CANCER
AND THE ROLE OF CELL CYCLE CHECKPOINTS
}
Will Renthal
University of Texas - Austin
Communicated By: Dr. Eva Lee
University of Texas Health Science Center at San Antonio, Institute
of Biotechnology
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SUMMARY
There exists an intricate network of proteins that continuously monitor
each phase of the cell cycle to ensure proper replication. This network
of proteins, termed checkpoints, first detects cellular abnormalities,
and then coordinates their repair before the cell divides. The malfunction
of these checkpoints often results in the proliferation of potentially
damaged cells, and thus a tremendous susceptibility to cancer. This
review will focus on the mechanisms by which checkpoints prevent the
proliferation of damaged cells through each phase of the cell cycle,
and how this understanding can provide novel targets for anticancer
therapy.
INTRODUCTION
The classic definition of cancer is "uncontrolled cell division."
In a large, multi-cellular organism, uncontrolled cell division will
soon result in large masses of rapidly growing cells (tumors), which
causes significant damage to surrounding tissues. When tumors spread,
they can damage vital organs and eventually cause death. In fact, cancer
is currently the second leading cause of death in the United States,
and thus cures for it would be of incalculable value. Current treatments
of cancer involve exposing the patient to relatively nonspecific toxins,
chemotherapy, in the hope that it will kill more cancer cells than normal
cells. This type of medicine is a modern equivalent of 18th century
bleeding treatments for bacterial infections. However, if clear biochemical
differences between cancer cells and normal cell are discovered, chemotherapy
could be improved considerably. Much as antibiotics only harm bacteria,
novel anticancer drugs that only harm cancer cells can be developed
through research. Since cancer is essentially the loss of cell division
control, it seems prudent to search in these regulatory mechanisms for
distinguishing characteristics of cancer cells. This review will present
the general mechanisms which drive the cell cycle and what is currently
known about the regulatory pathways that control it. It will then discuss
how current anticancer therapies are taking advantage of cell cycle
research.
THE CELL CYCLE
The Nobel Prize in Medicine or Physiology was recently awarded to three
men, Leland Hartwell, Tim Hunt, and Paul Nurse, "for their discoveries
of key regulators of the cell cycle" (www.nobel.se).
Essentially every topic discussed in this review was in some way pioneered
by these three men. The details they helped uncover may seem at first
glance rather cumbersome, but it is these very details that will eventually
enable the development of targeted anticancer therapies.
Many of our cells can be triggered to divide upon the proper mitogenic
stimulus (Cross and Dexter, 1991). A growth factor for example, can
trigger a highly regulated unidirectional program that, when run successfully,
results in the proper replication and division of the cell. This program,
the cell cycle, must copy the parent cell's chromosomes and seal them
in a safe daughter cell with all of the essential components needed
for the daughter cell to function on its own. The human cell cycle accomplishes
this in approximately 24 hours through four major phases: G1 (gap 1),
S (DNA Synthesis), G2 (gap 2), and M (mitosis) (Lodish et al., 2000).
Each phase serves a specific function to ensure proper cell division.
G1 - Gap 1
G1 takes about 9 hours to prepare the cell for DNA synthesis (S-phase)
(Lodish et al., 2000). Though preparing the cell for S-phase may seem
relatively uneventful, G1 is actually the most important regulatory
phase in the in the cell cycle. It is in this phase that the cell
decides whether to irreversibly continue the cell cycle through mitosis,
or to enter G0, a quiescent phase during which the cell can function
but not divide (Figure 1). This decision, called the restriction point
in mammalian cells, is made in late G1 based predominantly on external
growth signals (Lodish et al., 2000). Another key feature of late
G1, which contributes to the unidirectionality of the cell cycle progression,
is the priming of replication origins with MCM (minichromosome maintenance)
proteins. The binding of these proteins to origins of replication
is required for the initiation of DNA synthesis, but they can only
bind to DNA in late G1 (Young and Tye, 1997). Thus, DNA synthesis
is only initiated once - right at the G1/S transition.
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Figure
1: The Cell Cycle
The cell
progresses through its division cycle, G1->S->G2->M,
in the presence of growth signals and active CDK/cyclin complexes
specific for each cell cycle stage. G0, a non-dividing but functional
phase, occurs in the absence of growth signals. Finally, cyclin
degradation is illustrated by the S-phase cyclin/Cdk complex
(cyclin E/Cdk2) and the G2/M cyclin/Cdk complex (cyclin B/Cdc2),
where each must be synthesized and degraded systematically for
proper cell cycle progression into the next phase.
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S - DNA Synthesis
Once the cell has passed the restriction point, proteins synthesized
in late G1 initiate the DNA replication machinery of S-phase. This
delicate process of copying the parent cell's genome takes approximately
10 hours, and yields a single cell with two sets of each chromosome
(sister chromatids) (Lodish et al., 2000).
G2 - Gap 2
G2 lasts about 4.5 hours and serves as a buffer to ensure the completion
of DNA synthesis before the cell physically divides in mitosis (Lodish
et al., 2000). Now with two sets of each chromosome, cell growth continues
in order to double its size such that upon division two fully functional
cells will result. A large amount of information is known about the
G2/M transition, and it is discussed in detail below. This phase serves
to synthesize proteins required for nuclear envelope breakdown, chromosome
condensation, spindle formation, and other processes required for
entry into mitosis (Maller et al., 1989). Many of these mitosis-promoting
functions cannot be initiated until DNA synthesis is completed, thus
serving as a buffer phase to prevent premature cell division.
M - Mitosis
Mitosis (nuclear division) is comprised of four substages during which
specific events occur to separate daughter chromosomes from the parent's
and enclose them in a new cell. Mitosis typically takes about 30 minutes
in human cells, which is rather fast considering the complexity of
this phase (Lodish et al., 2000). The first substage to occur is Prophase,
in which the proteins synthesized in G2 break down the nuclear envelope
of the parent cell, condense its chromosomes, and initiate spindle
formation. Prometaphase follows as a transition period during which
the sister chromatids shuffle until they align in the middle of the
cell, which is then termed Metaphase. The sister chromatids then separate
to opposite poles of the cell in Anaphase, which is followed its physical
division in Telophase.
Cyclins and Cyclin-Dependent
Kinases
The proper transition from each cell cycle phase to the next is dependent
on two classes of proteins called cyclins and cyclin-dependent kinases.
As the name suggests, cyclins are a class of proteins which are periodically
synthesized and degraded, and the coordination of proper cyclin levels
at the right time is essential for successful cell cycle progression.
Cyclin-dependent kinases (Cdk) are a class of kinases whose catalytic
activity is dependent on complexing with an appropriate cyclin. Once
this complex is formed, the Cdk kinase activity is activated which
results in the phosphorylation of many downstream effectors (reviewed
in Udvardy, 1996). This phosphorylation serves to regulate the activity
of the downstream effector, typically by activating or inhibiting
it. One of the best studied cyclin/Cdk complexes involves cyclin B
and Cdc2, also called MPF (Maturation Promoting Factor), and was first
discovered by Yoshio Masui and Clement Market (Masui and Markert,
1971). It has been shown to serve many crucial roles in cell cycle
progression such as nuclear envelope degradation and sister chromatid
condensation in early mitosis (Maller et al., 1989) (Figure 1). These
MPF-dependent processes are essential for the cell to efficiently
divide the genetic information into daughter cells, and thus the activity
of MPF is tightly regulated. If the cell allowed MPF to remain active
through the latter stages of mitosis when the nuclear envelopes are
reforming, cell division would be prevented altogether. The way the
cell deals with this problem is by ubiquitin-mediated proteolysis
of cyclin B in late mitosis (Murray et al., 1989). Thus, the actively
regulated levels of cyclin B mediate mitotic entry and exit. Without
the synthesis of cyclin B prior to the G2/M transition, the kinase
activity of Cdc2 remains inactive, and the cell can not enter mitosis.
Without the subsequent degradation of cyclin B, the kinase activity
of Cdc2 remains active and prevents the exit from mitosis.
CELL CYCLE CONTROL:
CHECKPOINTS
A cell cycle checkpoint is a general term used to describe a cellular
process that stops or slows the cell cycle in conditions unfavorable
for cell division (Hartwell and Weinert, 1989). This review will focus
on the DNA damage cell cycle checkpoint. Since our cells undergo continuous
bombardment by DNA damaging agents such as UV light and by-products
of cellular metabolism, there exists an elaborate and evolutionarily
conserved cellular DNA damage response that coordinates cell cycle progression
with the repair of potentially mutagenic DNA damage (reviewed in Zhou
and Elledge, 2000). Many of the proteins involved in the mammalian DNA
damage response act as tumor suppressors and suggest that there are
newly evolved repair and/or checkpoint genes critical in the maintenance
of genome integrity, which highlights an additional importance of checkpoint
control in mammals. The DNA damage response, like most cellular signaling
pathways, involves first sensing a signal and then transducing it to
downstream effectors that elicit the appropriate response. This review
will present the recent studies, including some previously unreviewed,
which have provided tremendous insight into many key components of this
signal transduction pathway (outlined in Table 1).
Table1:
Components of the DNA damage response
Cell cycle checkpoint proteins and their respective functions. Putative,
but not yet proven functions are followed by question marks. |
Name
|
Function
|
PCNA |
Trimeric
DNA clamp, holds pols on DNA |
RFC1-5 |
Pentameric
complex, loads PCNA onto DNA |
CSC
- Checkpoint Sliding Clamp(Rad1, Rad9, Hus1) |
DNA
damage sensor? Structurally similar to PCNA |
Rad17 |
DNA
damage sensor? Complexes with RFC2-5 to load CSC onto DNA |
Rad26 (fission
yeast)
ATRIP (humans)
|
DNA
damage sensor? |
Rad3
(fission yeast)
ATR (humans) |
DNA
damage transducer |
ATM |
DNA
damage transducer |
p53 |
Transducer
for G1/S checkpoint and apoptosis |
p21 |
Cdk4,6
Inhibitor, involved in G1/S checkpoint |
Cdc25A |
Initiation
of DNA synthesis, S-phase checkpoint |
Nbs1 |
S-phase
checkpoint transducer, DNA repair |
Chk1 |
G2/M
checkpoint transducer |
Cdc25C |
G2/M
checkpoint transducer |
MPF
- Mitosis Promoting Factor(Cyclin B/Cdc2) |
Necessary
for G2/M transitionDNA damage effector, G2/M checkpoint |
Cyclin
D/Cdk4,6 |
Necessary
for G1/S transitionDNA damage effector, G1/S checkpoint |
Sensors
It is still unclear precisely how the cell senses damaged DNA, but
a group of four fission yeast checkpoint proteins, Rad1, Rad9, Hus1,
and Rad17, have been implicated along with their respective homologues
in other organisms (reviewed in Lowndes and Murguia, 2000). It has
been proposed that Rad1, Rad9, and Hus1 form a trimeric checkpoint-sliding
clamp (CSC) similar in structure to the DNA polymerase clamp PCNA
(Proliferating Cell Nuclear Antigen) (Venclovas and Thelen , 2000).
By analogy to the loading of PCNA onto DNA by the RFC1-5 pentamer,
the CSC is loaded onto DNA by Rad17/RFC2-5, where Rad17 is a checkpoint
protein that replaces RFC1 in the pentamer (Venclovas and Thelen ,
2000). Once the CSC is loaded onto DNA, it could serve not only to
recruit DNA polymerase but also to signal the activation of downstream
DNA damage checkpoint proteins. In addition to the CSC and Rad17,
the checkpoint protein Rad26 has also been implicated as a DNA damage
sensor because it binds to and is phosphorylated by the Rad3 kinase
independently of all other checkpoint proteins (Edwards et al., 1999).
Thus, Rad26 has been proposed to be a sensor or at least far upstream
in the DNA damage response. A recent model, the substrate recruitment
model (Melo et al., 2001), provides some insight into the behavior
of the putative DNA damage sensors Rad26/Rad3 and CSC/Rad17 in budding
yeast (Figure 2). The model proposes that each complex is independently
recruited to the same sites of DNA damage, and work in tandem for
proper DNA damage checkpoint activation (Kondo et al., 2001; Melo
et al., 2001). After the budding yeast CSC homologue is loaded onto
DNA near sites of damage, it recruits various substrates of its Rad3
homologue to drive the signal transduction pathway of the DNA damage
response. Though this model seems quite convincing in budding yeast,
little is known about DNA damage sensing in mammalian cells (Melo
et al., 2001). Recently, it was shown that the activation of both
G1/S and G2 DNA damage checkpoints requires the phosphorylation of
hRad17 by ATR (a Rad3 related kinase in mammals) and possibly ATM
as well (Bao et al., 2001; Post et al., 2001). However, unphosphorylated
hRad17 can still load hRad9 of the CSC onto chromatin (Zou et al.,
2002), so the significance of this phosphorylation remains unclear.
Also, a recently cloned human protein, ATRIP, has some homology to
the putative DNA damage sensor Rad26 (Cortez et al., 2001). ATRIP
also seems to have many functional similarities to Rad26 including
its tight association with and its phosphorylation by ATR (Rad3 related
kinase), and a role in the G2/M checkpoint. Thus, human homologues
of both of the putative DNA damage sensors in yeast seem to play similar
roles in the human DNA damage response.
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Figure
2: Substrate recruitment model for sensing DNA damage
Both ATRIP
(Rad26) and the checkpoint sliding complex (CSC) Rad1, Rad9,
Hus1, bind to the same sites of DNA damage. The CSC is loaded
onto chromatin after DNA damage in a Rad17/RFC2-5 catalyzed
reaction. Once chromatin bound, the CSC recruits substrates
of the ATR (Rad3) signal transducer kinase to propagate the
DNA damage response.
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Transducers
Once DNA damage is sensed, the cell must transduce this signal down
to its appropriate effector. In human cells, the activation of two
kinases is essential for the proper transduction of DNA damage: ATM
(Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3 Related) (reviewed
in Elledge, 1996; Zhou and Elledge, 2000). Little is known about precisely
how ATM and ATR are activated, but a great deal has been uncovered
about their respective roles in coordinating the DNA damage response.
ATM was identified from a rare mutation found in the disease ataxia
telangiectasia and leads to chromosomal instability and a high susceptibility
to cancer (Savitsky et al., 1995). There are no known pathologies
with ATR mutations, and ATR knockout mice die in early embryogenesis
(Brown and Baltimore, 2000; de Klein et al., 2001). Upon activation,
these similar kinases phosphorylate a number of target proteins, which
transmit the DNA damage signal downstream, eventually arresting the
cell cycle, initiating DNA repair, or, if necessary, causing cell
death (apoptosis) (reviewed in Zhou and Elledge SJ, 2000) (Figure
3).
 |
Figure
3: The DNA Damage Response
DNA damage
is first detected by sensor proteins which in turn activate
transducers in the signal cascade. These transducers then mediate
the activation or inhibition of downstream effectors which can
arrest the cell cycle or cause apoptosis.
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Depending on when the DNA
damage is sensed, ATM/ATR will activate a different axis of proteins.
For G1 damage, ATM/ATR will phosphorylate p53, which then acts as
a transcription factor for the synthesis of p21 (Cdk inhibitor) (Canman,
et al., 1998; Banin et al., 1998; Tibbetts et al., 1999). Upon p21
expression, the G1/S transition is inhibited, preventing the synthesis
of damaged DNA (Li et al., 1994). If damaged DNA is sensed during
S-phase, the cell needs to slow DNA synthesis to provide time for
repair. An ATM dependent pathway exists for an intra S-phase checkpoint
in which ATM activation leads to the degradation Cdc25A (Falck et
al., 2001). Since Cdc25A drives the initiation of DNA synthesis (Vigo
et al., 1999), its degradation would allow synthesis to slow during
S-phase and provide the necessary time for repair. This intra-S phase
checkpoint is also controlled by the ATM phosphorylation of Nbs1 (Lim
et al., 2000; Zhao et al., 2000; Wu et al., 2000), but the precise
molecular mechanism remains elusive. Interestingly, Nbs1 is a protein
involved in DNA repair as well, so this functional link between ATM
and Nbs1 provides evidence of a high level of coordination between
cell cycle progression and DNA repair. DNA damage incurred after S-phase
results in the activation of the G2/M checkpoint to prevent entry
into mitosis with damaged chromosomes. ATM/ATR also mediate this pathway
by phosphorylating Chk1 in response to DNA damage (Chen et al., 1999;
Zhao et al., 2001). This phosphorylation of Chk1 appears to enhance
its kinase activity, which in turn phosphorylates Cdc25C (Sanchez
et al., 1997). Cdc25C is inactivated by this phosphorylation and can
no longer mediate entry into mitosis.
These signal transduction pathways (Figure 3) are just a few examples
of the complex interacting network of proteins actually involved in
processing DNA damage signals. The main idea embedded in these vast
yet important details is that proteins (ATM/ATR) are activated upon
DNA damage, and then trigger phase-specific cell cycle arrest and
DNA repair.
Effectors
The halting of the cell cycle is typically elicited by deactivating
the Cyclin/Cdk complex involved in a specific phase transition (G1/S
or G2/M). For example, p21 which is synthesized in response to DNA
damage in G1, directly inhibits Cdk4,6 and thus prevents the transcription
of proteins required for DNA synthesis. The final effector in the
G2/M checkpoint is the CyclinB/Cdc2 complex (MPF) described earlier
as being essential for the transition from G2 into mitosis. Upon DNA
damage, the Cdc25C phosphatase can no longer remove inhibitory phosphates
from Cdc2, and thus prevents the CyclinB/Cdc2 complex from breaking
down the nuclear envelope, condensing chromosomes, and other events
that occur in early mitosis.
CELL CYCLE CONTROLLERS
AS ANTICANCER DRUG TARGETS
Thus far this review has focused on the details underlying the control
of cell division, but it is important not to lose sight of the exciting
applications of this knowledge. For example, about half of all tumors
have a damaged copy of the tumor-suppressor protein p53. Now that much
of the detailed mechanism by which p53 inhibits tumor growth is understood,
drugs can be developed to take advantage of its action. There are several
biotechnology companies currently attempting to develop p53 therapies
by reconstituting functional p53 back into tumor cells. This would restore
the broken signal transduction pathway, and thus prevent tumorigenesis.
The DNA damage checkpoint consists of many more pathways than those
introduced in this review, and it is difficult to say at this phase
which pathways will be of greatest utility in anticancer therapies.
Thus, current cancer biology research is directed at better characterizing
known pathways and elucidating novel ones involved in the DNA damage
response.
ABOUT THE AUTHOR
Will Renthal is currently a third-year Biochemistry Honors student at
The University of Texas at Austin. There he conducts research as an
Arnold and Mabel Beckman Scholar on the mechanism by which a novel antibiotic
kills bacteria and as an NSF Fellow on MAP Kinase signal transduction.
For the previous two summers, he has researched cell cycle checkpoints
with Dr. Eva Lee at the University of Texas Health Science Center at
San Antonio, Institute of Biotechnology. His research focused specifically
on characterizing the functions of a protein which is commonly mutated
in Nijmegen Breakage Syndrome (NBS) patients. This is a disease in which
patients have an extremely high susceptibility to cancer and chromosomal
instability. In his two summers of research, he has helped to clarify
some of the subtle points about the NBS gene product, which is involved
in both cell cycle checkpoints and DNA repair.
After his undergraduate education he plans to attend either an MD/PHD
program or graduate school where he will further study the mechanisms
underlying cell growth and development. Following this graduate training
and a brief postdoctoral position, he aspires to become a professor
at a medical center. There he hopes to conduct high quality basic research
with a focus on drug discovery while teaching the next generation of
scientists.
ACKNOWLEDGEMENTS
I would like to thank Dr. Eva Lee for providing me with two incredible
summers of exciting research; Dr. Song Zhao for serving as my mentor
and friend; Sean Post for stimulating discussions; and the rest of the
lab for their support. I also thank Drs. Ann and Robert Renthal for
their critical reading of this manuscript.
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