BIOC3900 ASSESSED COURSEWORK

1. Mouse mammary tumour virus (MMTV) is an oncogenic retrovirus that causes breast
cancer in mice when it integrates into the genome. You want to know whether it carries its
own oncogene or generates an oncogene upon integration. You isolate 26 different breast
cancers from mice that were exposed to MMTV and determine the sites at which the
retroviruses are integrated. In 18 of 26 tumours the viruses are found at a variety of sites that
are all located within a 20-kb segment of the mouse genome. Upon closer examination of
these 18 tumours, you find that an RNA is expressed from the region of the mouse genome
near the integrated virus, but not from the corresponding region in normal mouse breast
cells.
Do these observations argue for MMTV carrying an oncogene or for it generating an
oncogene upon integration? Explain your reasoning. (5 marks)
These observations argue for MMTV generating an oncogene upon integration. Firstly, as
the unique transcript is generated in the region of the mouse genome near the integrated
virus, this suggests that a gene is activated by the neighbouring virus upon integration via
the process of insertional mutagenesis. Also, the chances of MMTV carrying on oncogene
and integrating so often in the same region in 18 tumours, the 20kb segment, are slim,
further supporting the argument that MMTV generates an oncogene upon integration.

2. Retinoblastoma is an extremely rare cancer of the nerve cells in the eye. The disease
mainly affects children up to the age of five years because it can only occur while the nerve
cells are still dividing. In some cases, tumours occur only in one eye, but in other cases,
tumours develop in both eyes. The bilateral cases all show a familial history of the disease;
most of the cases affecting only one eye arise in families with no previous history of the
disease.

An informative difference between unilateral and bilateral cases becomes apparent when the
fraction of still undiagnosed cases is plotted against the age at which diagnosis is made
(Figure 1). The regular decrease with time shown by the bilateral cases suggests that a
single chance event is sufficient to trigger onset of bilateral retinoblastoma. In contrast, the
presence of a 'shoulder' on the unilateral curve suggests that multiple events in one neuron
are required to trigger unilateral retinoblastoma. (A shoulder arises because the events
accumulate over time. For example, if two events are required, most affected cells at early
times will have suffered only a single event and will not generate a tumour. With time, the
probability increases that a second event will occur in an already affected cell and, therefore,
cause a tumour).

A possible explanation for these observations is that tumours develop when both copies of
the critical gene (the retinoblastoma, Rb, gene) are lost or mutated. In the inherited (bilateral)
form of the disease, a child receives a defective Rb gene from one parent: tumours develop
in an eye when the other copy of the gene in any nerve cell in the eye is lost through somatic
mutation. In fact, the loss of a copy of the gene is frequent enough that tumours usually
occur in both eyes. If a person starts with two good copies of the Rb gene, tumours arise in
an eye only if both copies are lost in the same cell. Since such double loss is very rare, it is
usually confined to one eye.

To test this hypothesis, you use a cDNA clone of the Rb gene to probe the structure of the
gene in cells from normal individuals and from patients with unilateral or bilateral
retinoblastoma. As illustrated in Figure 2, normal individuals have four restriction fragments
that hybridise to the cDNA probe (which means each of these restriction fragments contains
at least one exon). Fibroblasts (non-tumour cells) from the two patients also show the same
four fragments, although three of the fragments from the child with bilateral retinoblastoma
are present in only half the normal amount. Tumour cells from the two patients are missing
some of the restriction fragments.

A) Explain why fibroblasts and tumour cells from the same patient show
different band patterns.
The fibroblasts and tumour cells show different band patterns in the same patient
because the tumour cells lose the normal copy of the Rb gene early in their
transformation and therefore do not show the normal band pattern.
The fibroblasts do not lose this normal copy of the Rb gene and therefore have at
least one normal gene copy, showing the normal band pattern. (5 marks)

B) What are the structures of the Rb genes in the fibroblasts from the two
patients? In the tumour cells from the two patients?
In the fibroblasts of the patient with bilateral retinoblastoma, one chromosome has
4 normal restriction fragments and is therefore normal. The other chromosome is
abnormal and only has the 6.2kb fragment remaining as the rest have been lost in
the mutant copy of Rb
In the fibroblasts of the patient with unilateral retinoblastoma, all four normal
restriction fragments are present at full intensity, as there are two normal copies of
the Rb gene present.
In the tumour cells of the patient with bilateral retinoblastoma, the three restriction
fragments of half intensity are gone and the 6.2kb restriction fragment is only
showing half intensity. This tells us that the tumour cells have lost the one normal
copy of the Rb gene leaving only the mutated copy.
In the tumour cells of the patient with unilateral retinoblastoma, there has been a
deletion of part of the Rb gene, with one allele containing both the 7.5kb and 5.3kb
fragments and the other showing only the 7.5kb fragment. (10 marks)

C) Are these results consistent with the hypothesis that retinoblastoma is due
to the loss of the Rb gene?
Yes, in both patients the retinoblastoma has a direct link to the loss of normal
copies of the Rb gene, as Rb is a tumour suppressor gene. Retinoblastoma
requires the loss of both Rb genes. (5 marks)

(iv) Suggest a plausible explanation for how the loss of the Rb gene product
might cause retinoblastoma.
As mentioned above, the Rb gene is a tumour suppressor gene and it is closely
linked to the control of the cell cycle. Rb causes cell cycle arrest by binding to
E2F transcription factors, recruiting them away from target genes to prevent cell
proliferation and the resulting transition of phases G1 to S. Rb is also involved in
the control of cell differentiation during embryogenesis, regulating apoptosis,
maintaining permanent cell cycle arrest and preserving chromosomal stability.
The Rb-E2F complexes formed serve to restrict expression of MAD2, which is a
spindle checkpoint component. Without this complex therefore, MAD2 is
overexpressed, inducing aneuploidy and tumour formation. Apoptosis can also
occur unregulated and chromosomal stability is at risk.
(5 marks)
Figure 1: The time of onset of unilateral and bilateral cases of retinoblastoma.
A population of children who, ultimately, developed retinoblastoma are represented in this
graph. The fraction of the population that is still retinoblastoma free is plotted against the
time after birth.

Sizes and order of HindIII restriction fragments in the retinoblastoma gene

Figure 2: Patterns of blot hybridisation of restriction fragments from the
retinoblastoma gene
(A) Hybridisation patterns (Southern blot) for normal individuals and for patients with
unilateral and bilateral retinoblastoma. Lighter shading of some bands indicates half the
normal number of copies. (B) The order of the restriction fragments. Fragments that contain
exons (shown as rectangles) hybridise to the cDNA clone that was used as a probe in these
experiments
3. The formation of tumours is a multistep process that may involve the successive activation
of several oncogenes. This notion is supported by the discovery that certain pairs of
oncogenes, of which Ras and Myc are among the best-studied examples, transform cultured
cells more efficiently than either alone. Similar experiments have been carried out in
transgenic mice. In one experiment, the Ras oncogene was placed under the control of the
MMTV (mouse mammary tumour virus) promoter and incorporated into the germ lines of
transgenic mice. In a second experiment, the Myc oncogene under control of the same
MMTV promoter was introduced into the mouse germ lines. In a third experiment, mice
containing the individual oncogenes were mated to produce mice with both oncogenes.
All three kinds of mice developed tumours at a higher frequency than normal animals.
Female mice were most rapidly affected because the MMTV promoter, which is responsive
to steroid hormones, turns on the transferred oncogenes in response to the hormonal
changes at puberty. In Figure 1 the rate of appearance of tumours is plotted as the
percentage of tumour-free females at different times through puberty. Assume that the lines
drawn through the data points are an accurate representation of the data.
A) How many events in addition to expression of the oncogenes are required to
generate a tumour in each of the three kinds of mice? (5 marks)
Only one other event is required in all kinds of mice to generate a tumour, shown by the
linear line which demonstrates a decrease in the percentage of tumour free females from
day 0 after puberty, most evident in the combined Myc + Ras mice. However, despite there
being a linear line for all three types, the individual Myc and Ras mice develop tumours at a
significantly slower rate than the combined mice, with the Myc mice being the slowest to
develop tumours. Nonetheless, the linear line shows that only one other event is required (if
there were more than one event the line would show a shoulder indicating multiple events).
B) Is activation of the cellular Ras gene the event required to trigger tumour formation
in mice that are already expressing the MMTV-regulated Myc gene (or vice versa)? (5
marks)
No, there must be another event involved in activation that occurs randomly over time
because not all the combined Myc and Ras mice develop tumours as they pass through
puberty. This tells us that the action of Myc and Ras together is not sufficient enough to
generate a tumour and that there must be another random event or events occurring over
time. If all combined mice did develop tumours as soon as they passed through puberty then
it could be said that one gene activates the other, and that that process was indeed sufficient
for the generation of tumours.
C) Why do you think that the rate of tumour production is so high in the mice
containing both oncogenes? (5 marks)
In the combined mice, I think that the two oncogenes interact together in a synergistic
manner to complement and promote the action of the other, thereby increasing the rate of
tumour production as opposed to the mice with the individual genes. However, as was
suggested in part B, the action of the two oncogenes alone is not enough to trigger tumour
formation and so I think that the synergistic action of the two oncogenes must serve to open
up a pathway to tumour production that is further triggered by an additional event or events
occurring randomly over time.

Figure 3: Fraction of tumour-free female mice as a function of time after puberty.