1. Compare and contrast the two hit hypothesis, haploinsufficiency and continuum model of tumor suppressor genes.
2. Compare and contrast oncogenes (Chapter 2) and Tumor Surpressor Genes.
3a. What do you think would be more detrimental to a cell (ie: more likely to cause transformation) a loss of a tumour suppressor gene or a gain of an oncogene?
3b. which event do you think is more likely?
Gatekeeper and Caretaker:
1. What is the difference between a gatekeeper gene and a caretaker gene? Do all tumor suppressor genes fall under one category or another?
2. Do caretaker gene mutations directly, or indirectly induce tumorigenesis? Explain.
1. Compare and contrast the mechanism and regulation of Rb and p53 as tumor suppressor genes.
2. Serines and threonines are residues typically phosphorylated in proteins for purposes of controlling protein activity. If a cell has both versions of the Rb gene mutated, such that multiple serines and threonines are glycines and alanines, what would the phenotype of the cell be? Explain your rationale.
1. Compare and contrast BRCA1 and BRCA2 and their roles.
2. Extended thought question: Given what you know about BRCA1, what do you think is the role of BRCA1 in the S phase?
1. Describe what APC is and explain its' significance in causing cancer. What would happen if you block the action of APC?
2. Describe a hypothetical oncogenic element which is part of the Wnt signaling pathway that could allow the pathway to circumvent the inhibitory action of APC. What changes could occur in this element that would allow it to possess oncogenic properties?
1. What is the significance of XP-A in cancer development?
2. If a patient has mutations in XP-A that render it defective, is the patient guaranteed to develop cancer?
1. What is the significance of BLM/WRNH in cancer development?
2. BLM, WRNH and XP-A are involved in some aspect of DNA repair in general. Compare and contrast BLM, WRNH and XP-A. How are they the same and how are they different?
1. Jerry the geneticist has identified a gene to be consistently mutated in hereditary brain cancers which have an early onset (ie. prior to the age of 5).
a) Is the gene likely to be a tumour suppressor gene or an oncogene? Why?
b) How can Jerry further support the notion that the gene is in fact, a tumour suppressor gene?
c) Given that Jerry's gene is a tumour suppressor gene, he now wishes to uncover the model by which it acts. How could he approach doing this?
d) A tumour sample which Jerry received from his friend, Frank the physician, showed lowered expression levels of the gene of interest via a microarray analysis. However, when Jerry subjected the tumour sample to sequencing, there were no mutations detected. Jerry suspects that promoter hypermethylation may have caused this. How could he test this?
2. Oncogenes are mutated genes that cause the transformation of normal cells into cancer cells. Tumor Surpressor Genes are responsible for the maintenance and homeostasis of a normal cells proliferative state.
Gatekeeper and Caretaker Genes
1. Gatekeeper genes directly affect the cell cycle at the cell cycle checkpoints and caretaker genes maintain the genomic integrity of cells and is important throuout the cell cycle. This means that all TSG fall under the category of caretaker, but there are specific ones that also belongs to the group of gatekeeper gene. For instance, p53 belongs to both of the categories. Hence the two are not mutually exclusive.
2. Caretaker genes indirectly induce tumorigenesis. This is because caretaker genes are responsible for maintaining the integrity of the genome. If a caretaker is mutated, then other genes that directly responsible for cell cycle maintenance may be mutated as well. It is the mutation of the genes responsible for cell cycle maintenance that causes tumorigenesis.
1. This question is quite open ended in the sense that many aspects of Rb and p53 can be compared. The answer should including comparisons on the main like where they function, what function they do and how the regulation is conduct. When the the two proteins are being differentiated, the answer should give clear points that distinguish the too.
2. A complete answer must explain the link between serine and threonine phosphorylation and the function of the Rb protein. Furthermore, the answer must name the consequences of the change in function of the Rb protein at the cellular level. The Rb protein is phosphorylated at multiple serine and threonine sites to regulate its activity. If these serine and threonine sties are mutated to alanine and glycine, the cyclin-dependent kinases which phosphorylate Rb will likely not be able to do so anymore. If this occurs, Rb would likely be constitutively active because phosphorylation of Rb results in its deactivation. At the cellular level, an active, hypophosphorylated Rb will (1) bind and inhibit E2F from acting as a transcription factor, and (2) recruit histone deacetylase complexes and histone methylases such as HDAC1 and SUV39H1 which will modify histone H3 ultimately repressing gene expression via HP1 binding to the histone and downregulating expression of S phase promoting factors. These functions inhibit progression of the cell into S phase and arrest the cell cycle. If the cell cycle arrests due to a constitutively active Rb, it will not progress further through the cycle and will become quiescent (ie. will not divide).
1. The answer should include what BRCA1 and BRCA 2 are, what they do and how they conduct their function especially in repair by HR. Again, this question is quite open ended but many aspects can be considered. When the two are being distinguished, the points have to be very clear.
2. This is thought question that applies what you know about BRCA1 and its' mechanism. Many variations are expected on this answer but the proposed mechanism has to be elucidated clearly.
1. APC is a tumour supressor gene that acts as a gatekeeper by controlling cell growth through action on other proteins. When talking about the structure of APC, the prominent regions of APC like beta catenin binding site should be mentioned. A diagram will help explain this part also. APC functions in preventing beta-catenin from activating downstream effectors of the Wnt pathway by binding on to beta-catenin and bringing it to the destruction complex where iti s phosphorylated by GSK-3β which destabilizes beta-catenin. This will target beta-catenin for ubquitination and eventually degredation by the proteosome. When APC is no longer functional either by inactivation of mutation, destabilization of beta-catenin does not occur and beta-catenin will eventually accumulate in the nucleus and target genes which lead to proliferation of crypt stem cells and eventual formation of microadenomas via binding on to TCF/LEF protein complex.
2. A hypothetical gain of function mutation in the Wnt signaling pathway which would predispose a cell towards malignancy would be considered the oncogenic component we are interested in describing. Furthermore, this component must be further downstream than the action of the APC protein. In the Wnt pathway, β-catenin is the component which is inhibited by the APC protein. Furthermore, β-catenin is the last element in the pathway before the regulation of transcription. Therefore, β-catenin is the most obvious element which may be able to circumvent the regulation of APC protein thereby activating the pathway in the absence of Wnt. The APC protein regulates β-catenin by recruiting it for destruction in a protein scaffold. This is achieved through the action of axin, casein, kinase1 and GSK-3β, which phoshporylates β-catenin, thus targeting it for degradation by a proteosome. If the reside which GSK-3β phosphorylates β-catenin at is mutated to a different residue, targeting for degradation would not occur. Therefore, a scenario would arise where β-catenin is not broken down. This would result in β-catenin dysregulation and constitutive stimulation of the signaling pathway in the absence of a Wnt signal. Furthermore, another answer to this question could be a mutation causing β-catenin to change conformation such that APC does not bind it efficiently, resulting in β-catenin not being recruited for degradation.
1. A suggested answer talking about XP-A's significance would talk about its' mechanism and how it can apply to causing cancer. The answer has to make logical sense but various aspects could be considered.
2. Having a defective XP-A does increase your risk for getting cancer but it does not guarantee you will get cancer. XP-A follows the two-hit model hypothesis so a second mutation in a gene involved with cancer is needed for a patient to develop cancer. Remember it most cases, there were defects in genes ike p53 in tumour samples with defective XP-A.
1. A suggested answer can covere many aspects of BLN/WRNH and how they can lead to cancer. The answer at least must talk about the mechanisms of BLN/WRNH and their role in DNA repair and then link it to cancer.
2. This question is quite open ended since many aspects of all three proteins can be compared. The answer must at least consider where they act, what their function is and the mechanism and can include points about their structure. When distinguishing them, the points made have to be very clear.
a) The gene is likely to be a tumour suppressor gene. At the cellular level, tumour suppressor genes tend to act in a recessive fashion. In contrast, oncogenes tend to act in a dominant fashion. Oncogene activation is therefore active at the cellular level during development of the individual. Oncogenes tend to dysregulate cell growth, apoptotic processes and cell signaling pathways; these are components critical to development of an individual prior to birth. Therefore, an individual who has inherited an oncogene in the germline is likely to die before birth. On the other hand, an individual who has inherited only one working copy of a tumour suppressor gene is more likely to live, it being the case that the phenotype is masked at the cellular level during most of development.
b) Jerry can perform a cell fusion assay. This is done by isolating a line of cells homozygous for the allele of interest and a normal cell line. The cells are then treated with a fusogenic agent such as polyethylene glycol, such that they fuse to become a tetraploid cell. This tetraploid cell is then observed to see if it develops cancer cell-like properties. If the cell develops cancer cell properties, the gene is likely to be an oncogene, because it works in a gain-of-function fashion at the cellular level; however, if the cell does not develop cancer cell properties, the gene is likely a tumour suppressor gene, because it works in a recessive fashion at the cellular level.
c) The three models presented in the introduction chapter must be considered possibilities; that is, the Two-hit model, the One-hit model and the Continuum model. To distinguish between the three, it would be best to first contrast if the gene followed a two-hit versus a one-hit model. This could be done by producing a gene knockdown cell line, and then profiling malignancy of cells which were heterozygous for the loci in comparison to a wild-type cell line. If the cell line propagated was as malignant as wild-type cells, this would support the notion that the gene follows a two-hit model. In contrast, if the cell line propagated was more malignant than the wild-type cells, this would support the notion that the gene follows a one-hit model. To see if the gene was following a continuum model pattern, he could create knockdown cell lines homozygous for the defective allele, and then see if these were more or less malignant than the heterozygous cell line, as the continuum model suggests that with no expression of the particular gene, the cell would be more likely to apoptose or be subject to self-imposed constraints on growth and division. As such, if the TSG follows a continuum model, the double knockdown would show less malignancy than the heterozygous cell line.
d) Jerry could utilize a bisulphite treatment to convert unmethylated cytosines to uracil via a chemical reaction which the bisulphite treatment induces. The sample can then be amplified using PCR with specific primers amplifying the promoter region. The amplified sample can be sequenced and compared to sequences of non-cancerous samples to contrast methylation patterns.