Chapter 6

Cell Cycle Responses to Hyperthermia

Yukihiro Furusawa

Abstract For decades, it has been well known that hyperthermia induced by heat
stress (HS) exerts anti-tumor activities through the inhibition of DNA repair,
induction of cell death, and arrest or delay of the cell cycle. HS as well as ionizing
radiation and anti-cancer agents exert multiple effects on cell proliferation, likely
by activating the cell-cycle checkpoint pathway. One of the most important molec-
ular mechanisms by which HS drives the cell-cycle checkpoint machinery is by the
activation of the DNA damage response pathway probably by generating reactive
oxygen species or through protein denaturation. DNA damage initiator and effector
proteins in this pathway are modulated by HS as well as by anti-cancer agents and
ionizing radiation, thus resulting in the delay of progression to the S-phase and cell
cycle arrest at the G1 and/or G2/M phases of the cell cycle. Here, I summarize the
cell cycle response provoked by HS with an aim to provide significant information
for the potential of hyperthermia for use in cancer treatment.

Keywords Cell-cycle checkpoint • Replication • DNA damage response • Cyclin-

dependent kinase inhibitor • Checkpoint kinase

6.1 Introduction

For a better understanding of how heat stress affects the cell cycle, I first describe
the cell cycle and cell-cycle regulating proteins. Since there are many excellent
textbooks and reviews describing the cell cycle [1], I will only briefly describe it
with a focus on mammalian cells (in particular human cells).
A cell has the capability to duplicate itself. When a cell reproduces, it needs to
duplicate its contents, including DNA and other organelles, and then divides in two
cells. Cells also need time to check the quality of the contents being duplicated for
cell division. After successful cell division, the newly reproduced cells perform
these steps again when they receive appropriate signals for proliferation. This

Y. Furusawa (*)
Department of Liberal Arts and Sciences, Faculty of Engineering, Toyama Prefectural
University, 5180 Kurokawa, Toyama 939-0398, Japan
e-mail: furusawa@pu-toyama.ac.jp

© Springer Science+Business Media Singapore 2016 61

S. Kokura et al. (eds.), Hyperthermic Oncology from Bench to Bedside,
DOI 10.1007/978-981-10-0719-4_6
62 Y. Furusawa

CDC25A

p21 p21
CDK
2

CDK Cyclin E CDK

4/6 2
Cyclin D Cyclin A

G1 S

M G2
CDK
1
Cyclin A
CDK p21
1
Cyclin B CDC25A
Wee1
CDC25C PLK1

Fig. 6.1 Cell cycle regulation by CDK-cyclin complexes, CDC25 phosphatases, and p21 CKI.
CDK4/6-cyclinD, CDK2-cyclin E, CDK2/1-cyclin A, and CDK1-cyclinB complexes are associ-
ated with the G1, G1/S, S-G2, and G2/M phases of the cell cycle, respectively. CDC25 phosphatases
promote cell cycle progression by dephosphorylating inhibitory phosphates on CDKs, whereas
p21 inhibits cell cycle progression by directly binding to CDK-cyclin complexes

orderly sequence of events is known as the “cell cycle,” which usually consists of
4 phases—the G1, S, G2, and M phases (Fig. 6.1), and each phase has its own
checkpoint machinery [2]. The most dynamic phase is the “M phase” in which cell
division occurs (“M” stands for mitosis). The chromosome is not stabilized through
the M phase and cells in the M phase thus show high sensitivity to heat stress
(HS) and ionizing radiation (IR). Previous studies have shown that HS causes
microtubule disorganization and multipolar mitosis, resulting in mitotic catastro-
phe, which is a cell death mode represented by cell destruction during mitosis
(including pre-mature mitosis) [3]. Heat shock protein 70, a 70-kDa molecular
chaperone protein, localized to the mitotic centrosome and protected against abnor-
malities in cell division, thus suggesting that the denaturation of proteins induced
by HS may cause damage to the mitotic centrosome [4].
The “S phase” is also a dynamic phase of the cell cycle where in chromosome
duplication for cell division occurs (“S” stands for synthesis). DNA replication is
initiated by the unwinding of the DNA double helix by DNA helicase, and the
unwound DNA is then stabilized by the associated single-strand DNA-binding
proteins known as replication protein A (RPA). The RPA complex, composed of
the RPA70, RPA32, and RPA14 subunits in humans, is an essential component of
the DNA replication process. The positions at which the DNA helix is opened are
6 Cell Cycle Responses to Hyperthermia 63

generally called replication origins, and thus this step is called “origin firing.” The
replication complexes composed of several proteins such as DNA polymerase for
nucleotide triphosphate polymerization and an accessary protein that forms a
sliding clamp (known as PCNA in mammalian cells) for keeping the polymerase
on the DNA strand are loaded onto the replication origin. Previous studies have
shown that HS preferentially led to the killing of cells at the S phase, probably due
to the thermal denaturation of the replication complex, which led to interference in
the initiation and elongation of the DNA [5, 6].
A cell has two gap phases called the G1 phase occurring between the M and S
phases, and the G2 phase occurring between the S and M phases. During the two gap
phases, the cell prepares components for entry into the next phase and monitors
whether the cellular condition is suitable for the transition to the next phase. If not,
the premature cell in the gap phase operates machineries to block the progression to
the next phase of the cell cycle. This step is called the “cell-cycle checkpoint.” [1, 2]
It should be noted that cell-cycle checkpoints are not restricted to only the G1 and
G2 phases but are also present in the S phase; the cell-cycle checkpoint of the S
phase is called the “intra-S phase checkpoint,” but the mechanism of action of the
intra-S phase checkpoint is less clear compared to that of the G1 and G2 phase
checkpoints [7–9]. Cells exposed to HS are also known to activate the G1, intra-S,
and G2/M checkpoint pathways, as described in following sections.

6.2 Cell Cycle Regulation by Cyclins, Cyclin-Dependent

Kinases (CDKs), Cell Cycle Division (CDC)
Phosphatase 25, and Cyclin-Dependent Kinase
Inhibitors (CKIs)

A cell commonly harbors a cell-cycle control system composed of members of the

protein kinase family, called CDK(s) [1]. Increase in CDK activity promotes cell-
cycle progression by regulating the phosphorylation of multiple downstream cell-
cycle regulators. CDK activity is controlled by the binding of cyclin, and a specific
pair of cyclin-CDK complexes contributes to the promotion of each step of entry
and progression into the next phase of the cell cycle (Fig. 6.1). For instance,
cyclin B, known as M-cyclin, forms a complex with CDK1 and stimulates mitotic
entry at the G2 phase, whereas cyclin D, known as G1 cyclin, forms a complex with
CDK4 or CDK6 and promotes cell-cycle progression into the S phase [11]. Among
these, the CDK1-cyclinB complex phosphorylates key proteins such as Polo-like
kinases (PLKs) and Aurora kinases (e.g., Aurora-A and -B), which contribute to the
assembly and stabilization of the mitotic spindle, respectively [12]. The CDK4-
cyclinD complex in the G1 phase promotes the phosphorylation and inhibition of
retinoblastoma protein (Rb), leading to E2F-dependent transcription of genes
required for S-phase entry [13].
CDK activity is controlled by the proteolysis or transcription of cyclins. In
addition, other cell-cycle regulating molecules such as Wee1 kinase and CDC25
64 Y. Furusawa

phosphatases contribute to cell cycle regulation through the phosphorylation or

dephosphorylation of CDKs [14]. Phosphorylation of the CDK inactivation site by
Wee1 changes the conformation of CDK-cyclin complexes, resulting in the inhi-
bition of CDK activity. On the contrary, dephosphorylation by CDC25 counteracts
the inhibition. Among CDC25 phosphatases, CDC25A is required for the progres-
sion of both the S- and the M-phases, whereas CDC25C is only required for mitotic
entry [15]. Phosphorylation of the CDC25 family of proteins by checkpoint kinases
(detailed in Sect. 6.3) as well as p38 mitogen-activated kinases (MAPKs) and
extracellular signal-regulated kinase (ERK) block S-phase entry through the phos-
phorylation and following degradation of CDC25A in response to extra-cellular
stress [16, 17].
CDK activity is also regulated by conformational changes in protein structure
caused by protein-protein interactions. CKIs, such as p21 and p27, bind to
CDK-cyclin complexes and inhibit their activities by alteration of the structure of
the complexes [18].
Negative regulation of the cell cycle has been well investigated in cells exposed
to genotoxic stress. Genotoxic stress caused by IR, hydroxyurea, and hyperthermia
induces cell-cycle arrest by activating cell-cycle checkpoint machinery, as
described below.

6.3 Cell-Cycle Checkpoint Activation Through Ataxia

Telangiectasia Mutated (ATM)-Checkpoint Kinase
2 (Chk2) and Ataxia Telangiectasia Mutated and Rad3-
Related (ATR)-Checkpoint Kinase 1 (Chk1) Axes

As shown in Chap. 3, HS itself is known to induce multiple types of DNA damage

such as single-strand breaks (SSBs), replicative stress in cells at the S phase, and
perhaps DNA double-strand breaks (DSBs) only in cells at the G1 and G2 phases
[19]. Whether HS induces DSBs in the same way as IR is a highly debated topic
[20, 21], and I hence will not refer to the type of DNA damage induced by HS in the
current section; however, there is no doubt that HS as well as genotoxic agents
activate the DNA damage response (DDR) pathway, thus promoting cell-cycle
arrest [22, 23]. To understand how the DDR pathway activated by HS negatively
regulates cell-cycle progression, I will shortly introduce the molecular functions of
proteins involved in the DDR pathway.
ATM and ATR are members of the phosphatidylinositol 3-kinase-related kinase
family that act as sensor proteins for DNA damage (reviewed in [24, 25]). ATM is
recruited to DNA DSB sites in conjunction with the MRE11/RAD50/ NBS1 (MRN)
complex, which recognizes the double-stranded end of DNA and contributes to
DNA repair (Fig. 6.2) [24]. Therefore, ATM is strongly activated by DNA damage
agents that directly induce DNA DSBs, such as IR or bleomycin. Auto-
phosphorylation of ATM at Ser1981 is often utilized for the activation of ATM
6 Cell Cycle Responses to Hyperthermia 65

DSBs (e.g., Ionizing radiation)

53BP1
MRE11 NBS1
RAD5
0
ATM

Phosphorylation of downstream proteins (e.g., p53 and Chk2)

Replicative stress (e.g., UV light or Hydroxyurea)

Hus RPA RPA RPA RPA

RAD1
1
TopBP1 ATRIP
RAD9
ATR
Claspin

Phosphorylation of downstream proteins (e.g., Chk1)

Fig. 6.2 Model of the cellular response to DNA DSBs and replicative stress. In response to DSBs,
ATM recognizes DSBs and activates itself, a process associated with the MRN complex and
53BP1. In response to replicative stress, RPA stabilizes the stalled replication fork followed by
ATR activation, which is associated with the 9-1-1 complex, ATRIP, TopBP1, and Claspin on the
replication fork. See Sect. 6.3 for details

and the full activation of ATM in response to DSBs requires the MRN
complex [24].
On the other hand, ATR is strongly activated by replicative stress elicited by
ultraviolet light or hydroxyurea [25]. When replication is impeded, DNA polymer-
ase uncouples from DNA helicase, resulting in the formation of ssDNA ends. Under
replicative stress, ssDNA is coated with the RPA complex to stabilize the stalled
replication fork. ATRIP forms a complex with RPA70, one of the components of
the RPA complex, as described above [26]. Association of the ATR-ATRIP com-
plex with the RPA-coated ssRNA at the stalled replication fork is required for the
activation of ATR. Activation of ATR also requires the Rad9-Hus1-Rad1 (9-1-1)
complex and topoisomerase binding protein (TopBP1) [27]. The 9-1-1 complex, a
heterotrimeric ring similar in structure to PCNA, is loaded onto RPA-coated
ssDNA by Rad17 [28]. TopBP1 interacts with Rad9, which brings the activation
domain of TopBP1 close to the RPA-bound ATR, thus allowing the activation of
ATR. Claspin, which associates with the active replication fork, is also required for
the activation of ATR [29]. ATR phosphorylation at Ser428 is a marker of ATR
activation, but unlike ATM, the auto-phosphorylation of ATR has not yet been
66 Y. Furusawa

demonstrated. ATR is activated by replicative stress, but it should be noted that

ATR is also activated in response to DSBs because ssDNA is generated during the
repair of DSBs [30]. Conversely, ATM is also activated by replicative stress,
probably due to DSB formation at the stalled replication fork [31].
ATM preferentially phosphorylates Chk2 at Thr68 rather than Chk1 at Ser317/
345, and both ATM and Chk2 kinases can phosphorylate and stabilize p53 (at Ser15
by ATM and at Ser20 by Chk2) [32]. p53 is well known to up-regulate p21 and thus
contribute to G1/S, intra-S phase, and weak G2/M checkpoint activation [9, 10]. The
primary role of the ATM-Chk2 pathway seems to be p53-dependent arrest at G1,
because mice deficient in Chk2 showed impaired G1 arrest in response to DNA
damage [33].
G2/M checkpoint activation following IR exposure is also largely dependent on
the ATR-Chk1 pathway. ATR phosphorylates Chk1 and phospho-Chk1 facilitates
the degradation or inactivation of CDC25 family proteins [34]. Chk1 contributes to
intra-S phase checkpoint activation by phosphorylating CDC25A and thus inhibits
DNA replication [8]. In addition, Chk1 phosphorylates CDC25C as well as Wee1
kinase and PLK1, leading to the inactivation of CDK1 and consequently to G2/M
arrest. [35, 36]. Chk2 also has the potential to phosphorylate CDC25 family pro-
teins; however, the role of Chk2 in G2/M arrest is less clear than that of Chk1
because Chk2-deficient MEFs and HCT116 cells do not exhibit defects in G2/M
checkpoint activation [33, 37]. However, inhibition of Chk1 results in G2/M
checkpoint defects, which is not observed in the case of Chk2 inhibition [38]. There-
fore, it is considered that Chk2 exists for compensation of G2/M checkpoint
activation by Chk1. Conversely, ATR and Chk1 can phosphorylate p53 [39, 40];
however, the mechanism of p53-mediated G1 arrest by ATR-Chk1 is less under-
stood than that by ATM-Chk2, because ATR-Chk1 activation seems to be restricted
to the S and G2 phases of the cell cycle [41].
In summary, both the ATM-Chk2 and the ATR-Chk1 pathways redundantly
activate downstream molecules associated with cell cycle regulation; however,
ATM-Chk2 preferentially regulates the p53-mediated activation of G1 and intra-S
phase checkpoints, whereas ATR-Chk1 regulates intra-S and G2/M checkpoint
activation.

6.4 Heat-Induced G1/S Checkpoint Activation

It may be difficult to simply compare the evidence showing heat-induced cell cycle
responses, because the effect of heat seems to be dependent on the duration of
heating at a given temperature and the cell type utilized in the studies. Therefore,
the information about both temperature and the duration of heating in addition to
the p53 phenotype must be described in order to accurately interpret the results
obtained by several groups (also see Table 6.1 and Fig. 6.3).
p53 response is well known to be augmented in cells damaged not only by IR but
also by HS [42, 43]. The phosphorylation of p53 at Ser15, leading to the
6 Cell Cycle Responses to Hyperthermia 67

Table 6.1 Heat-induced cell-cycle arrest associated with temperature, exposure time, and p53
phenotype
p53 Temp.
Cell phenotype ( C) Duration Arrest References Note
HEK293 Wild type 42 20 min G1 [53] p38-dependent Cdc25A
degradation (even in the
absence of pChk2)
Human Wild type 43 45 min G1 [46] p53-dependent p21
fibroblast induction
HL-60 Null 45 10 min G1 [51] HS for 20 min caused G2/
M arrest
A172 Mutated 45 15 min G1 [48] p53-independent p21
induction
T98G Mutated 45 15 min G1 [48] p53-independent p21
induction
U373- Mutated 43 120 min G2/M [47] Transfection of wild-type
MG p53 counteracts
HS-induced G2/M arrest
U251- Mutated 43 120 min G2/M [47] Transfection of wild-type
MG p53 counteracts
HS-induced G2/M arrest
Jurkat Mutated 44 30 min G2/M [52] Activation of ATR-Chk1
HL-60 Null 45 20 min G2 [51] HS for 10 min caused G1
arrest

stabilization of p53, seems to be dependent on ATM even in cells exposed to HS

[44]. HS induces p21 expression markedly in cells harboring wild-type p53, leading
to G1 arrest and a decrease in the population of cells in the S phase [45]. A previous
study showed that HS (43  C, 45 min) induced cell cycle arrest at the G1 phase in
normal human fibroblasts following p21 induction [46].
In general, it is widely believed that p53 deficiency weakens p21 induction and
causes preferential G2 arrest rather than G1 arrest in cells under genotoxic stress. A
previous study showed that U373-MG and U251-MG human glioma cells harboring
defective p53 showed increased numbers of cells at the G2/M phase in response to
HS (43  C, 120 min) [47], but this effect was not observed in cells in which p53 was
over-expressed using adenoviral transfection, thus indicating that p53 contributes to
G1 arrest in cells exposed to HS.
However, G1 arrest but not G2 arrest was found to be predominant in the human
glioblastoma cell lines A172 and T98G harboring defective p53 after HS exposure
(44  C, 15 min) [48]. p53-independent p21 induction, which may be explained by
the p53-independent p21 transcription pathway reviewed in [10], might be respon-
sible for the G1 arrest induced by HS in these p53-deficient cells.
Although the detailed mechanisms underlying the p53-independent induction of
p21 expression by HS remain unknown, we have previously observed induction of
CDKN1A mRNA (coding p21) expression in p53-deficient U937 cells exposed to
68 Y. Furusawa

MRE11 NBS1 Heat stress

(Replicative stress)
Translocation
RPA from nucleus (SSBs & DSBs?)
Nucleolin

+ ROS-induced bisulfite bond ? Rad9/Rad17/TopBP1/Claspin-dependent

+ Conformational change ? but RPA-independent
Nucleolin/RPA
translocation
ROS ? ATM ATR
Protein unfolding ?

(*): The pathway has not yet

Chk2 been directly demonstrated
* in cells exposed to HS
p38 *
p53 Chk1
*
Short-term Long-term
* *

CDC25A p21 * CDC25C Wee1 PLK1

M G1 S G2 M

Fig. 6.3 Model of the cellular response to HS. In brief, ATM and ATR are activated even if
NBS1, MRE11, and RPA are translocated from the nucleus under HS. ATM phosphorylates and
stabilizes p53, perhaps leading to the induction of p21 expression and cell cycle arrest at the G1
phase. p38 MAPK-dependent G1 arrest is also induced by CDC25A degradation. Chk2-dependent
CDC25A phosphorylation also occurs if the heat treatment is prolonged. HS activates the intra-S
phase checkpoint through translocation of the RPA-nucleolin complex, although it has not yet
been demonstrated that p21 and Chk1 contributes to the inhibition of S-phase progression in cells
exposed to HS. ATR rather than ATM phosphorylates Chk1 and causes G2/M arrest, probably
thorough phosphorylation of downstream cell-cycle regulators such as CDC25C, PLK1, and
Wee1, even in cells exposed to HS. See Sects. 6.4, 6.5 and 6.6 for details

HS (44  C, 15 min) [49]. In addition, Tabuchi et al. showed p53-dependent

induction of CDKN1A mRNA expression even in cells exposed to HS at a lower
temperature (41  C, 30 min) [50] (also see GEO Accession: GSE10043). These
results indicate that p21 expression can be responsive to HS at a broad temperature
range even in the absence of p53.
The duration of heating rather than the p53 phenotype seems to determine the
next cell-cycle stage. An impressive study in HL-60 cells showed that heating at
45  C for 10 min preferentially increased the number of cells at the G1 phase,
whereas heating for 20 min increased the number of cells at the G2 phase [51],
suggesting that prolonged heat exposure switches G1 arrest to G2/M arrest. Consis-
tent with this report, another study also showed that the long-term exposure to HS
(44  C, 30 min) causes G2/M arrest in Jurkat cells harboring mutated p53 [52],
whereas hyperthermia for a short duration (42  C, 20 min or 45  C, 15 min) induces
6 Cell Cycle Responses to Hyperthermia 69

the accumulation of cells at the G1 phase but not at the G2 phase, not only in p53-
proficient HEK293 cells but also in p53-mutated A172 and T98G cells [48, 53]. In
another study using HEK293 cells, Madlener et al. showed that the p38-dependent
degradation of CDC25A is also responsible for the G1 arrest induced by exposure to
HS for 20 min [53]. The authors reported that the degradation of CDC25A was also
dependent on Chk2 but not on Chk1 under HS for 60 min. Notably, the phosphor-
ylation of both Chk1 and Chk2 was not observed in cells exposed to HS for 20 min,
indicating that short-term exposure to HS induces p38-dependent but Chk2-
independent G1 arrest. Considering that the p38 MAPK is present even in
p53-deficient cells, heat-induced G1 arrest in cells deficient in p53 gene might be
explained by p38-dependent CDC25A phosphorylation as well as p53-independent
p21 induction.
Taken together, it appears that the heat durations used in Ref. [48] and Ref. [51],
showing heat-induced G1 arrest in p53-mutated or -deficient cells, may be not
enough to activate checkpoint kinases and thus failed to activate the following
G2/M checkpoint (See Sect. 6.7). p53 was also found to be important but dispens-
able for G1 arrest in cells exposed to HS. The G1 arrest in p53-mutated or -deficient
cells, probably through p53-dependent p21 induction and p38 MAPK-dependent
CDC25A degradation, seems to be largely dependent on the duration and temper-
ature of heat treatment.

6.5 Heat-Induced Intra S-Phase Checkpoint Activation

The intra S-phase checkpoint contributes to a transient decrease in the rate of DNA
synthesis, probably through slowing down of the elongation of the replication fork
or inhibition of origin firing. Among DDR proteins, p21 and Chk1 are implicated in
the regulation of the intra-S phase checkpoint. p21 blocks DNA synthesis as well as
G1-S transition by interacting with PCNA, which is necessary for the formation of
the replication complex [10]. In addition, Chk1 phosphorylates and thus degrades
CDC25A, resulting in the inactivation of the CDK2-Cyclin A complex [8, 54].
To the best of my knowledge, however, direct evidence showing the involve-
ment of p21 and Chk1 in intra-S phase checkpoint activation in cells exposed to HS
is not yet available. On the other hand, a previous study revealed that HS inhibited
S-phase progression by directly inhibiting the process of replication, including the
initiation of new replicons and the elongation of the replication fork [55]. The
molecular mechanism underlying heat-induced intra-S phase checkpoint activation
is the translocation of RPA from the nucleus. HS causes a translocation of
nucleolin, which forms a complex with RPA and leads to RPA dysfunction,
resulting in the inhibition of DNA replication [55]. We also confirmed that HS
did not cause nuclear RPA32 foci formation, which is a marker of replication fork
stalling in cells treated with a replication inhibitor such as hydroxyurea [52],
probably due to the release of the RPA-nucleolin complex from the nucleus
[55, 56].
70 Y. Furusawa

These results raised a new question: How does HS activate the ATR pathway
even in the absence of RPA? The RPA complex accumulates in the single-stranded
end on a stalled replication fork (thus forming foci in the nucleus), not only leading
to the stabilization of the replication fork but also providing a platform for ATR
phosphorylation [25, 56]. However, HS was found to induce ATR phosphorylation
despite RPA foci formation being attenuated [52].
Tuul et al. reported that the heat-induced ATR phosphorylation was also depen-
dent on TopBP1, Rad9, Rad17, and Claspin, suggesting the presence of a mecha-
nism underlying the RPA-independent ATR activation by HS [57]. Supporting this
idea, although in a Xenopus egg experiment, it was reported that ATR activation
occurred even in the absence of RPA32 but not in the absence of ATRIP or
Claspin [58].
Other possible mechanism involved in ATR activation might be the conforma-
tional change in proteins or the production of reactive oxygen species (ROS)
evoked in cells exposed to HS (see Chap. 2 for ROS production by HS). Hunt
et al. proposed that the conformational change in ATM caused by HS led to its
phosphorylation because HS induced ATM phosphorylation even in the absence of
obvious DNA DSBs [21]. Guo et al. also revealed that an ATM dimer formed
disulfide bonds in the presence of massive oxidative stress induced by hydrogen
peroxide, thus inducing the conformational change in ATM [59].
Therefore, it might be possible that the activation of ATR as well as ATM occurs
as a consequence of conformational changes in proteins caused by HS. The detailed
mechanism by which HS activates ATR and the role of p21 and Chk1 in intra-S
phase checkpoint activation should be further elucidated.

6.6 Heat-Induced G2/M Checkpoint Activation

Unlike that of p53-dependent G1/S checkpoint activation, the molecular mechanism

underlying G2/M checkpoint activation in cells exposed to HS had not been studied
until recent years. Recently, based on the DDR-mediated cell-cycle regulation
pathway, we aimed to evaluate the role of the ATM-Chk2 and ATR-Chk1 pathways
in G2/M checkpoint activation in cells exposed to HS. We found that HS (44  C,
30 min) activated the ATR-Chk1 pathway rather than the ATM-Chk2 pathway
[52]. ATR phosphorylation at Ser428 rapidly occurred, but ATM phosphorylation
at Ser1981 was slightly delayed after heat treatment in human leukemia Jurkat cells
harboring mutated p53. In addition, ATR phosphorylation induced by HS was not
suppressed in the presence of the ATM inhibitor Ku55933, which was different
from that observed in the case of SSBs ends of DNA during DSB repair [41], thus
indicating that ATR does not act downstream of ATM in cells exposed to HS.
The molecular mechanisms involved in ATR and ATM phosphorylation induced
by HS were similar but slightly different from that induced by genotoxic agents
such as IR or hydroxyurea. In the case of ATR activation, as mentioned above,
RPA32 was not required in cells exposed to HS. Similarly, heat-induced ATM
6 Cell Cycle Responses to Hyperthermia 71

activation is not accompanied with the formation of the MRN complex and 53BP1
because HS induces the transport of MRE11 and NBS1 from the nucleus into the
cytoplasm and suppresses 53BP1 recruitment to the damaged DNA site [21, 60, 61].
Although the mechanism underlying the activation of ATR and ATM needs to
be further elucidated, it seems certain that cells can respond to HS through these
kinases. In particular, the role of ATR-Chk1 in G2/M checkpoint activation was
obvious, unlike that of ATM-Chk2. An ATR-specific inhibitor, Schisandrin B,
preferentially suppressed Chk1 phosphorylation than Ku55933, supporting evi-
dence showing that ATR is the primary kinase of Chk1. Inhibition of Chk1 by a
selective Chk1 inhibitor SB218078 abrogated the accumulation of cells at the G2/M
phase, and increased the population of Sub- G1 cells and the cleavage of caspase-3
(apoptotic markers) not only in Jurkat cells, but also in other p53-deficient human
cancer cells such as HeLa (cervical carcinoma), HSC-3 (squamous carcinoma), and
PC3 (prostate cancer) cells exposed to HS. This suggested a critical role of Chk1 in
G2/M checkpoint activation and apoptosis evasion. However, whether cells with
dysfunctional Chk1 undergo apoptosis because of checkpoint abrogation and fol-
lowing chromosomal aberration needs to be investigated in the future.
The role of the ATM-Chk2 pathway in checkpoint activation and cell survival is
less clear than that of the ATR-Chk1 pathway. One study indicated that Chk2
contributes to CDC25A degradation and the subsequent induction of G1 arrest as
described in Sect. 6.4 [53]. However, in our study, inhibition of ATM or Chk2
showed marginal effects on apoptosis in Jurkat cells exposed to HS, indicating that,
at least under the conditions of HS employed in our study, G1 arrest induced by
Chk2-dependent CDC25A phosphorylation might not be involved in the promotion
of cell survival. One of the possible roles of the ATM-Chk2 pathway in response to
HS is in p53-dependent cell-cycle checkpoint activation because both ATM and
Chk2 have the potential to phosphorylate and thus stabilize p53.
Currently, to the best of my knowledge, whether the abrogation of ATM and/or
Chk2 elicits dysfunction of cell cycle regulation in p53-proficient cells exposed to
HS has not been elucidated. It will be interesting to elucidate whether ATM and
Chk2 contribute to p53-dependent cell-cycle checkpoint activation under
HS. Efforts to elucidate the role of ATM-Chk2 in heat response are currently
underway.

6.7 Sensitization of Tumor Cells to Hyperthermia by

Targeting Cell-Cycle Checkpoint Machineries: A
Future Strategy for Combination Therapy

Hyperthermia has been used to sensitize cells to DNA-damaging agents (combina-

tion therapy) because HS has the potential to inhibit DNA repair (see Chaps. 8, 9,
and 10 and Chaps. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 on combination
therapy). Inhibition of the DNA damage response pathway by small molecules, in
72 Y. Furusawa

particular inhibition of DNA repair or cell-cycle checkpoint activation, is currently

being investigated as an approach to boost the effect of DNA-damaging agents in
cancer therapy [62]. In particular, a combination of DNA-damaging agents and
checkpoint kinase inhibitors has been considered as a promising strategy for cancer
therapy because many cancer cells exhibit defective p53 thus rely on G2/M check-
point for cell survival [54].
Our group and Tuuls et al. showed that abrogation of the G2/M checkpoint
pathway by inhibition of ATR-Chk1 promoted heat-induced apoptosis and loss of
clonogenicity [52, 57], indicating that targeting cell-cycle checkpoint machinery
may be one of the strategies for hyperthermia. An ATR-specific inhibitor, VE-821
(IC50 is 13 nM in Ref [63]), which inhibits ATR more strongly than does
Schisandrin B, was recently developed; however, the clinical use of ATR inhibitors
has some problems. First, ATR is essential for accurate DNA replication in normal
cells and ATR inhibition can exert unexpected side effects by affecting the
ATM-p53 pathway [28]. Although Chk1 is also essential for replication, currently,
the small molecule inhibitor of Chk1 rather than that of ATR is being developed for
use in combination with chemotherapy [64].
Further, as shown in Table 6.2, several checkpoint kinase 1 inhibitors (some of
which are not selective only to Chk1 but also inhibit Chk2) are currently under
preclinical or clinical trial phase I/II trials but have not yet been approved for use as
drugs. Before considering the application of Chk1 inhibitors in hyperthermia,
whether Chk1 inhibition can be applied clinically in the chemotherapy needs to
be further investigated.

Table 6.2 Chk1 inhibitors under preclinical (Pre) or clinical trials (-Oct, 2015)
Compound name Phase of clinical
(Alias) IC50 Other targets (IC50) Trial Reference
LY2606368 1.5 nM Chk2 (4.7 nM) II https://
(MK-1775) clinicaltrials.gov
SCH900776 3 nM CDK2 (160 nM), Chk2 II https://
(MK-8776) (1500 nM) clinicaltrials.gov
LY2603618 7 nM PDK1 (893 nM) II https://
clinicaltrials.gov
GDC-0425 N.D. N.D. I https://
clinicaltrials.gov
GDC-0575 (Arry- 2 nM Rks3 (39 nM), MYLK I https://
575) (48 nM) clinicaltrials.gov
XL-844 2.2 nM Chk2 (200 nM) I https://
clinicaltrials.gov
PF-00477736 0.5 nM Chk2 (47 nM) I https://
clinicaltrials.gov
SAR-020106 13 nM CDK1 (7500 nM) Pre [65]
CCT-244747 8 nM N.D. Pre [66]
CEP-3891 4 nM TrkA (9 nM), MLK1 Pre [67]
(42 nM)
N.D.: Not disclosed
6 Cell Cycle Responses to Hyperthermia 73

References

1. Gra~na X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent
kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs).
Oncogene. 1995;11:211–19.
2. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature. 2004;18:316–23.
3. Vidair CA, Doxsey SJ, Dewey WC. Heat shock alters centrosome organization leading to
mitotic dysfunction and cell death. J Cell Physiol. 1993;154:443–55.
4. Hut HM, Kampinga HH, Sibon OC. Hsp70 protects mitotic cells against heat-induced centro-
some damage and division abnormalities. Mol Biol Cell. 2005;16:3776–85.
5. Warters RL, Stone OL. The effects of hyperthermia on DNA replication in HeLa cells. Radiat
Res. 1983;93:71–84.
6. Mondovi B, Strom R, Rotilio G, Agro AF, Cavalliere R, Rossi FA. The biochemical mecha-
nism of selective heat sensitivity of cancer cells. Eur J Cancer. 1969;5:129–36.
7. Seiler JA, Conti C, Syed A, Aladjem MI, Pommier Y. The intra-S-phase checkpoint effects
both DNA replication initiation and elongation: single-cell and -DNA fiber analyses. Mol Cell
Biol. 2007;27:5806–18.
8. Sørensen CS, Syljuåsen RG, Falck J, Schroeder T, R€ onnstrand L, Khanna KK, et al. Chk1
regulates the S phase checkpoint by coupling the physiological turnover and ionizing
radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell. 2003;3:247–58.
9. Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer.
2009;9:400–14.
10. Jung Y-S, Qian Y, Chen X. Examination of the expanding pathways for the regulation of p21
expression and activity. Cell Signal. 2010;22:1003–12.
11. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol.
1999;39:295–312.
12. Barr FA, Silljé H, Nigg EA. Polo-like kinases and the orchestration of cell division. Nat Rev
Mol Cell Biol. 2004;5:429–40.
13. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995;81:323–30.
14. Coleman TR, Dunphy WG. Cdc2 regulatory factors. Curr Opin Cell Biol. 1994;6:877–82.
15. Donzelli M, Draetta GF. Regulating mammalian checkpoints through Cdc25 inactivation.
EMBO Rep. 2003;4:671–7.
16. Isoda M, Kanemori Y, Nakajo N, Uchida S, Yamashita K, Ueno H, et al. The extracellular
signal-regulated kinase–mitogen-activated protein kinase pathway phosphorylates and targets
Cdc25A for SCFβ-TrCP-dependent degradation for cell cycle arrest. Mol Biol Cell.
2009;20:2186–95.
17. Wagner EF, Nebreda ÁR. Signal integration by JNK and p38 MAPK pathways in cancer
development. Nat Rev Cancer. 2009;9:537–49.
18. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of
cyclin kinases. Nature. 1993;366:701–4.
19. Velichko AK, Petrova NV, Kantidze OL, Razin SV. Dual effect of heat shock on DNA
replication and genome integrity. Mol Biol Cell. 2012;23:3450–60.
20. Takahashi A, Matsumoto H, Nagayama K, Kitano M, Hirose S, Tanaka H, et al. Evidence for
the involvement of double-strand breaks in heat-induced cell killing. Cancer Res.
2004;64:8839–45.
21. Hunt CR, Pandita RK, Laszlo A, Higashikubo R, Agarwal M, Kitamura T, et al. Hyperthermia
activates a subset of ataxia-telangiectasia mutated effectors independent of DNA strand breaks
and heat shock protein 70 status. Cancer Res. 2007;67:3010–17.
22. Velichko AK, Markova EN, Petrova NV, Razin SV, Kantidze OL. Mechanisms of heat shock
response in mammals. Cell Mol Life Sci. 2013;70:4229–41.
23. Turner T, Caspari T. When heat casts a spell on the DNA damage checkpoints. Open Biol.
2014;4:140008–8.
24. Lavin MF, Kozlov S. ATM activation and DNA damage response. Cell Cycle. 2007;6:931–42.
74 Y. Furusawa

25. Nam EA, Cortez D. ATR signalling: more than meeting at the fork. Biochem
J. 2011;436:527–36.
26. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA com-
plexes. Science. 2003;300:1542–8.
27. Lee J, Kumagai A, Dunphy WG. The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction
of TopBP1 with ATR. J Biol Chem. 2007;21:28036–44.
28. Wagner JM, Kaufmann SH. Prospects for the use of ATR inhibitors to treat cancer. Pharma-
ceuticals. 2010;3:1311–34.
29. Jeong SY, Kumagai A, Lee J, Dunphy WG. Phosphorylated claspin interacts with a phosphate-
binding site in the kinase domain of Chk1 during ATR-mediated activation. J Biol Chem.
2003;278:46782–8.
30. Cuadrado M, Martinez-Pastor B, Toledo LI, Gutierrez-Martinez P, Lopez E, Fernandez-
Capetillo O. ATM regulates ATR chromatin loading in response to DNA double-strand breaks.
J Exp Med. 2006;203:297–303.
31. Unno J, Takagi M, Piao J, Sugimoto M, Honda F, Maeda D, et al. Artemis-dependent DNA
double-strand break formation at stalled replication forks. Cancer Sci. 2013;104:703–10.
32. Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell.
2003;3:421–9.
33. Takai H, Naka K, Okada Y, Watanabe M, Harada N, Daito S, et al. Chk2‐deficient mice exhibit
radioresistance and defective p53‐mediated transcription. EMBO J. 2002;21:5195–205.
34. Blasina A, de Weyer IV, Laus MC, Luyten WH, Parker AE, McGowan CH. A human
homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase. Curr Biol.
1999;9:1–10.
35. O’Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk 1 is a wee 1 kinase in the G2 DNA
damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 1997;16:545–54.
36. Tang J, Erikson RL, Liu X. Checkpoint kinase 1 (Chk1) is required for mitotic progression
through negative regulation of polo-like kinase 1 (Plk1). Proc Natl Acad Sci.
2006;103:11964–9.
37. Jallepalli PV, Lengauer C, Vogelstein B, Bunz F. The Chk2 tumor suppressor is not required
for p53 responses in human cancer cells. J Biol Chem. 2003;278:20475–9.
38. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, et al. Chk1 is an essential kinase
that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev.
2000;14:1448–59.
39. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, et al. A role for
ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 1999;13:152–7.
40. Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of checkpoint kinases
Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes
Dev. 2000;14:289–300.
41. Jazayeri A, Falck J, Lukas C, Bartek J, Smith GCM, Lukas J, et al. ATM- and cell cycle-
dependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol.
2006;8:37–45.
42. Ohnishi K, Ohnishi T. Heat-induced p53-dependent signal transduction and its role in hyper-
thermic cancer therapy. Int J Hyperthermia. 2001;17:415–27.
43. Ohnishi T. The role of the p53 molecule in cancer therapies with radiation and/or hyperther-
mia. J Cancer Res Ther. 2005;1:147–50.
44. Miyakoda M, Suzuki K, Kodama S, Watanabe M. Activation of ATM and phosphorylation of
p53 by heat shock. Oncogene. 2002;21:1090–6.
45. Abe T, Tamiya T, Ono Y, Salker AH, Akiyama K, Ohmonot T. Accumulation of cell cycle
regulatory proteins, p21 and p27, induced after hyperthermia in human glioma cells. Int J
Hyperthermia. 2001;17:499–507.
46. Niitta M, Okamura H, Aizawa S, Yamaizumi M. Heat shock induces transient p53-dependent
cell cycle arrest at G1/S. Oncogene. 1997;15:561–8.
47. Nashimoto T, Komata T, Kanzawa T, Aoki H, Endo S, Kon T, et al. Mild hyperthermia plus
adenoviral p53 over-expression additively inhibits the viability of human malignant glioma
cells. Int J Hyperthermia. 2005;21:615–29.
6 Cell Cycle Responses to Hyperthermia 75

48. Fuse T, Yamada K, Asai K, Kato T, Nakanishi M. Heat shock-mediated cell cycle arrest is
accompanied by induction of p21 CKI. Biochem Biophys Res Commun. 1996;225:759–63.
49. Furusawa Y, Tabuchi Y, Takasaki I, Wada S, Ohtsuka K, Kondo T. Gene networks involved in
apoptosis induced by hyperthermia in human lymphoma U937 cells. Cell Biol Int.
2009;33:1253–62.
50. Tabuchi Y, Takasaki I, Wada S, Zhao QL, Hori T, Nomura T, et al. Genes and genetic
networks responsive to mild hyperthermia in human lymphoma U937 cells. Int J Mol Med.
2011;28:143–51.
51. Lim CU, Zhang Y, Fox MH. Cell cycle dependent apoptosis and cell cycle blocks induced by
hyperthermia in HL-60 cells. Int J Hyperthermia. 2006;22:77–91.
52. Furusawa Y, Iizumi T, Fujiwara Y, Zhao Q-L, Tabuchi Y, Nomura T, et al. Inhibition of
checkpoint kinase 1 abrogates G2/M checkpoint activation and promotes apoptosis under heat
stress. Apoptosis. 2012;17:102–12.
53. Madlener S, Rosner M, Krieger S, Giessrigl B, Gridling M, Vo TP, et al. Short 42 C heat shock
induces phosphorylation and degradation of Cdc25A which depends on p38MAPK, Chk2 and
14.3.3. Hum Mol Genet. 2009;18:1990–2000.
54. Zhang Y, Hunter T. Roles of Chk1 in cell biology and cancer therapy. Int J Cancer.
2013;134:1013–23.
55. Iliakis G, Krieg T, Guan J, Wang Y, Leeper D. Evidence for an S-phase checkpoint regulating
DNA replication after heat shock: a review. Int J Hyperthermia. 2004;20:240–9.
56. Wang Y, Guan J, Wang H, Wang Y, Leeper D, Iliakis G. Regulation of DNA replication after
heat shock by replication protein A-nucleolin interactions. J Biol Chem. 2001;276:20579–88.
57. Tuul M, Kitao H, Iimori M, Matsuoka K, Kiyonari S, Saeki H, et al. Rad9, Rad17, TopBP1 and
claspin play essential roles in heat-induced activation of ATR kinase and heat tolerance. PLoS
One. 2013;8:e55361.
58. Kim SM, Kumagai A, Lee J, Dunphy WG. Phosphorylation of Chk1 by ATM- and Rad3-
related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable
DNA-binding or coiled-coil domains of ATRIP. J Biol Chem. 2005;280:38355–64.
59. Guo Z, Kozlov S, Lavin MF, Person MD, Paull TT. ATM activation by oxidative stress.
Science. 2010;330:517–21.
60. Takahashi A, Mori E, Ohnishi T. Phospho-Nbs1 and Mre11 proteins which recognize DSBs
co-localize with γH2AX in the nucleus after heat treatment. Ann Cancer Res Ther.
2007;15:50–3.
61. Laszlo A, Fleischer I. Heat-induced perturbations of DNA damage signaling pathways are
modulated by molecular chaperones. Cancer Res. 2009;69:2042–9.
62. Jekimovs C, Bolderson E, Suraweera A, Adams M, O’Byrne KJ, Richard
DJ. Chemotherapeutic compounds targeting the DNA double-strand break repair pathways:
the good, the bad, and the promising. Front Oncol. 2014;4:86.
63. Reaper PM, Griffiths MR, Long JM, Charrier J-D, Maccormick S, Charlton PA, et al. Selective
killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol.
2011;7:428–30.
64. Toledo LI, Murga M, Fernandez-Capetillo O. Targeting ATR and Chk1 kinases for cancer
treatment: a new model for new (and old) drugs. Mol Oncol. 2011;5:368–73.
65. Walton MI, Eve PD, Hayes A, Valenti M, Brandon A, Box G, et al. The preclinical pharma-
cology and therapeutic activity of the novel CHK1 inhibitor SAR-020106. Mol Cancer Ther.
2010;9:89–100.
66. Walton MI, Eve PD, Hayes A, Valenti MR, De Haven Brandon AK, Box G, et al. CCT244747
is a novel, potent and selective inhibitor of CHK1 with oral efficacy alone and in combination
with genotoxic anti-cancer drugs. Clin Cancer Res. 2012;18:5650–61.
67. Syljuåsen RG, Sørensen CS, Nylandsted J, Lukas C, Lukas J, Bartek J. Inhibition of Chk1 by
CEP-3891 accelerates mitotic nuclear fragmentation in response to ionizing radiation. Cancer
Res. 2004;64:9035–40.