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Chapter 20




In This Chapter











Cancer stem cells




Primary tumor


Tumor progression


Genetically unstable




Replicative cell senescence







False. A carcinoma consists of a variety of normal cells, along with the cancer cells. The fibroblasts in the supporting connective tissue, the associated
inflammatory cells, and the cells of the newly formed blood vessels all are
normal cells that are present because they are in contact with the cancer cell
mass or have been recruited into the tumor. These normal cells do not
evolve from the cancer cell population.


True. Genes can be shut off, for example, by changes in chromatin structure;
specific covalent modifications of histones can attract complexes of chromatin-binding proteins that are stably maintained following DNA replication. Gene transcription can also be eliminated by methylation of CpG dinucleotides in the promoter region. Specific histone modifications and DNA
methylation are often linked.


False. There is an optimum level of genetic instability for the development of

cancer. A cell must be mutable enough to evolve rapidly, but not so mutable
that it accumulates too many harmful changes and dies.


True. Many cancers appear to be maintained by a small population of stem

cells. These cancer stem cells usually divide more slowly than the cells in the
bulk of the tumor, and they are less sensitive to treatments aimed at rapidly
dividing cells. If the stem cells are not killed, the cancer is likely to return.



Chapter 20: Cancer


The incidence of cancer increases dramatically with age because it takes

mutations in several critical genes to disable a cells normal mechanisms for
controlling its growth. Since growing cells continually accumulate mutations, which they pass on to their progeny cells, the chance that a cell will
accumulate a critical set of mutations increases with age. The steep rise in
cancer incidence in older women seen in Figure 201 reveals that colon cancer increases as the sixth power of age, suggesting that it arises only after
mutations have occurred in six or so genes that regulate cell growth in the


The key difference in the incidences of colon cancer and osteosarcomas is

the size of the population of cells at risk for the disease. Colon cancer arises
from the population of proliferating cells in the colon, which are present in
roughly the same number throughout life. This population can accumulate
mutations over time, giving rise to the age dependence seen in Figure 201.
By contrast, the cells responsible for osteosarcomas are present in much
greater numbers during adolescence, when their proliferation is required to
increase the size of the skeleton, than they are in young children or adults.
In this case, it is the number of cells at risk that is the most important determinant of the frequency of cancer.
Reference: Knudson AG (2001) Two genetic hits (more or less) to cancer. Nat.
Rev. Cancer 1, 157162.


The time at which the death rates due to breast and cervical cancer slow corresponds to the menopause, at which time the production of estrogen
declines. Estrogen normally promotes the proliferation of cells in the breast
and uterus. Thus, the decline in estrogen would reduce the population of
proliferating cells, thereby reducing the risk of cancer in these tissues.
Reference: Armitage R & Doll R (1954) The age distribution of cancer and a
multi-stage theory of carcinogenesis. Reprinted in (2004) Br. J. Cancer 91,


A tumor that arose from 50 cell doublings would contain 250 cells, which is
1.1 1015 cells. If 108 cells is 1 gram, then the tumor would weigh 1.1 107 g,
which is 24,000 pounds. Thus, a limit of 50 cell divisions, by itself, does not
provide much protection against cancer. Even in a 40-year-old, whose
fibroblasts divide about 40 times in culture, a tumor arising from 40 cell divisions would weigh more than 20 pounds.
This calculation assumes that all cells survive. It is likely, however, that
many cells die by apoptosis, especially at early stages in the evolution of cancer. In combination with extensive cell death, a limit of 50 cell divisions
could protect against cancer.

A. The cell-cluster model for cancer formation predicts an exponential relationship between carcinogen concentration and cancer formation. In the
equation, Nxn, the concentration of mutagen, would influence the probability of mutation, x, so that 1, 2, and 4 times the carcinogen concentration
would give 1x, 2x, and 4x probabilities of mutation, respectively. The numbers of cancers should then be N(1x)n, N(2x)n, and N(4x)n, respectively. To
illustrate this with numbers, let N = 109, x = 102, and n = 5, as they are in the
body of the problem. Substituting these numbers gives 0.1, 3.2, and 102.4


cancer clusters for 1, 2, and 4 times the carcinogen concentration. This predicted exponential dependence on carcinogen concentration does not
match the linear dependence observed experimentally.
B. The tumor-progression model for cancer formation predicts a linear relationship between carcinogen concentration and cancer formation, so long
as the frequency of mutation is low. A slowly progressing tumor presents a
moving target for mutation. At an early time, say when it has one mutation,
carcinogen treatment can stimulate the acquisition of a second mutation in
one of its cells, allowing that cell to progress to the next stage. It is unlikely,
however, to introduce two mutations into one cell in the small population of
cells that constitutes the early tumor. At a later stage, when the tumor cells
have, say, three changes, the same argument applies: the carcinogen can
realistically induce an additional mutation in only one of its cells. Induction
of one mutation at a time gives a linear dependence on carcinogen concentration.
References: Fisher JC & Hollomon JH (1951) A hypothesis for the origin of
cancer foci. Cancer 4, 916918.
Armitage P & Doll R (1954) The age distribution of cancer and a multi-stage
theory of carcinogenesis. Reprinted in 2004 in Br. J. Cancer 91, 19831989.


Although the numbers are small, there seems to be a reasonable correlation

between smoking and lung cancer. Statistical analysis shows that these differences are significant (P = 0.04). Similarly, the consistent upward trend in
heart attacks with increased smoking also turns out to be statistically significant (P = 0.03). Follow-up studies on this cohort (the most recent after 50
years) have confirmed the initial impression conveyed by the original, preliminary study. Compared to lifelong nonsmokers, lung cancer is 8-fold, 14fold, and 25-fold more prevalent among smokers of 114, 1524, and more
than 25 cigarettes per day, respectively. Moreover, in these longer studies it
is clear that other cancersfor example, mouth, nose, throat, stomach, liver,
kidney, and bladderare also significantly elevated in smokers, although
not so dramatically as the incidence of lung cancer.
References: Doll R & Hill AB (1954) The mortality of doctors in relation to
their smoking habits: A preliminary report. Reprinted in 2004 in Br. Med. J.
328, 15291533.
Doll R, Peto R, Boreham J & Sutherland I (2004) Mortality in relation to
smoking: 50 years observations on male British doctors. Br. Med. J. 328,


Development of most cancers seems to require a gradual accumulation of

mutations in a number of different genesat least five or six. In the ongoing
presence of cigarette smoke these mutations evidently accumulate at an
increased rate (over their accumulation in the absence of cigarette smoke).
By stopping smoking, an individual returns to the normal, slower rate of
mutation accumulation. Thus, whatever mutations remain to be generated
in a reformed smoker are generated at a slower rate than in a continuing
smoker. The slower rate of accumulation of mutations translates into a lower
cumulative risk.
Reference: Peto R, Darby S, Deo H, Silcocks P, Whitely E & Doll R (2000)
Smoking, smoking cessation, and lung cancer in the UK since 1950: combination of national statistics with two case-control studies. Br. Med. J. 321,



Chapter 20: Cancer



Tumor promoter




True. The modifications introduced into dietary carcinogens by the action of

the P-450 enzymes of the liver can convert them from relatively inert compounds to reactive ones that damage DNA. The normal function of these
enzymes is to convert ingested toxins to harmless compounds for easy
excretion. Unfortunately, their action on some chemicals renders them
highly mutagenic.


False. Infectious agents are thought to participate in the formation of

roughly 15% of human cancers. In most cases, the specific mechanism is
unknown, but there are strong associations, for example, between hepatitis
viruses type B and type C and liver cancer, between H. pylori infection and
stomach cancer, and between blood fluke infection and bladder cancer.


False. Although it is popular to think so, there is scant evidence to support

those ideas. However, we certainly know of specific instancessuch as 2naphthylamine and asbestoswhere industrial products cause human


These data are consistent with the idea that cancer is a multi-step process in
which cancer-causing changes accumulate over time. The 25-year delay
between exposure and cancer reflects the time it takes for lung cells to accumulate a sufficient number of changes to become cancerous. From other
studies it is known that cigarette smoke contains tumor initiators and tumor
promoters, both of which contribute to the progression from normal to cancerous cells.
Your uncles suggestion that there is a genetically predisposed fraction of
the population that is prone to lung cancer does not match the data. If a
fixed fraction of the population were genetically predisposed, the incidence
of lung cancer would be relatively constant over time. It would not be
expected to track with per capita smoking.


The highly rearranged karyotypes and their similarity from tumor to tumor
suggest that the cancer cells themselves are being transmitted from devil to
devil. It is extremely unlikely that an infectious agent such as a virus or a
microorganism could induce the same set of complicated rearrangements
in different animals. Most importantly, the existence of a chromosome-5
inversion in one Tasmanian devil, which is not present in chromosome 5 of
its tumor cells, argues strongly that the tumors are not generated from the
host devils own cells. It appears that this cancer has arisen from a rogue line
of cancer cells, from a tumor of unknown origin, that has acquired the capability for parasitic existence. This is one of just two examples of natural
transmission of cancer by tumor cells, the other being a venereal disease in
dogs. A special case of such transmission occurs occasionally during organ
transplantation in humans. But the requirements for organ transplantationmatching tissue and immune suppressionhighlight just how


unusual natural transmission is. The cancer cells responsible for facial
tumors in Tasmanian devils must somehow evade the new hosts immune
Reference: Pearse A-M & Swift K (2006) Transmission of devil facial-tumour
disease. Nature 439, 549.
A. These results, which were reported in 1933, were the first indication that the
tendency to form breast tumors was maternally transmitted in mice. The
results of the experiments in Table 201 cannot be readily explained on the
basis of standard types of chromosomal mutations, be they recessive, dominant, or X-linked. If you assume that the high strains are homozygous for
a recessive mutation and the low strains are homozygous for the wild-type
allele, all the F1 progeny would be heterozygous and unaffected. For a dominant mutation, the males, as well as the females, should transmit the mutations to their offspring. X-linked mutations would be expected to give
affected males in every other generation. Although it was unknown at the
time these experiments were carried out, the pattern of inheritance shown
in Table 201 matches the expectations for mitochondrial mutations, which
are inherited via the egg cytoplasm. (See Problem 14110 for more discussion of these patterns of inheritance.)
B. The key to understanding how the tendency to form breast cancers is inherited is the foster-mother experiment. Although presented as an accident in
this problem, it represented a critical insight at the time. Because foster
mothers could pass this trait on to mice with which they shared no genetic
connection, inheritance could not be due to chromosomal (or mitochondrial) mutations. The link between mothers and daughters was identified as
the milk. The so-called milk factor was later identified as a virus, which we
now call mouse mammary tumor virus (MMTV).
References: Little CC (1933) The existence of non-chromosomal influence in
the incidence of mammary tumors in mice. Science 78, 465466.
Bittner JJ (1936) Some possible effects of nursing on the mammary gland
tumor incidence in mice. Science 84, 2172.
Paigen K (2003) One hundred years of mouse genetics: An intellectual history. I. The classical period (19021980). Genetics 163, 17.











Cancer-critical gene


Tumor suppressor gene


False. Oncogenes, which are mutated, overactive forms of proto-oncogenes,

can be detected in this way. Their addition to the genome can convert a cell



Chapter 20: Cancer

to a cancer cell. By contrast, tumor suppressor genes have their effects
because they are inactive; it is their absence that causes cancer. One cannot
use the same sort of transformation assay to detect something that is not


False. It is not that DMBA is a specific mutagen, but rather that the Ras gene
is converted to its activated, cancer-causing form by a particular A-to-T
alteration that leads to a very specific amino acid change. DMBA causes
mutations throughout the genome, but only those at the specific site in the
Ras gene give rise to cells that have cancerous properties and thus are identified in the assay.


True. An inherited predisposition to cancer often occurs because one copy

of a tumor suppressor gene is mutated. Heterozygous cells, which retain one
wild-type copy of the gene, are fine, but they are at increased risk for cancer
because a single event (rather than the two in a normal individual) can inactivate the remaining good copy, causing loss of heterozygosity.


Oncogenes correspond to stuck accelerators. In their mutated, overactive

form they drive a cell to proliferate in a way that is not responsive to normal
controls. Defective tumor suppressor genes correspond to broken brakes.
They normally function to inhibit steps in signaling pathways; that is, to act
as brakes. When tumor suppressor genes are defective, signaling pathways
are unrestrained. Defective DNA maintenance genes correspond to bad
mechanics. These genes normally operate to maintain the genome during
its propagation and in the face of DNA damage. Defective DNA maintenance
genes lead to genetic rearrangements or increased point mutations, either of
which can convert a proto-oncogene to an oncogene, or eliminate a tumor
suppressor gene.
Reference: Vogelstein B & Kinzler KW (2004) Cancer genes and the pathways
they control. Nat. Med. 10, 789798.


Antiproliferative genes such as Rb encode proteins that stop the cell cycle.
During normal cell division, these proteins must be turned off. If they were
overexpressed in all cells, it is likely that the machinery that keeps these proteins turned off would be overwhelmed, and cell division would stop. Thus,
this cure for cancer might be successful but the patient would be dead.


Cancer cells have additional changes that typically disable cell-cycle checkpoints and apoptotic mechanisms. In the absence of these regulatory controls, which are fully operational in normal cells, overexpression of Myc
drives cell growth and proliferation of cancer cells.

A. If the integration events were random, then 0.00005 [fi = (100 kb/2 106 kb)]
would be expected to occur in the target sequence. The fraction of integration events expected to occur outside the target would be 0.99995 (fo = 1 fi).
B. The probability of not finding (PN) a second integration at Il2rg in a survey
of 600 tumors with a retroviral integration at Lmo2, is the probability of not
finding it in one, raised to the power of 600. Thus, PN = (0.99995)600 = 0.97.
This means that in 97 out of 100 times you survey a new set of 600 tumors,
you would not expect to find a second integration at Il2rg.
C. The probability of finding (PY) a second integration at Il2rg in a survey of 600
tumors is 1 minus PN, or 0.03. This means that 3 times out of 100, you would
expect to find a second integration at Il2rg.


D. Given that only 2 out of 600 tumors actually had a retrovirus integrated at
Lmo2, the probability of finding a tumor with dual integrations at Lmo2 and
Il2rg would be 0.0001 [0.03 (2/600)], or 1 chance in 10,000.
E. The calculation in part D depends on the specific assumptions. If the target
size were 10 kb (instead of 100 kb), the calculation in part D would decrease
to 1 chance in 100,000. If there were, on average, fewer than 2 retroviral integrations per tumor, the calculation in part D would decrease; if there were
more than 2 integrations per tumor, the calculation would increase.
The assumption of randomness of retroviral integration is the most difficult to evaluate in terms of this calculation. Retroviruses are decidedly nonrandom in their integration, with most varieties showing some degree of
preference for actively transcribed genes. An entirely different approach to
this question takes such preferences into account. If 2 out 600 tumors had a
retroviral integration at Lmo2, and 2 out of 600 had a retroviral integration at
Il2rg, then the chance of having both is (2/600) (2/600) = 0.000011, or
about 1 in 100,000 tumors. The chance of not finding a dual integration in
600 tumors would be PN = (0.999989)600 = 0.993. Thus, there would be a
7/1000 chance of finding a dual integration. This differs by a factor of 100
from the result calculated in part D. Yet, both suggest that dual integration
should be a relatively rare event.
References: Dave UP, Jenkins NA & Copeland NG (2004) Gene therapy insertional mutagenesis insights. Science 303, 333.
Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I, Villeval J-L, Fraser CC, Cavazzana-Calvo M & Fischer A
(2003) A serious adverse event after successful gene therapy for severe combined immunodeficiency syndrome. N. Engl. J. Med. 348, 255256.

A. Fibroblasts and tumor cells from the same patient have different patterns of
hybridization because the tumor cells have lost portions of the Rb gene. Loss
of this gene is a very rare somatic event that affects less than one in a million
cells. Only in the retina does its loss cause uncontrolled growth and tumor
formation. No doubt the same proportion of fibroblasts also lose the Rb
gene, but its loss from fibroblasts has no known biological consequence, so
its absence cannot be readily detected.
B. The fibroblasts from the patient with unilateral retinoblastoma appear to be
identical to those from normal cells, suggesting that the patient with unilateral retinoblastoma inherited two good Rb genes. Fibroblasts from the
patient with bilateral retinoblastoma are not normal. Three of the four
restriction fragments are present at half the normal intensity, suggesting
that one of the Rb genes contains a deletion that encompasses those three
restriction fragments. Note that the three affected fragments are adjacent on
the map of the Rb gene (see Figure 208B).
The tumor cells from both patients are abnormal. The patient with unilateral retinoblastoma is missing two fragments entirely and a third is present
at half the normal intensity. This pattern indicates that each copy of the Rb
gene has undergone deletion: one deletion encompasses the 9.8-kb and the
6.2-kb fragments; the other encompasses these two fragments and the 5.3-kb
fragment. The patient with bilateral retinoblastoma is missing three fragments
entirely and the remaining fragment is present at only half the normal intensity. This pattern indicates that the one good Rb gene has been entirely
deleted, leaving only the 6.2-kb fragment from the original inherited deletion.
C. These results are exactly what is expected from the hypothesis that
retinoblastoma is due to the loss of both copies of the Rb gene. Many cases
of retinoblastoma have now been examined, and they all show loss or alteration of the Rb gene. Thus, retinoblastoma develops in the absence of functional Rb.



Chapter 20: Cancer

References: Fung Y-KT, Murphree AL, TAng A, Qian J, Hinrichs SH & Benedict WF (1987) Structural evidence for the authenticity of the human
retinoblastoma gene. Science 236, 16571661.
Knudson AG (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. U.S.A. 68, 820823.

A. Most of the 463,248 sequence changes that remained after the reading errors
were removed have nothing to do with cancer. The authors of this study
applied six additional filters to eliminate sequence changes that are unlikely
to contribute to the functional differences between the normal cells and the
tumor cells. See if your suggestions are included in this list.
1. Filter out changes that do not alter the encoded amino acid sequence; for
example, mutations to synonymous codons. (259,957 changes were
eliminated by this criterion.)
2. Filter out changes that are also present in the DNA from the two normal
individuals that were included in the analysis. (163,006 changes were
eliminated by this criterion.)
3. Filter out changes that correspond to known sequence polymorphisms
in the human population. (11,004 changes were eliminated by this criterion.)
4. Filter out changes that cannot be confirmed upon reamplifying and resequencing the sample. (Of the 29,281 sequence differences that remained
after applying the above filters, 9295 were not confirmed and therefore
5. Filter out changes that are also present in normal tissue from the same
individual that had the tumor. (18,414 of the 19,986 sequence differences
that remained after applying filter 4 were eliminated were eliminated by
this criterion.)
6. Filter out changes in sequences that have closely related sequences elsewhere in the genome. There can be problems deciding which genomic
location is the true source of the sequence read. (265 of the remaining
1572 sequence differences were eliminated by this criterion, leaving 1307
potential cancer-relevant mutations.)
B. Deciding which of these 1307 mutations are likely to contribute to the cancers is not an easy task. One approach is to look for mutant genes that are
found in multiple breast tumors or in multiple colorectal tumors. The
underlying assumption is that similar cancers should have similar sets of
causative mutations. The authors used this sort of analysis to identify
roughly 12 cancer-related mutations in breast tumors, and about 9 in colorectal tumors. (The rest of the mutations are likely to be passenger mutations.) Because only about half the genes in the genome were analyzed
(13,023/25,000), the real number of cancer-relevant mutations in these
tumors may be closer to 20.
C. The sequencing strategy used hereamplifying and sequencing exons
was designed to detect small changes in sequence: point mutations and
short deletions. Larger deletions and gene rearrangements would not be
detected because they would not give an informative PCR product.
Reference: Sjoblom T et al. (2006) The consensus coding sequences of
human breast and colorectal cancers. Science 314, 268274.








Colorectal cancer


True. That is why oncogenes in their overactive, mutant form tend to drive
cell growth and proliferation, and why the loss of tumor suppressor genes
removes natural blocks in these pathways, which also promotes cell growth
and proliferation.


False. Activation of a single oncogene is generally not sufficient to convert a

normal cell into a cancer cell. Typically, in mice engineered to express an
oncogene such as Myc or Ras, cells in some tissues that express the oncogene show enhanced proliferation, and, over time, occasional cells will
undergo further changes to give rise to cancers.


False. Cancer cells rarely have mutations in multiple components of the

same regulatory pathway. Mutation in any one component of a pathway is
usually sufficient to inactivate the pathway and promote cancer. Inactivation of more than one component in a pathway would have no benefit for
the cancers evolution.


True. p53 protein normally acts to limit the harm done by DNA damage.
Cells that are severely damaged are driven to commit suicide by apoptosis;
mildly damaged cells are prevented from dividing until the damage is
repaired. In the absence of p53 these two safeguards are eliminated, allowing some cancer cells to proliferate even when exposed to damaging effects
of irradiation and many anticancer drugs.


False. In general, carcinomas show either chromosome instability or defective mismatch repair, but not both. This represents another example of the
general principle that cancers have one mutation per pathway: in this case
the pathway for genetic stability. Genetic instability can arise either by
increased rates of chromosome rearrangement or by increased rates of point
mutation. It seems that increasing genetic instability in either way serves the
needs of the evolving cancer cell. Evidently, stimulating both chromosome
instability and point mutations does not speed up the evolution of the cancer cell.


Mutant B-Raf with glutamate at position 599 is thought to be active because

it carries a negative charge (COO) in the same region of the protein at
which a negative charge (PO4) is normally introduced by Ras-induced
phosphorylation. Evidently, the negative charge alters the conformational
equilibrium of B-Raf, shifting it toward the active form. It is common practice these days to test whether a phosphate at a site activates (or inactivates)
a protein by engineering a version of the protein with a glutamate at that
position. Often (but not always) the glutamate change will mimic the effect
of the phosphate.
Reference: Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ,
Boardman LA, Burgart LJ, McDonnell SK, Schaid DJ & Thibodeau SN (2003)
BRAF mutations in colon cancer are not likely attributable to defective DNA
mismatch repair. Cancer Res. 63, 52095212.


These observations argue strongly that MMTV generates an oncogene upon

integration into the mouse genome. It is extremely unlikely that MMTV
would integrate so often in the same region of the genome by chance. Moreover, a unique transcript is generated in the region of the integrated virus,



Chapter 20: Cancer

suggesting that a gene is turned on in response to the neighboring viral
These results summarize the initial characterization of the Int1 locus in
mice. It was later shown to be homologous to the wingless locus in
Drosophila, both of which are now referred to as Wnt genes. These genes
secrete a signaling molecule that triggers a signaling pathway in other cells
that activates expression of a set of Wnt-responsive genes, some of which
promote cell proliferation.
Reference: Nusse R & Varmus HE (1982) Many tumors induced by the mouse
mammary tumor virus contain a provirus integrated in the same region of
the host genome. Cell 31, 99109.

A. Four different haploid gametes can combine to give 16 different diploid
products. Of the several different ways of representing these possibilities,
the most concise is the Punnett square, a time-honored tradition with
geneticists (Table 203). However the possibilities are represented, the 16
combinations of gametes form 9 distinct genotypes. Four genotypes are represented once (the ones along the diagonal from upper left to lower right in
Table 203). One genotype is represented four times (the one along the diagonal from lower left to upper right). The remaining genotypes are each represented twice (symmetrically arrayed about the diagonal from upper left to
lower right). The ratios of the genotypes, along with their expected frequencies among 36 progeny, are given in Table 204).
Progeny with the genotypes p53 +/+ Mdm2 / and p53 +/ Mdm2 / are
under-represented in these experiments: none were found. More detailed
experiments have shown that these mice do not survive because they die
early in embryonic development.
B. It is striking that p53 +/+ Mdm2 / and p53 +/ Mdm2 / do not survive,
whereas p53 / Mdm2 / mice do. In the absence of Mdm2, it seems that
even a haploid amount of p53 is lethal. Since Mdm2 is a ubiquitin ligase, one
reasonable explanation for this result is that Mdm2 normally keeps the cellular concentration of p53 very low. In the absence of Mdm2, p53 is no
longer destroyed at the proper rate and accumulates to levels that are toxic.
The basis for cell toxicity in this instance is not entirely clear, but it is thought
that the abnormally high amounts of p53 arrest the cell cycle and promote

Table 203 A Punnett square showing all possible genotypes resulting from random
assortment of p53 Mdm2 gametes (Answer 2055).





























Table 204 Actual and expected genotypes of progeny mice from crosses between
doubly heterozygous p53+/ Mdm2+/ mice (Answer 2055).
p53+/+ Mdm2+/+
p53+/+ Mdm2+/
p53+/+ Mdm2/
p53+/ Mdm2+/+
p53+/ Mdm2+/
p53+/ Mdm2/
p53/ Mdm2+/+
p53/ Mdm2+/
p53/ Mdm2/






2 14
4 12
2 14
4 12
4 12
2 14
4 12
2 14

References: de Oca Luna RM, Wagner DS & Lozano G (1995) Rescue of early
embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378,
Jones SN, Roe AE, Donehower LA & Bradley A (1995) Rescue of embryonic
lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206208.

A. An Arf-knockout mouse would be expected to be more prone to cancer than
a wild-type mouse. In the absence of Arf, Mdm2 would be more active than
in a wild-type mouse. Overactive Mdm2 would, in turn, tend to repress p53
activity more than normal. Thus, the consequence of an Arf knockout would
be reduced p53 activity. If this lowered activity impaired the ability of p53 to
force abnormal cells to undergo cell cycle arrest or apoptosis, more precancerous cells would escape detection and more tumors would form.
B. A p53 +/+ Mdm2 / mouse will not be rescued by knockout of the Arf gene. In
the absence of Mdm2, the activity of p53 will be maximized. Because Mdm2
is absent, the link between Arf and p53 is missing. Thus, no change in Arf
levels can affect p53 activity, and p53+/+ Mdm2 / Arf / mice will die just like
p53 +/+ Mdm2 / mice do.
C. Mice that express the Myc oncogene will overstimulate Arf activity, which
will decrease Mdm2 activity, which will cause an increase in p53 activity.
Increased p53 activity (so long as it is not increased to the point where it is
toxicsee Problem 2055) will tend to increase the mouses ability to force
abnormal cells into cell cycle arrest and apoptosis. This increased activity of
p53 operates in opposition to the proliferation-promoting activity of the Myc
oncogene. In an Arf +/ mouse there is less Arf, hence, less of a decrease in
Mdm2 activity and less of an increase in p53 activity. Because the proliferation-promoting activity of the Myc oncogene is opposed to a lesser extent
(by the lower p53 activity), Arf +/ mice generate tumors more quickly and die
at a younger age.
References: Quelle DE, Zindy F, Ashmun RA & Sherr CJ (1995) Alternative
reading frames of the INK4a tumor suppressor gene encode two unrelated
proteins capable of inducing cell cycle arrest. Cell 83, 9931000.
Zindy F, Williams RT, Baudino TA, Rehg JE, Skapek SX, Cleveland JL, Roussel
MF & Sherr CJ (2003) Arf tumor suppressor promoter monitors latent oncogenic signals in vivo. Proc. Natl Acad. Sci. U.S.A. 100, 1593015935.
A. As discussed in Problem 2043, the absence of a shoulder on any of the three
curves suggests that in all cases only a single event is needed to trigger


Chapter 20: Cancer

tumor production in mice that are already expressing one or both oncogenes.
B. Although the rate of tumor production is much higher in mice with both
oncogenes, activation of the cellular Ras gene cannot be a required event in
the production of tumors in mice that are already expressing the Myc oncogene. Nor can activation of the cellular Myc gene be a required event in triggering tumor formation in mice that are already expressing the Ras oncogene. As indicated in part A, even when mice express both Myc and Ras,
some additional event is required to produce a tumor. If expression of Myc
plus Ras were sufficient for tumor formation, then all mice containing both
oncogenes would develop tumors as soon as they passed through puberty
and turned on their expression.
C. The rate of tumor production in mice with both oncogenes is much higher
than expected if the effects of the individual oncogenes were additive. Thus,
the two oncogenes together have a synergistic effect on the rate of tumor
production. As argued in part B, activation of both oncogenes is not sufficient to generate a tumor. Thus, the two oncogenes acting together must
open up a pathway to tumor production that can be triggered by any one of
several low-frequency events or that can be triggered by one very common
event. The nature of the activating events is unclear for these transgenic
mice, but analysis of transformed cells suggests that inactivation of the p53
tumor suppressor pathway may be responsible.
Reference: Sinn E, Muller W, Pattengale P, Tepler I, Wallace R & Leder P
(1987) Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49, 465475.

A. The formation of the three types of patches observed in speckled kernels is
shown in Figure 2017. The formation of a colorless, nonwaxy (c-Wx) patch
results from a breakage that eliminates the dominant color (C) allele (Figure
2017A). In the absence of the dominant allele, the color of the patch is
determined by the recessive colorless (c) allele on the normal chromosome
(which is not shown in the figure).
The formation of a colorless, waxy (c-wx) spot in a colorless, nonwaxy (cWx) patch is due to a second breakage event that eliminates the dominant






C Wx



C Wx





C Wx


C Wx

C gene has been lost

Wx gene has been lost





extra copies of C gene



Figure 2017 Formation of three different types of patches observed in speckled kernels (Answer 2058).
(A) Formation of a colorless, nonwaxy (c-Wx) spot. (B) Formation of a colorless, waxy (c-wx) spot inside
a colorless, nonwaxy (c-Wx) spot formed as in (A). (C) Formation of an intensely colored, nonwaxy
(C-C-Wx) spot. Vertical arrows pointing to the dicentric chromosomes show the positions of the breaks
that lead to formation of the patches. In each case the upper half of the starting dicentric chromosome
gives rise to the new dicentric chromosome.


nonwaxy (Wx) allele (Figure 2017B). In the absence of the dominant allele
the spot is waxy (wx) due to the recessive allele on the normal chromosome
(not shown).
The formation of an intensely colored patch is due to a breakage event
that leads to a dicentric chromosome with multiple copies of the dominant
color allele (Figure 2017C). Thus, the genetic constitution of the intensely
colored patch is C-C-Wx.
B. You would never expect to see a colored spot within a colorless patch
because, once eliminated, the dominant color (C) allele cannot be regained
by further bridgebreakagefusion cycles.
C. You would expect to see colorless spots within an intensely colored patch
because the dominant color (C) allele could be lost by subsequent
bridgebreakagefusion cycles.
The demonstration by McClintock of bridgebreakagefusion cycles in
plants was one of the earliest indications that the broken ends of chromosomes are in some way stickyentirely different from natural chromosome
ends. It is clear now that cells have an active repair pathway for joining broken DNA ends together as a defense against potentially lethal double-strand
breaks. So long as breaks are rare, the correct ends are joined. But when multiple breaks are present, the wrong partners can be joined, leading to
translocations or other genetic rearrangements. In humans, such rearrangements are often associated with cancers.
Reference: McClintock B (1939) The behavior of successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl Acad. Sci. U.S.A. 25,





Multidrug resistance


Gene expression profile


True. Useful therapies selectively target cancer cells and leave normal cells
relatively unaffected. This selective action always depends on a key difference between normal cells and cancer cells. For example, most anticancer
drugs and ionizing radiation damage DNA. These treatments preferentially
kill cancer cells because the cancer cells have a diminished capacity to survive the damage.


True. Tamoxifen is an estrogen receptor antagonist. By preventing the binding of estrogen to the receptor, tamoxifen prevents the proliferation of breast
cancer cells, which is dependent on estrogen binding.


False. Although there are likely to be some cancer cells in a hypermutable

population that are resistant to a single drug, it is unlikely that cells will be
resistant to multiple drugs. For such a combination therapy to work, the
drugs must be designed so that a single mutation cannot render cells resistant. For example, if the multiple drugs in a cocktail can all be pumped out
of the cell by an amplified ABC transporter, then treatment of a cancer cell
population will select for cells that have amplified that geneand the treatment will fail. If, on the other hand, a single mutation cannot render a cell
resistant to all of the drugs in the cocktail, the treatment has the potential for
eliminating all the cancer cells and producing a cure.



Chapter 20: Cancer


Technological advances in our ability to detect cancers since 1970 means

that we can diagnose them at earlier stages in the course of the disease.
Thus, even in the absence of better treatment regimes, a patient might be
expected to survive somewhat longer now than in the past because they will
be at a slightly earlier stage of the disease at the 5-year mark.
Reference: Weinberg RA (2006) The Biology of Cancer, p726. New York: Garland Science.


The promyelocytes of APL are blocked at an intermediate stage in their

development, at a point where they still divide and increase in number. It is
this unchecked increase in number that causes problems for the cancer
patient. Normally, such precursor cells divide only a few times before they
terminally differentiate into a nondividing blood cell. By triggering the differentiation of promyelocytes into terminally differentiated neutrophils,
which no longer divide, treatment with all-trans-retinoic acid eliminates the
problems caused by unchecked proliferation.
APL arises by one of a few types of translocation that fuses the retinoic acid
receptor (RAR) gene on chromosome 17 with a gene on another chromosome to make a hybrid protein that interferes with the normal developmental program. It is not yet clear how the fusion protein blocks development,
although it likely does so by interfering with the function of the normal RAR
receptor. In some way, treatment with all-trans-retinoic acid allows APL cells
to move through the block.
Reference: Warrell RP Jr, de The H, Wang Z-Y & Degos L (1993) Acute promyelocytic leukemia. N. Engl. J. Med. 329, 177189.


The products of oncogenes are the only feasible targets for such small
molecules. The product of an oncogene has a dominant, growth-promoting
effect on the cell. Thus, if the growth-promoting oncogene product were
inhibited, the cell might return to a more normal state. This is the underlying rationale for searching for drugs that inhibit oncoproteins.
By contrast, the products of tumor suppressor genes and DNA maintenance genes are not targets for anticancer drug development. These two
classes of gene cause cancer by not making their product. Thus, there is no
abnormal product to be inhibited in cancer cells that arise by mutation of
tumor suppressor or DNA maintenance genes.

A. The 1-hour incubation allows the binding reactionstest compound to
kinase and ATP analog to kinaseto come to equilibrium. Making the measurements at equilibrium gives a much more reproducible assay and allows
comparisons between test compounds to be made on an equal footing.
B. At equilibrium, some of the phage-attached kinases will be bound to the test
compound and some will be bound to the ATP-analog-coated magnetic
beads. The relative proportions depend on how strongly the test compound
binds and its concentration. In the presence of a weakly binding test compound, most of the phage will be attached to the magnetic beads, which will
yield a high count in the plaque assay. In the presence of the same concentration of a strongly binding test compound, most of the phage will be
attached to the test compound and will be washed away at the end of the
incubation. Thus, strongly binding test compounds will give a low count in
the plaque assay.
C. The assay is set up to measure competition between the ATP analog and the
test compound for the ATP-binding site on the protein kinase. Thus, it is
expected that the test compounds that score well in this assay will bind at or
near the ATP-binding cleft on the kinase. This assay can also identify
molecules that bind to other sites (allosteric sites) but alter the conformation of the ATP-binding site, so that the kinase no longer binds ATP. One


might imagine other sites where a useful inhibitor might bind to a kinase to
inhibit its functionat a key proteinprotein interface, for examplebut
those types of inhibitors will not be picked up in this assay system.
D. Although all protein kinases bind ATP in an evolutionarily conserved binding site, the binding sites are not identical. The amino acid differences in
and around the binding site provide slightly different binding surfaces that
can be exploited to develop compounds that selectively bind to one kinase
but not others.
References: Griffin JD (2005) Interaction maps for kinase inhibitors. Nat.
Biotechnol. 23, 308309.
Fabian MA et al. (2005) A small molecule-kinase interaction map for clinical
kinase inhibitors. Nat. Biotechnol. 23, 329336.

A. The Kd values can be estimated directly from the graph in Figure 2015, as
the concentration of test compound that give a half-maximal response. The
Kd values for BIRB-796, VX-745, and SB203580 are about 0.3 nM, 3 nM, and
15 nM, respectively.
B. The phage concentration is 17 pM, well below the Kd(test) values for the compounds tested, the lowest of which is 0.3 nM (300 pM). Thus, the estimates
in part A are valid. Phage concentration = (1010 phage/mL) (mole/6 1023
phage) (103 mL/L) (1012 pmol/mole) = 17 pmol/L = 17 pM.
Reference: Fabian MA et al. (2005) A small molecule-kinase interaction map
for clinical kinase inhibitors. Nat. Biotechnol. 23, 329336.

A. Iressa, which is being used clinically in the treatment of non-small-cell lung
carcinomas, and Gleevec have similar specificities. Iressa has fewer off-target binding interactions, and binds only one of those with an affinity than is
within a factor of 10 of the main target. BIRB-796 binds to more off-target
proteins than Gleevec or Iressa, and it binds three of them with only 10-fold
lower affinity. Staurosporine, which is the least specific of all, is a potent
inhibitor of many protein kinases.
B. The clustering of the binding targets on the kinome is expected. After all, the
kinases that are most closely related are the closest together on the tree.
Closely related kinases should have more similar binding sites, and thus
might be expected to bind an inhibitor similarly. Indeed, it is the similarity
of all kinases, and especially of closely related kinases, that makes drug
development for them such a challenge.
C. There is no way that you (or anyone) could predict from these data that
BIRB-796 would bind Abl(T315I). Resistant variants arise by such subtle
changes that direct measurements must be made.
D. This high-throughput screen can be adapted to look for drugs that bind resistant variants of protein kinases. If the particular mutant versions responsible
for resistance are identified, they can be included in the screen to find drugs
that are active against them. It is hoped that screens such as the one
described here can be used to identify a second generation of drugs that will
prove beneficial in the treatment of cancers resistant to the primary drug.
Reference: Fabian MA et al. (2005) A small molecule-kinase interaction map
for clinical kinase inhibitors. Nat. Biotechnol. 23, 329336.