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Chapter 13
Molecular Genetics of Cell
Cycle and Cancer
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The Cell Cycle
•
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There are two major parts in the cell cycle:
 Mitosis: M
 Interphase: G1 = gap1, S = DNA synthesis, G2 =
gap2
There are two essential functions of the cell cycle:
 To ensure that each chromosomal DNA
molecule is replicated only once per cycle
 To ensure that the identical replicas of each
chromosome are distributed equally to the two
daughter cells
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Figure 13.1: Major events in the cell cycle
Adapted from L. H. Hartwell and M. B. Kastan, Science 266 (1994):
1821-1828.
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The Cell Cycle
• The cell cycle is under genetic control
• A fundamental feature of the cell cycle is that it is a
true cycle, it is not reversible
• Many genes are transcribed during the cell cycle
just before their products are needed
• Mutations affecting the cell cycle have helped to
identified the key regulatory pathways
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The Cell Cycle
• Progression from one phase to the next is propelled
by characteristic protein complexes, which are
composed of cyclins and cyclin-dependent protein
kinases (CDK)
• Expression of mitotic cyclins E, A, and B are
periodic, whereas cyclin D is expressed throughout
the cell cycle in response to mitosis stimulating
drugs (mitogens)
• The cyclin-CDK complexes phosphorylate targeted
proteins, dramatically changing their activity
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Figure 13.8: Fluctuations of cyclin levels during the cell cycle
Adapted from C. J. Sherr, Science 274 (1996): 1672-1677.
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The Retinoblastoma Protein
• The retinoblastoma (RB) protein controls the initiation
of DNA synthesis.
• RB maintains cells at a point in G1 called the G1
restriction point or start by binding to the transcription
factor E2F, until the cell has attained proper size
• If the cycling cell is growing properly and becomes
committed to DNA synthesis, several cyclin-CDK
complexes inactivate RB by phosphorylation
• After the cell enters S phase, E2F becomes
phosphorylated as well and loses its ability to bind
DNA
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Figure 13.10: Role of the retinoblastoma protein RB
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The Cell Cycle
• The progression from G2 to M is controlled by a
cyclin B-CDK2 complex known as maturationpromoting complex
• Protein degradation (proteolysis) also helps regulate
the cell cycle. The anaphase-promoting complex
(APC/C), which is a ubiquitin–protein ligase
responsible for adding the 76-amino-acid protein
ubiquitin to its target proteins and marking them for
destruction in the proteasome
• Proteolysis eliminates proteins used in the preceding
phase as well as proteins that would inhibit
progression into the next
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Checkpoints
• Cells monitor their external environments and
internal state and functions
• Checkpoints in the cell cycle serve to maintain the
correct order of steps as the cycle progresses;
they do this by causing the cell cycle to pause
while defects are corrected or repaired
• Checkpoints in the cell cycle allow damaged cells
to repair themselves or to self-destruct
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Three Main Checkpoints
• A DNA damage checkpoint
• A centrosome duplication checkpoint
• A spindle checkpoint
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Figure 13.12: Key cell-cycle checkpoints that act to maintain the genetic
stability of cells
Adapted from L. H. Hartwell and M. B. Kastan, Science 266 (1994):
1821-1828.
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A DNA Damage Checkpoint
• A DNA damage checkpoint arrests the cell cycle
when DNA is damaged or replication is not
completed.
• In animal cells, a DNA damage checkpoint acts at
three stages in the cell cycle: at the G1/S transition,
in the S period and at the G2/M boundary
• The p53 transcription factor is a key player in the
DNA damage checkpoint.
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A DNA Damage Checkpoint
• In normal cells, level of activated p53 is very low
• Protein Mdm2 keeps p53 inactivated by preventing
phosphorylation and acetylation of p53 and by
exporting p53 from the nucleus
• Damaged DNA leads to activation of p53 and its
release from Mdm2
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A DNA Damage Checkpoint
• Activated p53 triggers transcription of a number of
genes – p21, 14-3-3s, Bax, Apaf1, Maspin, GADD45
• DNA damage detected in G1 blocks cell G1/S
transition
• DNA damage in S phase reduces processivity of
DNA polymerase and gives the cell time for repair
• Processivity – number of consecutive nucleotides
that replicate before polymerase detaches from
template
• DNA damage detected in S or G1 arrests cells at
G2/M transition
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Figure 13.15: Role of activated p53 in the DNA damage checkpoint
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A DNA Damage Checkpoint
• DNA damage also triggers activation of a pathway
for apoptosis = programmed cell death
• When the apoptotic pathway is activated, a cascade
of proteolysis is initiated that culminates in cell
suicide
• The proteases involved are called caspases
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Centrosome Duplication Checkpoint
• Monitors spindle formation
• Functions to maintain the normal complement
of chromosomes
• Sometimes coordinates with the spindle
checkpoint and the exit from mitosis
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The Spindle Checkpoint
• Monitors assembly of the spindle and its
attachment to kinetochores
• The kinetochore is the spindle-fiber attachment
site on the chromosome
• Incorrect or unbalanced attachment to the spindle
activates spindle checkpoint proteins, triggers a
block in the separation of the sister chromatids by
preventing activation of the anaphase-promoting
complex (APC/C)
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Figure 13.18: The spindle checkpoint
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Cancer
• Cancer cells have a small number of mutations that
prevent normal checkpoint function
• Cancer is not one disease but rather many
diseases with similar cellular attributes
• All cancer cells show uncontrolled growth as a
result of mutations in a relatively small number of
genes
• Cancer is a disease of somatic cells
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Cancer
• 1% of cancer cases are familial: show evidence
for segregation of a gene in pedigree
• 99% are sporadic: the result of genetic changes
in somatic cells
• Within an organism, tumor cells are clonal, which
means that they are descendants from a single
ancestral cell that became cancerous
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Cancer Cells vs. Normal Cells
• In normal cells, cell-to-cell contact inhibits further
growth and division, a process called contact
inhibition
• Cancer cells have lost contact inhibition: they
continue to grow and divide, and they even pile
on top of one another
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Cancer Cells vs. Normal Cells
• Even in the absence of damage, normal cells cease
to divide in culture after about 50 doublings = cell
senescence
• Senescence of normal cells is associated with a
loss of telomerase activity: the telomeres are no
longer elongated, which contributes to the onset of
senescence and cell death
• Cancer cells have high levels of telomerase, which
help to protect them from senescence, making them
immortal
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Key Mutational Targets
• Many cancers are the result of alterations in cell
cycle control, particularly in control of the G1-to-S
transition
• These alterations also affect apoptosis through
their interactions with p53
• The major mutational targets for the multistep
cancer progression are of two types:
 Proto-oncogenes
 Tumor-suppressor genes
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Key Mutational Targets
• The normal function of proto-oncogenes is to
promote cell division or to prevent apoptosis
• The normal function of tumor-suppressor genes is
to prevent cell division or to promote apoptosis
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Oncogenes
• Oncogenes are derived from normal cellular genes
called proto-oncogenes
• Oncogenes are gain-of-function mutations
associated with cancer progression
• Oncogenes are gain-of-function mutations because
they improperly enhance the expression of genes
that promote cell proliferation or inhibit apoptosis
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Oncogenes
• Mdm2:
 Amplification of Mdm2 gene and overexpression
of Mdm2 protein leads to inactivation of p53 gene
 Amplification of Mdm2 gene has been found in
many tumors of adipose tissue, soft tissue
sarcomas, osteosarcoma, and esophageal
carcinoma
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Oncogenes
• Cyclin D and CDK4:
 Amplification and overexpression leads to
unscheduled entry to S phase
 Amplification and/or overexpression has been
found in many esophageal carcinomas,
bladder and breast cancers
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Oncogenes
• Growth-factor receptors:
 Cellular growth factors stimulate growth
by binding to a growth-factor receptor
at the cell membrane
 The binding activates a signal
transduction pathway that acts through
Ras, cyclin D, and its partner CDKs.
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Oncogenes
• Growth-factor receptors:
 Amplification and overexpression of the gene
that encodes the receptor for epidermal
growth factor (EGFR) has been found in
many malignant astrocytomas,
glioblastomas, breast and ovarian cancers,
head and neck cancers, and melanomas.
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• Ras:
Oncogenes
 The Ras protein acts as a switch in stimulating cellular growth
in the presence of growth factors.
 Certain mutant Ras proteins lack GTPase activity and remain
in the form of Ras–GTP. The signal for cellular growth is
transmitted constitutively–unrestrained growth and division
take place.
Figure 13.22: Function of the Ras protein
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Tumor-Suppressor Genes
 Tumor-suppressor genes normally negatively
control cell proliferation or activate the
apoptotic pathway
 Loss-of-function mutations in tumorsuppressor genes contribute to cancer
progression.
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Tumor-Suppressor Genes
•
p53:
– Loss of function of p53 eliminates the DNA
checkpoint that monitors DNA damage in G1 and
S
– The damaged cells survive and proliferate and
their genetic instability increases the probability
of additional genetic changes, thus progressing
toward the cancerous state
– p53 proves to be nonfunctional in more than half
of all cancers
– Mutant p53 proteins are found frequently in
melanomas, lung cancers, colorectal tumors,
bladder and prostate cancers.
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Tumor-Suppressor Genes
• p21:
 Loss of p21 function results in renewed rounds of DNA
synthesis without mitosis, and the level of ploidy of the
cell increases. Mutations in the p21 gene occur in some
prostate cancers.
• p16/p19ARF:
 The p16 and p19ARF proteins are products of the same
gene transcribed from different promoters. The p16
product can inhibit the cyclin D–Cdk4 complex and help
control entry into S phase The gene is deleted in many
gliomas, mesotheliomas, melanomas, nasopharyngeal
carcinomas, biliary-tract and esophageal carcinomas.
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Tumor-Suppressor Genes
• RB:
 The retinoblastoma protein controls the transition
from G1 to S phase by controlling the activity of the
transcription factor E2F Loss of RB function frees
E2F, hence, excessive rounds of DNA synthesis are
continuously being initiated.
 Loss of RB function is found in melanomas, small-cell
lung carcinoma, osteosarcoma, and liposarcomas
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Tumor-Suppressor Genes
• Bax:
 The Bax tumor-suppressor protein promotes
apoptosis
 Loss of Bax function is found particularly in
gastric adenocarcinomas and in colorectal
carcinomas associated with microsatellite
instability because of defective mismatch
repair.
 Cells that are defective in mismatch repair are
prone to undergo replication slippage leading
to deletions or additions of nucleotides in runs
of short tandem repeats
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Tumor-Suppressor Genes
• Bub l:
 Bub1 is a protein that is primarily involved
in the spindle checkpoint
 A subset of colon cancers show
chromosomal instability. Some of these
unstable lines are defective in Bub1.
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Familial Cancers
• Mutations that predispose to cancer can be
inherited through the germ line
• The presence of this mutation predisposes the
individual to cancer, because it reduces the
number of additional somatic mutations necessary
for a precancerous cell to progress to malignancy
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Li–Fraumeni Syndrome
• The Li–Fraumeni syndrome shows autosomal
dominant inheritance. However, the affected
individuals have a range of different tumors and
often have more than one
• A large fraction of Li–Fraumeni families show
segregation for a mutation in the p53 gene.
• A situation analogous to the human Li–Fraumeni
syndrome has been created in mice by
experimental knockout (loss of function) of the p53
gene via the germ-line transformation
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Figure 13.24: Pedigree of Li–Fraumeni syndrome
Data from W. A. Blattner, et al., J. Am. Med. Assoc. 241 (1979): 259-261.
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Retinoblastoma
• Retinoblastoma (RB) protein in animal cells holds
cells at restriction point by binding to and holding
E2F
• Alfred Knudson in 1971 suggested that loss of the
wildtype allele of a tumor-suppressor gene might
be the triggering event at the cellular level for
tumors in heterozygous genotypes, and that
genesis of a tumor in familial cases of RB required
a “single hit” in a somatic cell, whereas genesis of
a tumor in sporadic cases required “two hits”
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Retinoblastoma
• RB is inherited in pedigrees as a simple Mendelian
dominant. But Knudson’s hypothesis implied that
even in familial cases, there must be another
mutational event that triggers tumor development.
• Analysis of genetic markers around the gene in tumor
cells revealed that the triggering event is the loss of
the wildtype RB1 allele
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Retinoblastoma
• At the organismic level, the mutant gene is
dominant. At the cellular level, it is recessive.
• Several mechanisms can uncover the mutant allele:
chromosome loss, mitotic recombination, deletion,
and inactivating nucleotide substitutions
• Uncovering of the recessive allele by various
mechanisms is called loss of heterozygosity
• RB is an inherited cancer syndrome associated
with loss of heterozygosity in the tumor cells.
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Defects in DNA Repair
• Genetic instability clearly contributes to the origin
of tumor cells
• Some inherited cancer syndromes result from
defects in processes of DNA repair
• Inherited skin cancer syndromes are called
xeroderma pigmentosum
• Xeroderma pigmentosum cells are defective in
nucleotide excision repair
• Individuals with this syndrome are very sensitive to
ultraviolet light
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Acute Leukemias
• Acute leukemias are malignant diseases of the bone
marrow, spleen, and lymph nodes associated with
uncontrolled proliferation of leukocytes and their
precursors in the bone marrow
• Acute leukemias do not arise as a consequence of
alterations in cell cycle regulation or checkpoints,
nor are they familial
• Up to 60% of acute leukemias result from a
chromosomal translocation that fuses a
transcription factor with a leukocyte regulatory
sequence
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Acute Leukemias
• The translocations are of the two types:
 promoter fusion—the coding region for a gene that
encodes transcription factor is translocated near an
enhancer for an immunoglobulin heavy-chain gene
or a T-cell receptor gene
 gene fusion—found more frequently in acute
leukemia than a promoter fusion. The translocation
breakpoints occur in introns of genes for
transcription factors in two different chromosomes.
The result is a fusion gene called a chimeric gene
composed of parts of the original gene
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