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68
CHAPTER 10
3.
Metaphase.
The chromosomes align at the metaphase plate. The cells can be ar-
rested in this stage by microtubule inhibitors (e.g., colchicine). The cells arrested
in this stage can be used for karyotype analysis.
4.
Anaphase.
The centromeres split, the kinetochores separate, and the chromo-
somes move to opposite poles. The kinetochore microtubules shorten. The polar
microtubules lengthen.
5.
Telophase.
The chromosomes begin to decondense to form chromatin. The nu-
clear envelope re-forms. The nucleolus reappears. The kinetochore microtubules
disappear. The polar microtubules continue to lengthen.
6.
Cytokinesis.
The cytoplasm divides by a process called cleavage. A cleavage fur-
row forms around the middle of the cell. A contractile ring consisting of actin and
myosin filaments is found at the cleavage furrow.
Control of the Cell Cycle (Figure 10-2).
The control of the cell cycle involves three
main components which include
A. CDK-CYCLIN COMPLEXES. The two main protein families that control the cell cycle
are cyclins and the cyclin-dependent protein kinases (Cdks). A cyclin is a protein that
regulates the activity of Cdks and is so named because cyclins undergo a cycle of syn-
thesis and degradation during the cell cycle. The cyclins and Cdks form complexes
called Cdk-cyclin complexes. The ability of Cdks to phosphorylate target proteins is
dependent on the particular cyclin that complex with it.
1.
Cdk2-cyclin D and Cdk2-cyclin E
mediate the G
1
S
S phase transition at the G
1
checkpoint.
2.
Cdk1-cyclin A and Cdk1-cyclin B
mediate the G
2
S
M phase transition at the G
2
checkpoint.
B. CHECKPOINTS. The checkpoints in the cell cycle are specialized signaling mecha-
nisms that regulate and coordinate the cell response to DNA damage and replication
fork blockage. When the extent of DNA damage or replication fork blockage is beyond
the steady-state threshold of DNA repair pathways, a checkpoint signal is produced
and a checkpoint is activated. The activation of a checkpoint slows down the cell cycle
so that DNA repair may occur and/or blocked replication forks can be recovered. This
prevents DNA damage from being converted into inheritable mutations producing
highly transformed, metastatic cells.
1.
Control of the G
1
checkpoint.
There are three pathways that control the G
1
checkpoint which include
a.
Depending on the type of the DNA damage, ATR kinase and ATM kinase will
activate (i.e., phosphorylate) Chk1 kinase or Chk2 kinase, respectively. The
activation of Chk1 kinase or Chk2 kinase causes the inactivation of CDC25 A
phosphatase. The inactivation of CDC25 A phosphatase causes the down-
stream stoppage at the G
1
checkpoint.
b.
Depending on the type of the DNA damage, ATR kinase and ATM kinase will
activate (i.e., phosphorylate) p53, which allows p53 to disassociate from
Mdm2. The activation of p53 causes the transcriptional upregulation of p21.
The binding of p21 to the Cdk2-cyclin D and Cdk2-cyclin E inhibits their ac-
tion and causes downstream stoppage at the G
1
checkpoint.
c.
Depending on the type of the DNA damage, ATR kinase and ATM kinase will
activate (i.e., phosphorylate) p16, which inactivates Cdk4/6-cyclin D and
thereby causes downstream stoppage at the G
1
checkpoint.
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69
THE CELL CYCLE
2.
Control of the G
2
checkpoint.
Depending on the type of the DNA damage, ATR
kinase and ATM kinase will activate (i.e., phosphorylate) Chk1 kinase or Chk2
kinase, respectively. The activation of Chk1 kinase or Chk2 kinase causes the
inactivation of CDC25 C phosphatase. The deactivation of CDC25 C phosphatase
will cause the downstream stoppage at the G
2
checkpoint.
C. INACTIVATION OF CYCLINS. Cyclins are inactivated by protein degradation during
anaphase of the M phase. The cyclin genes contain a homologous DNA sequence called
a destruction box. A specific recognition protein binds to the amino acid sequence
coded by the destruction box that allows ubiquitin (a 76 amino acid protein) to be co-
valently attached to lysine residues of cyclin by the enzyme ubiquitin ligase. This
process is called polyubiquitination. Polyubiquitinated cyclins are rapidly degraded by
proteolytic enzyme complexes called a proteosome. Polyubiquitination is a widely oc-
curring process for marking many different types of proteins (cyclins are just a specific
example) for rapid degradation.
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70
CHAPTER 10
DNA
damage
ssDNA
ATR
ATM
CDC25A
CDC25C
ChK2
ChK1
p53
p53
p21
E2F
E2F
G
1
checkpoint
G
0
G
1
(5 hrs)
G
2
(3 hrs)
M
(1 hr)
S
(7 hrs)
Pro
p
has
e
Pro
m
etap
hase
Me
ta
phas
e
Ana
p
hase
TelophaseCyt
o
kines
is
G
2
checkpoint
+
+
RB
RB
cdk2-cyclin D
cdk2-cyclin E
cdk1-cyclin A
cdk1-cyclin B
cdk4/6-cyclin D
p16
Mdm2
Mdm2
DNA
damage
Double strand
DNA breaks
STOP
STOP
STOP
STOP
ChK2
ChK1
p16
PO4
PO4
PO4
PO4
PO4
Pathways:
1
2
3
● Figure 10-2 Diagram of the cell cycle with checkpoints and signaling mechanisms. ATR kinase responds to
the sustained presence of single-stranded DNA (ssDNA) because ssDNA is generated in virtually all types of DNA dam-
age and replication fork blockage by activation (i.e., phosphorylation) of Chk1 kinase, p53, and p16. ATM kinase
responds particularly to double-stranded DNA breaks by activation (i.e., phosphorylation) of Chk2 kinase, p53, and
p16. The downstream pathway past the STOP sign is as follows: Cdk2-cyclinD, Cdk2-cyclinE, and Cdk4/6-cyclinD phos-
phorylate the E2F-RB complex which causes phosphorylated RB to disassociate from E2F. E2F is a transcription factor
that causes the expression of gene products that stimulate the cell cycle. Note the location of the four stop signs.
S
activation; inactivation.
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71
The Development of Cancer (Oncogenesis).
In general, cancer is caused by muta-
tions of genes that regulate the cell cycle, DNA repair, and/or programmed cell death (i.e.,
apoptosis). A majority of cancers (so-called sporadic cancers) are caused by mutations of
these genes in somatic cells that then divide wildly and develop into a cancer. A minority
of cancers (so-called hereditary cancers) are predisposed by mutations of these genes in
the parental germ cells that are then passed on to their children. In addition, certain can-
cers are linked to environmental factors as prime etiological importance (e.g., bladder cancer/
aniline dyes, lung cancer/smoking or asbestos, liver angiosarcoma/polyvinyl chloride, skin
cancer/tar, or UV irradiation). From a scientific point of view, the cause of cancer is not
entirely a mystery but still remains in the theoretical arena which include the following:
● Figure 11-1 Standard Theory.
A. STANDARD THEORY (Figure 11-1). The
standard theory suggests that cancer is the
result of cumulative mutations in proto-
oncogenes (e.g., RAS gene) and/or tumor-
suppressor genes (e.g., TP53 gene) eventually
producing a cancer cell. However, if cancer is
caused only by mutations in these specific cell
cycle genes, it is very hard to explain the ap-
pearance of the nucleus in a cancer cell. The
nucleus in a cancer cells looks as if something
has detonated an explosion resulting in an ar-
ray of chromosomal aberrations (e.g., chromo-
some pieces, scrambled chromosomes, chromosomes fused together, wrong number of
chromosomes, chromosomes with missing arms, or chromosome with extra segments;
so-called karyotype chaos). The question is “Which comes first, the mutations in cell
cycle genes or the chromosomal aberrations?” The photograph (left side) shows a nor-
mal human karyotype. The photograph (right side) shows an abnormal human kary-
otype due to a mutation involving the RAD 17 checkpoint protein which plays a role
in the cell cycle. This mutation results in a re-replication of already replicated DNA
and an abnormal karyotype.
B. MODIFIED STANDARD THEORY (Figure
11-2). The modified standard theory suggests
that cancer is the result of a dramatically ele-
vated random mutation rate caused by envi-
ronmental carcinogens or malfunction in the
DNA replication machinery or DNA repair ma-
chinery. The random mutations eventually hit
● Figure 11-2 Modified Standard
Theory.
the proto-oncogenes (e.g., RAS gene) and/or tumor-suppressor genes (e.g., TP53 gene)
producing a cancer cell.
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72
CHAPTER 11
C. EARLY INSTABILITY THEORY (Figure 11-3).
The early instability theory suggests that can-
cer is the result of disabling (either by muta-
tion or epigenetically) of “master genes” that
are required for cell division. No specific mas-
ter genes have been identified. Therefore, each
time a cell undergoes the complex process of
● Figure 11-3 Early Instability Theory.
● Figure 11-4 All-Aneuploidy Theory.
aneuploid. The chromosomal aberrations get worse with each cell division eventually
producing a cancer cell.
E.
THE FORMATION OF CANCER STEM CELLS. All adult tissues contain adult stem
cells that are predominately dormant until they are activated when adult tissues re-
quire replenishment due to wear and tear or injury. However, the repair capacity of
adult stem cells is limited in comparison with embryonic stem cells. Consequently,
when the repair capacity of adult stem cells is exhausted, they may undergo transfor-
mation leading to oncogenesis.
A. HIGH LEVELS OF GENOMIC INSTABILITY. Genomic instability is broadly classified
into microsatellite instability (MIN) and chromosome instability (CIN).
1.
Microsatellite instability.
MIN refers to a condition whereby microsatellite DNA
is abnormally lengthened or shortened due to defects in various DNA repair
processes.
2.
Chromosome instability.
CIN refers to condition whereby chromosomal DNA
continuously forms novel chromosome mutations at a rate higher than normal cells.
CIN is typically associated with the accumulation of mutations in proto-oncogenes
and tumor–suppressor genes. The mechanisms of CIN involve chromosome break-
age, concurrent breaks in two chromosomes giving rise to translocations, and loss
of chromosomes.
B. DNA REPAIR. There are three types of DNA repair that may affect the mutation
phenotype.
1.
Nucleotide excision repair
2.
Base excision repair
3.
Mismatch repair (MMR)
C. ACCUMULATION OF MUTATIONAL EVENTS. Currently, it is believed that multiple
mutation events are required to transform normal cells to cancer cells. The current con-
sensus is that oncogenesis imparts six “superpowers” to a cancer cell as indicated below.
1.
A cancer cell can grow in the absence of normal growth-promoting signals (e.g.,
EGF [epidermal growth factor]) binding to the EGFR (EGF receptor).
cell division, some daughter cells get chromosomes fused together, the wrong number
of chromosomes, chromosomes with missing arms, or chromosome with extra segments
which will affect gene dosage of the proto-oncogenes and tumor-suppressor genes. The
chromosomal aberrations get worse with each cell division eventually producing a can-
cer cell.
D. ALL-ANEUPLOIDY THEORY (Figure 11-4).
The all-aneuploidy theory suggests that cancer
is the result of aneuploidy (i.e., abnormal
number of chromosomes) that occurs during
cell division. Although a great majority of ane-
uploid cells undergo apoptosis, the few sur-
viving cells will produce progeny that are also
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