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Cancer
20
In This Chapter
A457
DEFINITIONS
CANCER AS A
MICROEVOLUTIONARY
PROCESS
THE PREVENTABLE
CAUSES OF CANCER
A460
A461
201
Carcinogenesis
202
Malignant
203
204
Sarcoma
205
Primary tumor
206
Tumor progression
207
Genetically unstable
208
Benign
209
2010
Carcinoma
A469
TRUE/FALSE
2011
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.
2012
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.
2013
2014
A457
A458
THOUGHT PROBLEMS
2015
2016
2017
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,
19831989.
CALCULATIONS
2018
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.
2019
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
DATA HANDLING
2020
2021
A459
A460
Tumor promoter
2023
Carcinogen
TRUE/FALSE
2024
2025
2026
THOUGHT PROBLEMS
2027
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.
DATA HANDLING
2028
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
Oncogene
2031
Transformation
2032
Proto-oncogene
2033
Retinoblastoma
2034
Cancer-critical gene
2035
TRUE/FALSE
2036
A461
A462
2037
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.
2038
THOUGHT PROBLEMS
2039
2040
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.
2041
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.
CALCULATIONS
2042
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.
DATA HANDLING
2043
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.
A463
A464
2044
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
eliminated.)
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.
Papillomavirus
p53
2047
Colorectal cancer
TRUE/FALSE
2048
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.
2049
2050
2051
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.
2052
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.
THOUGHT PROBLEMS
2053
2054
A465
A466
CALCULATIONS
2055
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
apoptosis.
Table 203 A Punnett square showing all possible genotypes resulting from random
assortment of p53 Mdm2 gametes (Answer 2055).
MALE
FEMALE
GAMETES
p53+Mdm2+
p53+Mdm2
p53Mdm2+
p53Mdm2
p53+Mdm2+
p53+Mdm2+
p53+Mdm2+
p53+Mdm2+
p53+Mdm2
p53+Mdm2+
p53Mdm2+
p53+Mdm2+
p53Mdm2
p53+Mdm2
p53+Mdm2
p53+Mdm2+
p53+Mdm2
p53+Mdm2
p53+Mdm2
p53Mdm2+
p53+Mdm2
p53Mdm2
p53Mdm2+
p53Mdm2+
p53+Mdm2+
p53Mdm2+
p53+Mdm2
p53Mdm2+
p53Mdm2+
p53Mdm2+
p53Mdm2
p53Mdm2
p53Mdm2
p53+Mdm2+
p53Mdm2
p53+Mdm2
p53Mdm2
p53Mdm2+
p53Mdm2
p53Mdm2
A467
PROGENY MICE
(NUMBER)
PROGENY MICE
(EXPECTED RATIOS)
PROGENY MICE
(EXPECTED NUMBERS)
3
5
0
7
11
0
1
7
2
1
2
1
2
4
2
1
2
1
2 14
4 12
2 14
4 12
9
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,
203206.
Jones SN, Roe AE, Donehower LA & Bradley A (1995) Rescue of embryonic
lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206208.
DATA HANDLING
2056
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.
2057
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
A468
2058
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
(B)
(A)
(C)
Wx
Wx
C Wx
Wx
Wx
C Wx
BRIDGEBREAKAGE
FUSION
BRIDGEBREAKAGE
FUSION
BRIDGEBREAKAGE
FUSION
Wx
C Wx
Wx
C Wx
C-Wx
C-Wx
c-Wx
c-Wx
c-wx
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.
Gleevec
2060
Multidrug resistance
2061
TRUE/FALSE
2062
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.
2063
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.
2064
A469
A470
THOUGHT PROBLEMS
2065
2066
2067
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.
2068
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
CALCULATIONS
2069
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.
DATA HANDLING
2070
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.
A471