fraction is administered
exactly at the height of cell accumulation in the most
radiosensitive cell cycle phase (G2/M) (206, 207). This
suggests that the redistribution of cells in a particular phase
would determine the response of an initially asynchronous
population to fractionated high- and low-dose RT. Ngo et
al. (201, 205) showed that the sequential exposure of Chinese hamster cells to low- and high-linear energy transfer
gradually enriched the population of G2 cells, which showed
increased radiosensitivity to sequential radiation exposure.
Others have similarly shown that fractionated radiation can
effectively synchronize cells in a more sensitive state for
irradiation. Using human prostate cells, Geldof et al. (206)
showed that doses of 2 Gy or 4 Gy led to a shift toward a
predominance of cells in the G2/M phase, causing the prostate cells to be more sensitive to radiation.
Doses of ⬍2 Gy have also been studied and been shown
to increase considerably the G2/M phase fraction as well
(209). Doses of ⬍1 Gy have been shown to induce hyperradiosensitivity in a number of cell lines, including hamster
fibroblasts (210) and various human cancer cell lines (211–
214). As the most sensitive and immediate indicator of
cellular reactions to radiation (215, 216), the hyperradiosensitivity effect on the cell should be reflected in the cell
RT and the cell cycle
cycle. However, as Bartkowiak et al. (209) noted, one
would expect that, below a certain threshold of repair induction, cells would completely “ignore” damage and continue through the cell cycle unaltered. Nonetheless, Bartkowiak et al. (209) showed that cells are extremely sensitive
to the G2/M checkpoint and accumulate in a dose-dependent
manner with doses as low as 0.2 Gy. Others have shown that
the cell-cycle phase also has an important influence on the
response to low-dose radiation of human tumor cell lines
(217). Because the magnitude of hyperradiosensitivity appears to be greatest in the G2 phase, this also suggests that
tumors with larger cell growth fractions and/or an aggressive proliferative response to treatment may be the best
candidates for treatment using low-dose fractions (217).
Despite the overwhelming evidence that the cell phase
plays some role in radiosensitization, it cannot entirely
account for the increased radiosensitivity observed for fractionated RT (171, 201). Changes in repair fidelity or efficiency resulting from the induction of repair processes in a
dose- or damage-dependent manner may also play a role
(212, 218, 219). Another area that requires additional investigation is whether p53-deficient cells that have a foreshortened G2/M duration remain as sensitive to a fractionated RT regimen.
To better understand the molecular events that govern sensitivity to radiation damage in different phases of the cell cycle,
several investigators have examined cell cycle– dependent
DNA damage and repair mechanisms after exposure to ionizing radiation (220 –225). From work with synchronized populations of cells, it is clear that radiation-induced chromosomal
damage and micronuclei formation depend on the cell cycle
distribution. Ionizing radiation can produce both different
types of, and quantitative differences in, chromosomal aberrations at various stages of the cell cycle (222, 224, 226).
Illustrating the latter point, in Chinese hamster cells, the
frequency of chromosomal aberrations after irradiation was
about three times greater for G2 phase cells than for S and
G1 phase cells (227). However, in mouse cells, the frequency of translocations was significantly greater in G1 and
S phase cells, than in G2 phase cells (224). Furthermore,
Tallon et al. (221) reported that primary human lymphocytes undergo a cell cycle– dependent induction of aneuploidy after irradiation. Cells exposed to radiation during
the G1 phase exhibited a greater frequency of centromerepositive micronuclei than cells in the G2 phase at exposure.
In addition, G1 phase exposure induced a centromere-positive micronuclei dose– effect relationship that was not observed after G2 phase exposure (221). Paglin et al. (223)
examined breast cancer cells and noted that after the irradiation of G1 and S phase– enriched cell populations, S
phase cells were more prone to micronuclei formation than
G1 cells. Not only does the degree of radiation-induced
damage depend on the cell cycle, the nature of the cell cycle
repair varies with the phase of the cell (225). In particular,
Iliakis and Okayasu (225), who studied double-strand break
repair in CHO cells, observed faster kinetics in the G1/S and
mid-S phases than in the G1 phase. Taken together, these
● T. M. PAWLIK et al.
935
data suggest that the degree of chromosomal damage and
repair after irradiation also depends to some extent on the
cell cycle phase. However, this effect varies depending on
the cell lines examined and the radiation dose used.
Some of the variation in the molecular and cell cycle
response to ionizing radiation is believed to be due to the
intrinsic radiosensitivity of certain human tumor cells (228,
229). Specifically, Deschavanne and Fertil (229) showed
that in vitro radiosensitivity varied depending on the cell
type and organ (Fig. 4). Similarly, Biade et al. (228) examined the effects of radiation on ovarian OVCAR10 cells and
HT29 colon cells. They found that although all cells exhibited a maximal radioresistance near the G1–S phase boundary, the HT29 cells remained relatively radioresistant in the
G2 phase, but the ovarian cells became more radiosensitive
(228). Tutt et al. (230) also found in breast cells that the
BRCA2 and p53 status and the cell cycle phase all played a
critical role in determining the effect of ionizing radiation
on the cell. For example, the BRCA2 mutation had little
effect on cells irradiated in quiescence but sensitized proliferating cells to ionizing radiation on a p53 null background. They also showed that BRCA2’s role in mediating
cell survival after irradiation occurred in the S and G2
phases of the cell cycle (230). This all underscores the need
to consider not only any perturbations in a specific gene
status (i.e., p53, p21, BRCA2), but also the origin of the
tumor line being examined when determining the effect of
irradiation and cell cycle phase on radiosensitivity. That is,
cell-type and organ-specific variations in intrinsic radiosensitivity must be taken into consideration when examining
issues of cell cycle phase and radioresistance.
Although understanding the concept of the cell-cycle
phase is important to understanding ionizing radiation’s
effect on the cell, the therapeutic application of synchronization is extremely limited. Concerns about the therapeutic
efficacy of synchronization therapy have come from a number of sources (199, 231–234). For example, Tubiana et al.
(223, 224) has expressed the view that the timing of synchronization varies in different cell lines and that optimal
synchronization is, therefore, seldom achieved. In a review
of clinical data, Tannock (232) observed that synchronizing
chemotherapy schedules had seldom been compared for the
same drugs used with a different timing, and thus the actual
synchronizing effects of these agents were unsubstantiated.
A number of reasons have also been proposed for why
synchronization therapy may not work (199). One reason is
that the synchrony of cell populations is difficult to maintain
and many human tumors display a kinetic heterogeneity
even after synchronization (199, 235). The complete synchronization of a population of cells is also difficult and
inefficient, if not impossible. In addition, the synchronization of cell populations before each RT fraction would be
cumbersome and difficult to achieve, given the average cell
cycle time and the heterogeniety of most cancer cells (199,
235). Finally, blocked cells are often dead and not capable
of further proliferation. If synchronization is meant to prime
surviving resistant cells for treatment, the dead cells do not
936
I. J. Radiation Oncology
● Biology ● Physics
Volume 59, Number 4, 2004
Fig. 4. Intrinsic radiosensitivity of human cancer cell lines. Mean inactivation radiation dose varies depending on cell
type examined. Adapted from Deschavanne PJ, Fertil B. A review of human cell radiosensitivity in vitro. Int J Radiat
Oncol Biol Phys 1996;34:251–266, with permission (229).
matter. Some studies have shown that, although the lowdose irradiation of human tumor cells induced substantial
cell synchrony, the extent of cell blocking and cell killing
increased together, and thus cell cycle arrest probably was
not very important to the effectiveness of RT (236). These
studies point out that the use of cell synchronization as a
therapeutic tool is limited and most likely clinically untenable.
FUTURE DIRECTIONS
In the future, novel therapeutic regimens will not only
need to continue to maximize the radiosensitivity of tumor
cells, but also to identify those tumors most likely to respond to RT. As presented in this review, one possibility to
improve therapeutic radiosensitivity may be through modulation of the cell cycle. Not surprisingly, however, cellular
response to cell cycle modulation will ultimately be dictated
by the tumor cell’s genetic profile (p53, p21, ATM status),
as well as the integrity of the underlying checkpoint pathways. Attempts to increase radiosensitivity through alteration of the cell cycle must, therefore, take into account the
molecular profile of the target tumor cells. Future studies
should strive to implement clinical strategies that target
specific phases of the cell cycle according to the molecular
profile of the individual tumor being treated.
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