Final Project

The role of MDM2 oncogene in the regulation of tumour growth

Background

A wide variety of oncogenes and tumour suppressor genes are responsible for regulating cell proliferation and development. It has been known for decades that mutations in these genes give a predisposition to cancer. Murine double minute 2 (MDM2) encodes for MDM2 protein, a nuclear phosphoprotein that binds and inhibits transactivation by tumour protein p53, as part of an autoregulatory negative feedback loop (Freedman, D.A., et al. 1999). MDM2 gene is classified as an oncogene as it is widely overexpressed in a variety of human tumours.

MDM2 has several domains that are conserved between species, ranging from humans to zebrafish (Momand, J., et al. 1998). The amino-terminus of MDM2 encodes for the p53 negative regulator site. This site is necessary and sufficient for interaction with p53 tumor suppressor protein and inhibition of its transcriptional activation function. This is achieved via the interaction with the N-terminal p53 binding domain to promote p53 ubiquitylation, which targets p53 protein for proteasomal degradation (Chen J., et al. 1993). The primary function of MDM2 protein is to act as a repressor of p53 through this mechanism.

P53 is a well-studied tumour suppressor protein that is responsible cell cycle regulation by inducing growth arrest and apoptosis when DNA is damaged. The three major roles of p53 are cell cycle arrest, DNA repair, and programmed cell death (apoptosis)(Freedman, D.A., et al. 1999). P53 functions as a physiological barrier against clonal expansion or mutation accumulation in the genome. The gene also controls and arrest growth of the cells that hyperproliferate when subjected to oncogene activity. P53 repression and inactivation is strongly correlated to about 50% of cancers when knocked out, inactive or mutated in a wide range of mammals (Batinac, T., et al. 2003). P53 mutations typically occur on one allele, but this is usually sufficient for enough damaged genes in cells to escape the p53 surveillance and become immortalized cancer cells at some point.

When DNA is damaged, MDM2 is repressed and allows p53 to stop cell cycle proliferation to repairs. When MDM2 is present, p53 function is inhibited and the cell cycle continues. Thus, MDM2 is also known as an oncogene because an overabundance of MDM2 shuts down p53 completely, thereby compromising its cancer-preventing abilities (Perry M. E., et al. 1993). Although it is well known that MDM2 protein acts on p53, its independent functions and gene regulation are not well defined. A study by Reifenberger et al. (1993) show there has been shown to be MDM2 overexpression but no TP53 mutation indicative that MDM2 overexpression can take the place of inactivating TP53 mutations.

To facilitate transcription of MDM2, recruitment of TRRAP protein to the promoter of MDM2 by p53 is required, as part of the negative feedback loop. TRRAP functions as a transcriptional cofactor that recruits several other actyltransferase complexes, which catalyze the modification and acetylation of histones, associated with the MDM2 promoter (de Graaf, P., et al. 2003). In turn, MDM2 transcription is induced.

Relevance

Overexpression of MDM2 has been correlated in excessive inactivation of tumour suppressor protein p53(Freedman, D.A., et al. 1999). In this way, MDM2 could affect cell cycle regulation, apoptosis and tumorgenesis. MDM2 is overexpressed by gene amplification, increased transcription or enhanced translation. It has been shown to be amplified in a variety of tumours including soft tissue sarcomas, osteosarcomas and gliomas. The highest frequency, about 20%, of MDM2 amplification is found in soft tissue tumours, which includes Ewing’s sarcoma, leiomyosarcomas, lipomas, malignant Schwannomas and many other sarcomas. The second highest frequency of MDM2 amplification had been shown in osteosarcomas at 16% (Isreal, B. et al. 2010). Furthermore, Higher frequency of splice variants lacking p53 binding domain sequences was found in late stage and high grade ovarian and bladder carcinomas (Ladanyi, M., et al. 1993). Understanding how gene regulation of MDM2 occurs and its association with cancers may lead to MDM2 gene targeted therapy for specific cancers.

Research question

While p53 mutation typically occurs in one allele (Li-Fraumeni syndrome), MDM2 regulates all p53 activity through repression. Based on this, my question is whether MDM2 overexpression will allow for cancer cell proliferation and metastasis to happen more readily and efficiently than a single allele p53 mutation.

Hypothesis

Since a p53 mutation occurs on one allele, there is another functional copy of the tumour suppressor gene encoding for a functional tumour supressing protein, therefore there is “half” the normal amount of cell cycle modulation occurring. However, MDM2 overexpression would allow for faster degradation of all p53 proteins. I hypothesize that MDM2 overexpression would have a greater regulatory role in cancer cell proliferation and have greater tumour growth rates.

Materials and Methods

Mouse models:

·      For MDM2 overexpression mice: use a p53^+/+ and MDM2^+/+ mouse strain and create a model with an inducible promoter at an additional transposed MDM2 gene (explained in experimental approach)  

·      For a p53 mutant mouse: use a p53^+/-, MDM2^+/+ mouse model. 

·      For the positive control: use wildtype mice that are p53^+/+ and MDM2^+/+.

·      For the negative control: use p53 knockout mice (p53^-/-). MDM2 can be either wildtype or mutated, because without p53, MDM2 has nothing to act upon.

Use 5 replicates of the test mice, and 3 replicates for the control mice. The mouse strains are available at Jackson Laboratory mouse consortium.

Cancer cell lines:

To create mouse glioblastoma cancer cell, use Cre-loxP–controlled lentiviral vectors to generate a mouse glioblastoma multiforme cancerous tumour model as described and done by Marumoto, T., et al (2008). These mice can then be sacrificed and their cancer cells used for tumour injection.

Experimental approach

To create gain of function MDM2 mice, I would use tyrosine site specific recombinase to insert an MDM2 gene with an inducible promoter, in addition to the existing MDM2 gene. Using the Cre-loxP system as described by Anastassiadis et al. (2009), I would flank the transposed MDM2 gene with lox sites for Cre recombination to occur with a CrePBD (progesterone ligand binding domain) promoter in a F_o generation of mice. The PBD is inducible by synthetic anti-progestin RU 486, but not by endogenous progestins, making it a desirable promoter for this experiment. After breeding these mice so recombination can occur (using embryonic stem cell clones that are crossed with this reporter), embryos are screened for complete recombination using B-galactosidase staining (Anastassiadis, K., et al. 2009). These mice display the same phenotype as WT mice in the absence of RU 486, but can show overexpression (gain of function) MDM2 when RU 486 is introduced. I would allow these mice to mature into adulthood. 

Inject glioblastoma cancer cells into each mouse model, at the same site in the brain and same quantity. Ensure that the cells are injected in the same layer of the brain, since cancer cells grow more effectively at certain niches, to guarantee that the cells are surrounded by the same type of environment for equal opportunity growth between mice strains. For gain of function MDM2 mice, inject RU 486 to overexpress MDM2 gene transcription prior to cancer cell injection.

Use p53 protein antibodies and MDM2 protein antibodies to bind proteins present, and measure their quantities with immunofluorescence. These antibodies can also show localization of the proteins to ensure MDM2-p53 interaction. I can also check p53 and MDM2 mRNA levels with qt-PCR to measure the difference in gene expression between the mouse strains.

After allowing time for the tumours to grow, mouse models can be sacrificed at the same time and their tumour mass and volumes measured.

Challenges and Sources of Error

The failure to create a gain of function MDM2 gene is a huge problem since this is the basis of the project. Cre-loxp recombination is typically done to make a conditional knock-out mouse, however, it is possible to recombine genes to knock-in the inducible promoter at the MDM2 gene. This may cause other potential problems such as interfering with gene transcription of other genes, Also, Cre-loxp recombination has been known to have some toxic effects on the mice overtime (Anastassiadis, K., et al. 2009), which may contribute to more DNA damage and tumour formation.

There may be biological differences between the mice, which I would try to minimize by using a larger sample size. Furthermore, I can also genotype the p53 and MDM2 gene using DNA sequencing (the whole genome would take too long) to ensure these genes are the same, therefore functioning the same.

Possible outcomes and Discussion

Predicted outcome: the tumour growth rates of overexpressed MDM2 mice are higher than the p53^+/- mice.

If this outcome occurs, it would prove that MDM2 is a key oncogene and its repressing effects on p53. This indicates that MDM2 modulates all p53 activity as its repressor and its overexpression allows for cancer cells to proliferate more readily. If this is the case, immunofluorescence should show p53 proteins saturated with MDM2 binding, showing that they are being led to degradation and not preforming their cell cycle regulation duties.  

Null outcome: the tumour growth rates between the mice are relatively similar.

If this outcome occurs, it indicates that MDM2 represses p53 at a moderate level, and its overexpression does not affect all p53 activity. This indicates that there may be a threshold for MDM2 activity on p53 and the negative feedback loop between these proteins is sufficient to compensate for the overexpression of MDM2. If this is the case, the p53 and MDM2 protein antibodies should show a proportional amount of MDM2-p53 binding as the positive control WT mice. This way, there is still p53 preforming its cell cycle regulation duties. The additional translated MDM2 may be repressed by some mechanism to prevent them from binding p53.

Although testing MDM2 overexpression mice against p53^+/+ wildtype mice would be simpler than against p53^+/- mice, I chose the p53 mutant because it would be more relevant for human cancer studies. This is due to the fact that most single inactivating alleles in p53 have been associated with cancer. Furthermore, Li-fraumeni syndrome, an individual born with only one working p53 allele, has a huge predisposition to developing tumours later in life (Batinac, T., et al. 2003). It is already known that MDM2 affects p53, so this experiment helps to illustrate to what effect MDM2 plays a key role. Research typically associates a p53 mutation as the primary cancer causing factor, but this experiment will demonstrate whether MDM2 repression on p53 affects tumour growth more significantly.

Discovering how significant the role of MDM2 oncogene in tumour progression can lead to exciting new gene targeted cancer therapies. The purpose of this experiment is to learn how gene regulation of an oncogene contributes to cancer cell formation, thereby, we can learn how to manipulate gene regulation to slow down or even prevent gliomas and other cancers that MDM2 affects.

References

Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., Stewart, A.F., 2009. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Disease Models & Mechanisms, 2: 508-515. 

Batinac, T., Gruber, F., Lipozencic, J., Zamolo-Koncar, G., Stasic, A., & Brajac, I. (2003). Protein p53--structure, function, and possible therapeutic implications. Acta Dermatovenerologica Croatica : ADC / Hrvatsko Dermatolosko Drustvo, 11(4), 225-230.

Chen J., Marechal V. and Levine A. J. .1993. Mapping of the p53 and mdm-2 interaction domains. Molecular Cell Biology, 13: 4107–4114.

de Graaf, P., Little, N.A., Ramos, Y.F., Meulmeester, E., Letteboer, S.J., Jochemsen, A.G. 2003. Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2. J. Biol. Chem. 2003, 3: 38315-24.

Freedman, D.A., Wu, L., Levine, A.J. 1999. Functions of the MDM2 oncoprotein. CMLS Cellular and Molecular Life Sciences, 55: 96-107.

Isreal, B. 2010. Shedding Light OnGene Regulation-Causing Gene Regulation. Current Cancer.

Ladanyi, M., Cha, C., Lewis, R. 1993. MDM2 Gene Amplification in Metastatic Osteosarcoma. Cancer Research, 53:16-18.

Marumoto, T., Tashiro, A., Morvinski, D.F., Scadeng, M., Soda, Y., Gage, F.H., Cerma, I.M. 2008. Development of a novel mouse glioma model using lentiviral vectors. Nature Medicine, 15: 110-116.

Momand, J., Jung, D., Wilczynski, S., Niland, J. 1998. The MDM2 gene amplification database. Nucleic Acids Research, 26(15): 3453-3459.

Perry M. E., Piette J., Zawadzki J. A., Harvey D. and Levine A. J. 1993 The mdm-2 gene is induced in response to UV light in a p53-dependent manner. National Academy of Science USA, 90: 11623–11627.

Reifenberger, G., Liu, L., Ichimura, K., Schmidt, E.E., Collins, V.P. 1993. Amplification and Overexpression of the MDM2 Gene in a Subset of Human Malignant Gliomas without p53 Mutations. Cancer Research, 53:2736-2739.

Some (more) thoughts about genetics ...

Gene regulation has obviously always been one of the biggest controlling factors is our phenotypes, genetic predispositions and even social behaviour as shown in the Garfield et al. paper about Grb10. In this day and age, we have so many technologies at hand that allow us to manipulate genes and conduct insane studies on animal models. A particularly interesting study I recently heard about was trying to study the language center of the brain by transfecting mice with “language genes” in hopes that they could get these mice to “speak.” There has been much controversy against this due to the humanizing of mice. However, particularly in the study of Grb10, it would prove to be extremely beneficial if we could understand what the mice are expressing in social studies. This would provide a lot of insight to gene regulation and social behaviour; whether we are genetically determined to a certain personality. But is this too inhumane? Thoughts?

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071

Assignment #5 - News & Views

The last assignment I'm going to include is my news & views presentation report that I did with Arthi. We presented a paper from Nature, September 2010 by Rolf Ohlsson entitled The Coherent Mediator. This article can be found at http://www.nature.com/nature/journal/v467/n7314/full/467406a.html.

The Coherent Mediator - News and Views

            In this news and views article, a paper published by Kagey et al is discussed. The article details the findings of the experiment done. The article focuses on the mediator and cohesin complex. The mediator complex helps enhancers increase gene transcription by bringing together diffusible trans-acting factors and basal transcriptional machinery. The mediator complex also brings together cis-regulatory elements such as enhancers and promoter of active genes. The primary focus of the news and views article is to give evidence on how the mediator achieves this. The recent discovery made by Kagey et al is that the mediator enlists another protein complex cohesin to cohere the enhancer and promoter sequences. The mediator-cohesion complex work together to form a chromatin loop which allows for distal enhancers and promoters to be brought together prior to transcription. The mediator itself interacts with the promoter to stabilize the pre-initiation complex, which positions RNA polymerase II over the start sites of gene transcription. It was also discovered that genes encoding cohesin complex members are also regulators of Oct4 (a regulator for pluripotency in stem cells) expression in embryonic stem cells. Cohesin has been shown to insulate enhancer-promoter communications by aiding in the formation of repressing chromatin loops by CTCF (a transcriptional receptor); however findings by Kagey et al show cohesin localization to regions not possessing CTCF. The discovery of cohesin being involved with in may also have implications on diseases that involve mutations in the mediator complexes.

This article has to do with what we discussed in class in week 4 regarding DNA regulatory sequences. In class, we saw that enhancers, insulators, repressors and locus control regions worked on promoter regions. This was shown using examples that illustrated how enhancers increased gene transcription by acting on promoters and insulators decreased transcription by preventing enhancer-promoter interactions. Specifically, in the transcriptional elements paper by Maston et al that we read that week, we learned that distal upstream regulatory elements (as mentioned) were able to make contact to the core or proximal promoter via a mechanism that loops out intervening DNA between these elements. This article provides insight to the workings of such a mechanism; mediator complex involved in promoter interactions, whether it is with an enhancer or insulator. The article gives us a better understanding of what we learned in class to demonstrate to us how enhancers actually interact with the promoters and what other factors are involved.

            We found this article interesting because of the implications these findings (i.e. that cohesin interacts with the mediator complex) has on diseases involved with mediator mutations. As mentioned in the article, schizophrenia (as well as Opitz-Kaveggia (rare genetic syndrome linked to the X chromosome causing physical anomalies and developmental delays) and Lujan syndromes(X-linked genetic disorder that causes mild to moderate mental retardation)) involves mutations involved in the mediator complex. However, with these new findings, it could be shown that cohesin is actually involved in schizophrenia, leading to new treatments for the disorders.  As well, gene transcription is one of the most important functions in the human body, the more we know about how it works , the more we are able to find out about how things happen the way they do. 

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071

Assignment #4

Assigned articles:

1)  Schubiger, G. (1980) The development of animal segments.

(Note that this short article is an introduction to/presentation of the second article).

2)  Nüsslein-Volhard, C. and Wieschaus, E. (1980) Mutations affecting segment number and polarity in Drosophila.

3)  Haffter, P., et al. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.

Questions

1)  Briefly summarize articles #2 and #3 IN YOUR OWN WORDS (point form is fine… maximum 250 words in total for the two papers)

In articles 2 and 3, using Drosophila and Zebrafish, respectively, they explained experiments regarding mutations that affect development. The drosophila paper illustrated how segment polarity mutants, pair rule mutants and gap mutants cause phenotypic alterations. These 3 classes are responsible for embryo defects in their subregions on repeat length. With the zebrafish in paper 3, experiments were preformed to find the phenotypic alterations in embryos using mutagenic treatment to mutate specific genes in attempt to characterize genes responsible for segmentation and growth. This allowed the researchers to find mutants in 372+ genes, with a wide range of mutated phenotypes.

2)  Think about the kinds of experimental approaches that were discussed in class. What kind of approaches did the authors take in the experiments reported in papers #2 and #3?

Primarily loss of function experiments for papers 2 and 3:

     Paper 2: This was shown in deletion of segments which result in a mirror-image duplication -> fused, wingless, cubitus, inturruptus, gooseberry, hedgehog and patch mutants came from a loss of function experiment that deleted different segments of DNA.

     Paper 3: This was evident because they only mutated parts of the genome, resulting in loss of function of certain genes to find if some genes are necessary for function or development in embryogenesis.

No gain of function experiments were done for either papers.

3)  Refer to paper #2:

Back in 1980, what set the authors’ approach apart from other developmental biology/developmental genetics studies? (What’s novel about it?)

They were able to isolate and represent the majority of the loci affecting segmentation from the entire drosophila genome, thereby identifying all the genetic components involved in embryonic pattern formation

Now consider paper #3:

What’s novel, and what’s extremely valuable about paper #3?

They were able to conclude that the most severe limitation of detection of genes with important functions using mutational approaches (which is what their experiments were based on) was redundancy. They showed that there was complete or partial overlap in functions of two or more genes involved in the same process.

Prior to this paper (#3), Dr Nüsslein-Volhard publicly presented her intention to do with the zebrafish what she and Wieschaus had done with Drosophila in terms of identifying all the genes required for early development. People did not laugh in her face because she was extremely well-respected and well-known for achieving what she set out to do, but many a researcher quietly questioned her sanity, as the project seemed to be an extremely challenging one.

Why do you think other scientists were skeptical about the project’s chances of success?

Because the number of genes in zebrafish, compared to drosophila, is much larger. The sheer number of screenings and mutants that she and her researchers had to conduct was enormous in such an ambitious study. Of 3857 mutagenized genomes, all were extensively screened and only 1163 were kept and characterized.

4)  What general conclusion can be made from the results presented in papers #2 and #3?

A general conclusion can be that embryogenesis is extremely complex and characterized by many genes that each have both an individual function and functions as part of a larger group of up or downstream pathways.

5)  In paper #2, the authors’ hypothesis is not clearly stated. Nonetheless, they did have one. What do you think it was? (Hint: you may find valuable information in this sense in paper #3!).

I think their hypothesis was that loss of function phenotypes can show genes that are essential for development and pattern formation such as segmentation.

6)  In paper #3, the researchers decided to do a DIPLOID screen, as opposed to using haploids (as depicted in MGA’s Figure 12-24). What are the advantages and disadvantages of doing a diploid screen, as opposed to a haploid one?

(It may help to compare MGA’s figure 12-24 to Figure 3 in paper #3).

Doing a diploid screen was done in paper 3 because the identification and recovery of mutants of many phenotypic classes can be performed with more consistency and reliability. However, it’s much more laborious and time consuming.

7)  In both paper #2 and paper #3, the authors performed a set of complementation tests. Why did they test mutants with similar phenotypes against each other (as opposed to testing every single mutant against all of the mutants)?

They did this to identify the mutant genes, to see whether mutants that displayed the phenotype showed the same genotype because it’s more likely that there would be a correlation in such case. Furthermore, it would be much more time consuming to screen all mutants against one another.

8)  Study Paper #3’s Table 4. What does it show? Be prepared to discuss the information shown in the table in a lot of detail!

This table shows the number of alleles, number of genes and number of mutants that were found. They demonstrate that the allele frequencies that are observed were not random because there are significant numbers of mutants that correspond to each allele and gene.

9)  a) Genetic screens are probably the oldest genome-wide experiments around. What is another genome-wide type of experiment that you know of?

Reverse genetic screens are for discovering the function of a gene by analysing the phenotypic effects of specific gene sequences. This is like the opposite of genetic screening. An example is creating KO mice of any sort to analyse phenotype.

b) Compare the type of information provided by a genetic screen vs. your other genome-wide experiment.

Genetic screens show the genome of individuals with a particular phenotype, therefore you can find SNPs or other mutations in the gene. Reverse genetics show the phenotype of a gene that you alter so you can find the effects of mutating or silencing a particular gene.

c) Compare the obvious follow-up experiment(s) for a genetic screen and for your other genome-wide experiment.

Follow up to genetic screens would be to characterize the SNPs, and screen further subjects. For reverse genetics, alter the gene in another way, or transfect another individual (mouse) with the gene and see if the same effect occurs

d) Would you say that your other genome-wide experiment a “reverse genetics” or a “forward genetics” one?

It’s a forward genetics one because it identifies the genes responsible for a phenotype, like finding the underlying cause of a big picture, whereas reverse genetics starts with genes and finds phenotypes.

10)   Think about the types of mutations discussed in class, and also about the kinds of mutations described in Figure 12-38 of your background reading for this week. With respect to those examples, what kinds of mutations did the authors of article #2 identify?

They found segmentation mutants; segment polarity mutants, pair rule mutants and gap mutants cause phenotypic alterations in segmentation of the abdomen and thorax.

11)   In paper #3, several mutants have multiple phenotypic defects (see for example Table 5). For example, defects in the otic vesicles are reported to be often accompanied by other mutant phenotypes, such as lack of the pelvic fin. What does this suggest?  (Think about at least TWO possible explanations for this phenomenon).

It suggests that otic vesicles and the pelvic fin (for example) are dependent of each other. This could be due to the affected gene being an upstream of the other, therefore both are affected when the upstream gene is not functioning, or they are both part of a multi-system complex that cannot function independently.

12)   Imagine that it’s 1980, and you have just come into possession of one of the mutants described in article #2 (choose your favourite one). You are interested in the control of Drosophila development and are planning to apply for a new research grant. List 3 important questions relating to your mutant of choice that you’d like to tackle over the next couple of years.

[You are encouraged to post your answer to this question on the bulletin board and discuss it with your classmates!]

For gap mutants: At what stage in development is the A-P axis determined? Is kruppel dominant or recessive? Is kruppel a maternal-effect gene? What happens if there is overexpression of kruppel?

13)   Based on what you have learned from the assigned articles, and on your own experience and knowledge, what is the relevance of discovering the relationships between specific mutations in specific genes and the specific phenotypes associated with them?

This helps us to understand embryogenesis and what genes are important for this. It allows us to associate specific genes with certain phenotypes, and determine the necessary concentration, location, and timing for the transcription/translation of cretain genes in order for healthy development.

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071

Final Project Description

My project takes a look into gene regulation and what happens when it goes wrong. The most obvious and biggest problem when gene regulation is not maintained is the development of cancer. This happens when tumour suppressors are downregulated or knocked out, or when oncogenes are upregulated. Several factors can play a role in this: defects in promoters, enhancers, inhibitors, transcription factors, point mutations, and many more! To study this more specifically, I’ve chosen the MDM2 gene, which encodes for the oncogenic protein also named MDM2. MDM2 has shown to be upregulated in certain tumours, especially soft tissue sarcomas, osteosarcomas and gliomas. Its main function is to negatively regulate tumour suppressor protein p53, one of the major factors in cancer formation when it is mutated or non-functioning. Based on preliminary research, I hypothesize that when MDM2 is overexpressed, there will be unregulated tumour growth due to the repression of p53 protein. Several research studies have shown a correlation of MDM2 overexpression to cancer, yet the specific effects of intentional MDM2 overexpression have not been characterized. Furthermore, minimal research has been done on MDM2 knock out mice models, but it has been shown that KO MDM2 mice die in embryogenesis, suggesting this gene is essential for proper growth in mice. The importance of studying gene regulation in MDM2 is because this gene is highly conserved in a range of species, from humans to zebrafish to mice; therefore it is obviously quite important. Furthermore, different expression patterns of this gene shows linkage to various human cancers, thus analysis of its regulation can help lead researchers in finding a cure for cancer. 

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071

Annotated Bibliography

1. Zhang, X., Miao, X., Guo, Y., Tan, W., Zhou, Y., Sun, T., Wang, Y., Lin, D. 2006. Genetic Polymorphisms in Cell Cycle Regulatory Genes MDM2 and TP53 Are Associated With Susceptibility to Lung Cancer. Human Mutation, 27(1): 110-117.

·      Genotyping MDM2 showed several significant single nucleotide polymorphisms (SNPs). MDM2 promoter polymorphisms show a significant impact on the risk of developing lung cancer as they interact with smoking. A particular SNP of interest that has been shown to be strongly correlated to binding affinity is SNP309, a T to G change in the first intron. This results in a increased affinity for binding stimulatory protein 1 (SP1) which causes higher levels of MDM2 RNA and protein to be produced. At SNP309, there has been shown to be a 7.5-fold increased risk assocatied with the MDM2 GG mutated genotype compared to the MDM2 TT wildtype genotype in lung cancer. This is due to the G allele having a higher binding affinity to Sp1, thereby resulting in increased MDM2 expression, as described

2. Reifenberger, G., Liu, L., Ichimura, K., Schmidt, E.E., Collins, V.P. 1993. Amplification and Overexpression of the MDM2 Gene in a Subset of Human Malignant Gliomas without p53 Mutations. Cancer Research, 53:2736-2739.

·      Due to MDM2’s known binding and inhibiting capability on p53 tumour suppressor protein, there is a strong argument made for MDM2 overexpression as one of the underlying causes of malignancy of gliomas.

3. Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J., Pavletich, N.P. 1996. Structure of the MDM2 Oncoprotein Bound to the p53 Tumour Suppressor Transactivation Domain. Science, 274(5289): 948-953.

·      Higher frequency of splice variants lacking p53 binding domain sequences was found in late stage and high grade ovarian and bladder carcinomas. Four of the splice variants show loss of p53 binding.

4. Barak Y., Gottlieb E., Juven-Gershon T. and Oren M. 1994. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Genes Dev, 8: 1739–1749.

·      The MDM2 gene has two different promoters, leading to transcripts that may initiate translation at different start codons. There are 40 splice variants, but for the majority of these variants, it is presently unknown whether they are translated into proteins. There have been about seven unique transcripts that have been described in both mouse and human cells as of 1999; a result of alternative internal splice sites.

5. Reifenberger, G., Liu, L., Ichimura, K., Schmidt, E.E., Collins, V.P. 1993. Amplification and Overexpression of the MDM2 Gene in a Subset of Human Malignant Gliomas without p53 Mutations. Cancer Research, 53:2736-2739.

·      Measures MDM2 gene levels and its correlation to malignant gliomas.

6. Ladanyi, M., Cha, C., Lewis, R. 1993. MDM2 Gene Amplification in Metastatic Osteosarcoma. Cancer Research, 53:16-18.

·      Shows MDM2 gene levels and its correlation to osteosarcoma.

7. Bougeard, G., Baert-Desurmont, S., Tournier, I., Vasseur, S., Martin, C., Brugieres, L., Chompret, A., Bressac-de Paillerets, B., Stoppa-Lyonnet, D., Bonaiti-Pellie, C., Frebourg, T. 2006. Impact of the MDM2 SNP309 and p53 Arg72Pro polymorphism on age of tumour onset in Li-Fraumeni syndrome. Journal of Medical Genetics, 43:531-533.

·      Shows correlation for age of onset of tumors in patients with li-fraumeni syndrome (lacking 1 p53 allele, predisposition to cancer) and relative MDM2 gene levels.

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071