Brief intro to carcinogenesis and directions to further literature for 2nd year biochemists at Oxford uni.

1. Tutorial 2
Carcinogenesis
9th week Hillary Term
Tutorial for 2nd year biochemists
by Christiane Riedinger

2. Christiane Riedinger
2nd Year Biochemists, HT 2010
Tutorial 2: Introduction to Cancer/Carcinogenesis
The assignment
YOUR ESSAY QUESTION
Discuss briefly the function of six important cell-cycle related proteins implicated in
cancer. (Please no more than ~3 pages, so half a page per protein, keep it short and
to the point)
In your essay, please show that you have understood the most important aspects
of the topic, there is no need go into huge amounts of detail. Parts of your work
could also be bullet points and/or diagrams/flowcharts. Do not copy the answer
directly from the book, but read and understand the references, then process the
• any problems?
information in your head, read and understand the question, and then write the
best possible answer.
• favourites on reading list?
READING MATERIAL
Reviews: • your essays
• Lapenna et al. Cell cycle kinases as therapeutic targets for cancer, Nature
Drug Discovery, 2009, 8, 547 (ATTACHED)
• Kastan et al., Cell cycle checkpoints and cancer (ATTACHED), Nature, 2004,
432, 316 • aims for today
• Sherr C.J. Principles of Tumor Suppression, Cell 2004, 116, 235-246
(ATTACHED)
• Collings and Garrett, Targetting the cell division cycle in cancer: CDK and
cell cycle checkpoint kinase inhibitors (ATTACHED), Curr. Op. Pharm.,
2005, 5:366-373
• Please take a look around yourself for good papers/reviews/books on this
topic and report at least one reference you liked back to me
EXAM QUESTIONS TO KEEP IN MIND:
• "'Tumour cells contain mutations that facilitate transition through
cell cycle checkpoints.' Discuss evidence for this statement."
• "How does the tumour suppressor protein p53 respond to different stresses and
thus regulate the choice between alternative cell fates?"
• "What is the evidence that eukaryotic cells are dependent on checkpoints to
regulate progression through the cell cycle? Discuss, with examples, their
subversion in cancer."

3. Essay
• briefly: why are cell cycle proteins involved in cancer?
• briefly: what happens during carcinogenesis?
• possible proteins you might have chosen to discuss:
• p16/INK4
• ARF
• CDKs/cyclins: CDK4, cyclin E, cyclin D, CDK2 ....
• p53
• MDM2
• pRb
• ....

4. Cancer
• highly heterogeneous disease
• aberrant uncontrolled growth (= hence cell division!!!)
• result of gradual accumulation of mutations
• increase in activity of growth-enhancing proteins
• decrease in activity of growth-inhibiting/restraining proteins

5. Tissue
• wikipedia:
• “communities” of cells
• size, growth, division, death and differentiation highly controlled
• highly compartmentalised
• cancer: disruption of this organisation

6. Accumulation of Mutations
• tumour develops by accumulating new genetic traits that confer a competitive advantage
• through time, regulatory barriers are overcome
• most cancers result of 6-10 mutations (or genetic in the first place, then less mutations)
• prostate cancer: up to 30 mutations

7. Healthy Cell:
•

8. Imbalance as number of mutations increases:

9. Pathways/Proteins affected
by carcinogenic mutations
• tumour suppressors: growth inhibition impaired (recessive)
• proto-oncogenes: growth stimulation enhanced (dominant)
• proteins receiving signals for growth-stimulation by mitogens and
growth factors: no longer need those signals to stimulate growth
• proteins reacting to extracellular signals that inhibit proliferation:
develop resistance
• proteins important in apoptosis: apoptosis is no longer induced,
allowing the cell to survive under abnormal conditions
• proteins involved in telomere regulation: no longer a limit on
number of cell divisions (immortalisation)

10. How do mutations arise?
• 5-10% through inheritance (familial cancer syndrome)
• through errors in duplication, repair or segregation of DNA and chromosomes
• through environmental factors: radiation, carcinogenic substances
• though mutations that confer genetic instability : acceleration of accumulation of mutations

11. The cell cycle and its checkpoints
CDK11-cyclinL
CDK1-cyclinB
G2/M
CDK1-cyclinA START
CDK4-CyclinD
CDK6-CyclinD
CDK3-CyclinC (humans)
CDK2-cyclinE
CDK2-cyclinA
G1/S

12. Involvement of Cyclins/CDKs in cancer:
• cyclins: over-expression
• CDKs: overexpression and mutation
• also - kinase inhibitors often mutated, deleted or silenced:
INK4 - inhibitor of CDK4
CIP/KIP (CDK inhibitor protein / kinase inhibitor protein)

13. checkpoint kinases:
Chk1 and Chk2

14. p53
(picture taken from my thesis)

15. The p53 Tumor Suppressor: Structure and Function
Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations.
Because of its essential function in growth control, the p53 tumor suppressor is sometimes termed the “Guardian of the Genome” [12]. Currently, over
20,000 studies investigating p53 are available on PubMed; a result of nearly 30 years of dedicated research into the subject (for a historical perspective
of the p53 discovery, see Box 1).
p53 is a tetrameric 393 amino acid protein, composed of five functional domains connected by flexible linkers [13],[1]. The N-terminal transactivation
domain (TAD) is responsible for MDM2-binding and comprises residues 1-61 [14-17]. NMR and circular dichroism studies of a p53 fragment
comprising the transactivation domain and the adjacent proline rich region (residues 64-92) showed that this section is natively unfolded, lacking tertiary
and secondary structure elements in the apo-form [18]. The DNA binding domain comprises residues 102-292 [19] and is the region most affected by
mutations in cancer cells [20]. This domain is intrinsically unstable, which explains its high susceptibility to oncogenic mutations [21],[22]. The
oligomerization domain (residues 324-355) is responsible for the formation of a tetramer in vivo [23],[24]. The final C-terminal regulatory domain
(residues 367-393) is capable of binding to single-stranded DNA and RNA [25]. Models of full-length p53 in the free or DNA-bound state have been
obtained from X-ray scattering data [26]. For a full review of p53 structure, the reader is referred to Joerger et al. [27].
p53 has so far been best understood in its function as a tumor suppressor, responding to signals of DNA damage and aberrant growth, initiating DNA
repair, cell cycle arrest and, if necessary, apoptosis. These functions are mainly provided through p53’s activity as a transcription factor, controlling the
expression of dozens of genes [28]. Due to the potentially lethal response that can be triggered by p53, its levels are kept low during normal cell
development. Tight regulation of p53 levels is achieved in three different ways, affecting the protein’s activity, stability and sub-cellular localization.
Activity can be controlled through various post-translational modifications such as phosphorylation, acetylation or glycosylation or through direct
interaction with its antagonists. The effects of MDM2 and MDMX on p53 activity are described in more detail below. The stability of p53 levels is
mainly dependent on the rate of ubiquitin-mediated degradation, while localization of p53 in different sub-sections of the cell can be regulated through
shuttling the transcription factor in and out of the nucleus.
There are several pathways leading to p53 activation [29],[30], amongst them DNA damage and oncogene activation (Figure 1). If DNA damage is
caused by ionizing radiation, then the central signaling protein in DNA damage control, the kinase ATM (Ataxia telangiectasia mutated) activates p53
either through direct phosphorylation or indirectly via the transducer kinase Chk2 [31]. p53 activation can also be induced via the kinase ATR (Ataxia
telangiectasia and Rad3 related), which is activated by the presence of single stranded regions of DNA [32]. Oncogene activation, such as expression of
the Ras or Myc gene products [32] results in activation of p53 by the p14/p19ARF tumor suppressor, a negative regulator of MDM2 (see below).
One important transcriptional target of p53 is the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1, which serves to prevent entry from G1 to S
phase, as well as G2 to mitosis transitions (Figure 1). Other transcriptional targets include the apoptotic proteins Bax, PUMA and tumor necrosis factor
(TNF), reviewed in [28],[33]. Interestingly, among the genes controlled by p53, there are some targets which themselves modulate the activity of p53.
One of these genes is MDM2, p53’s main antagonist, described in more detail below.
More recently, important p53 functions distinct from its role as a transcription factor have been described [33]. Furthermore, new studies have
investigated the function of p53 beyond its involvement in cancer prevention, suggesting an important contribution of p53 to other aspects of cellular
life, such as cellular senescence, differentiation, angiogenesis etc. [20]
From: Riedinger et al., Future Medicine, 2009, 1(6), 1075-1094

16. Additional references:
Hanahan et al. The Hallmarks of Cancer. Cell (2000) 100(1) pp57-70
Knudson. Two genetic Hits (more or less) to cancer. Nat Rev Cancer 2001 vol 1(2) pp157-62
Stratton et al. The cancer genome. Nature 2009 vol. 458 (7239) pp719-24
Nature Reviews Cancer: http://www.nature.com/nrc/journal/v9/n12/index.html
Scientific American special edition about cancer: http://www.scientificamerican.com/special/toc.cfm?issueid=56