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LSH4813 ASSIGNMENT 3 SOLUTIONS 2023 UNISA PORTFOLIO MOLECULAR TECHNIQUES
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LSH4813ASSIGNMENT 3
563381
SOLUTIONS 2023
Unique number: *******
DUE DATE:8 SEPTEMBER 2023
MOLECULAR TECHNIQUES
Page 1 of 18
,Molecular Techniques
LSH4813
Assignment 3: 563381
Total Marks: 100
Name and Surname:__________________________________________________
Student Number:_____________________________________________________
Question 1 [40]
Read the article and supplementary information by Vassilev et al (2004) and answer
the following questions.
Vassilev, L.T., Vu, B.T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N.,
Kammlott, U., Lukacs, C., Klein, C. and Fotouhi, N. (2004) In vivo activation of the
p53 pathway by small-molecule antagonists of MDM2. Science, 303: 844-848.
1.1. Explain why cancer cell lines which have mutant p53 phenotypes were used as
negative controls in this study. (5)
1.1 P53 protein is very important in the proper functioning of the cell. They are
considered as the guardian of the genome.
• They prevent the damaged DNA to enter the cell cycle. So they arrest the
cell for DNA repair mechanisms.
• The cancer cell lines with p53 mutants are used as negative control.
Negative control gives negative results.
• The p53 mutant will not produce proper p53 phenotype. So normal p53
phenotype produced here. So negative results are seen.
• This mutant will not prevent the cell to enter the cell cycle even if they
have DNA damage. This results in cancer.
• Because of the lack of expression of p53, the p53 mutant is considered
as negative control.
1.2 1.2. Western blots are routinely used to quantify the level of protein expression
in cells. Given the relationship between p53 and MDM2, interpret the results seen in
Figure 2A (also shown below). Make sure you consider each cell line, the effect of
drug treatment on each protein and what this means for the cell on a cellular level.
Page 2 of 18
, (15)
p53 is the guardian of the genome and prevents the cell from becoming cancerous
by arresting it during cell cycle G1/S checkpoint for DNA repair. In human cancer,
this p53 is mostly inactivated. If there is cellular stress, then the level of p53 in the
cell increases, leading to either cell cycle arrest or apoptosis. In a normal
non0srtesedx situation, MDM2 binds to p53 and prevents the activity of p53.
MDM2 is an antagonist of p53 and it binds with p53 with very high affinity and due to
this it negatively affects the transcriptional ability of p53. if MDM2 is overexpressed in
a cancer cell line, then the p53 will stop functioning. Anti-cancerous drugs are
thought to be working by inhibiting the interaction between MDM2 and p53 and
therefore can be used as a potential cancer treatment option.
Nutlins are cis-imidazoline analogs that bind to p53 and prevent the interaction
between MDM2 and p53 and hence help in promoting the activity of p53 which will
help in arresting the uncontrollable cell division.
Now if a cell is treated with a Nutlin drug, then it will lead to three consequences -
stabilize the p53 protein by preventing it from nuclear export and degradation,
increase in MDM2 expression by an autoregulatory feedback loop, and activation of
other p53 associated regulatory pathways.
From the western blots, it can be seen that this Nutlin-1 which is an MDM2 inhibitor
when incubated with HCT116 and SW480 cell line, shows different behavior. In
HCT116, Nutlin-1 causes an increase in the expression of all these three proteins
(p53, p21, and MDM2) in a dose-dependent manner. On the other hand in SW480,
Nutlin-1 increases the expression of p53 but decreases or no expression of p21 and
MDM2. So this confirms that Nutlin-1 causes accumulation of p53 in cells and also
Page 3 of 18
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