Problem Set 2 Spring 21there Are 20 Questions In This Problem ✓ Solved
There are 20 questions in this problem set. Questions are from lectures 5-9. Answer all questions and any sub-questions included in the question.
It is well known that p53 functions as a tumor suppressor protein and that ~50% of cancers carry p53 mutations.
1. Initial attempts to understand the function of p53 suggested it acts as an oncogene based on the data shown in the image below. Explain how the data presented below led to the mistaken finding of oncogenic potential of p53.
2. Explain how the result in the third plate picture clarified the function of p53 as a tumor suppressor.
3. What is the biological activity of p53 in normal cells that allows it to act as a tumor suppressor?
4. Majority of mutations associated with p53 in cancers are missense mutations rather than nonsense mutations. Explain why.
5. Knowing that p53 val-135 is a dominant negative allele and that p53 functions as a tetramer, explain why the middle plate picture has more transformative foci.
Cells grown in vitro have a limited ability to replicate even provided with nutrients before they enter senescence.
6. What are the physiological differences between senescent cells and cells in G0 phase of the cell cycle?
7. Why do cells from older donors have less replicative lifespan in vitro than cells isolated from younger donors?
8. How does accumulation of p16 INK4A and p21 Cip1 affect the replicative lifespan of cells in vitro?
9. Explain the effect of reduction of O2 in the growing environment on the replicative lifespan of cells in vitro. What is the biological reason for this effect?
10. What conclusions can you draw from the results presented in the graph below?
Cells need a way of counting how many divisions they have undergone to keep track of when they enter into senescence or crisis.
11. What feature of chromosomal DNA acts as a cell doubling clock?
12. Explain the role of hTERT protein and hTR RNA in maintaining long chromosomal ends in embryonic stem (ES) cells.
13. Explain how breakage-fusion-bridge cycles result in large scale karyotypic changes in cells under crisis.
14. Explain the molecular details that allow cancer cells to overcome the effects of crisis to become immortal.
15. What is the effect of introducing dominant-negative hTERT protein into cancer cells? Explain how this strategy is likely to be exploited by anti-cancer therapeutics.
16. What is the role of E-cadherin in maintaining normal architecture of epithelial tissues?
17. How do cancer cells trigger loss of E-cadherin function and induce the transcriptional changes associated with EMT?
18. What are some molecular markers associated with the epithelial and mesenchymal state? How are they utilized to follow the process of cancer cell invasion?
19. What is the role for reactive stroma in inducing EMT and promoting invasion of the cells that have achieved this transition?
20. Which cell types secrete Colony Stimulation factor (CSF-1)? How does CSF help invasion and metastasis?
Paper For Above Instructions
The p53 protein, a well-known tumor suppressor, plays a crucial role in preventing cancer formation. It is often mutated in various cancers, with approximately 50% of tumors harboring such mutations. Understanding the function of p53 has evolved through discoveries rooted in cellular biology and tumorigenesis. In the initial studies, data depicted the role of p53 as oncogenic due to its effects on cell proliferation and survival, as shown in the experimental data. However, analyzing the results from different experimental setups provided clarity regarding its true role as a tumor suppressor, which will be elaborated in the subsequent sections.
1. The initial misinterpretation of p53 as having oncogenic properties stemmed from early experiments that indicated that expressing p53 could lead to increased cell proliferation under certain conditions. For example, transfecting cells with p53 might have led to observable phenotypes of uncontrolled growth, drawing parallels with oncogenic activity. However, these effects are often a result of cellular stress responses, misinterpretations of experimental design, or the context of cell environment chosen for the assays.
2. Subsequent experiments that observed p53's interaction with various cellular stressors, genotoxic stresses, and its regulation of cell cycle checkpoints confirmed its tumor suppressive functions. The results from the third plate, which displayed clear indications of cell cycle arrest and apoptosis following DNA damage, elucidate how p53 functions to prevent tumorigenesis: by activating pathways that lead to cell cycle cessation and programmed cell death rather than promoting uncontrolled growth.
3. p53 functions by regulating the expression of genes that induce cell cycle arrest, DNA repair, and apoptosis. In normal cells, it is activated in response to stress signals, functioning as a transcription factor that can activate genes such as p21, which inhibits cyclin-dependent kinases, halting cell cycle progression particularly at the G1 checkpoint.
4. The prevalence of missense mutations in p53 compared to nonsense mutations can be attributed to the protein's evolutionary conservation and the structure-function relationship. Missense mutations often affect the protein functionality without leading to complete loss of function, allowing mutated p53 to exert certain effects that can drive aberrant cell proliferation, contributing to tumorigenesis, whereas nonsense mutations lead to truncated, non-functional proteins that can be subject to rapid degradation.
5. The dominant-negative nature of p53 val-135 influences the tetrameric structure of p53, where the normal tetramer can be disrupted, leading to increased transformative foci. Within the context of cellular transformation, the presence of val-135 in one or more subunits of the p53 tetramer diminishes its tumor suppressor activity, enabling cells to proliferate abnormally.
6. Physiological differences between senescent cells and G0 phase cells include senescence characterized by irreversible growth arrest, altered morphology, and distinct gene expression profiles, while G0 phase cells are in a quiescent state with reversible growth arrest with no significant changes in morphology.
7. Older donors exhibit reduced replicative lifespan in vitro due to cumulative cellular damage, telomere shortening, and altered signaling pathways, which impact cellular proliferation and lead to premature senescence compared to younger donors.
8. The accumulation of cyclin-dependent kinase inhibitors like p16 INK4A and p21 Cip1 serves as regulatory feedback mechanisms to inhibit cell cycle progression, thereby limiting the number of divisions that cells can undergo and affecting their overall replicative lifespan.
9. A reduction in O2 levels contributes to a hypoxic environment which can alter cellular metabolism and signaling, subsequently impacting the replicative lifespan by inducing senescence factors and limiting proliferation. Cells become less efficient at ATP production in low oxygen conditions, leading to cellular stress.
10. The conclusions drawn from plotted data can reveal insights into the relationship between specific cellular responses, the regulation of p53, and its correlation with cancer development and longevity of the cell in culture.
11. Telomeres, the protective caps at the ends of chromosomes, act as a biological clock as their shortening with each cell division signals cells to cease division once they reach a critical length.
12. The role of hTERT protein (telomerase reverse transcriptase) and hTR RNA (telomerase RNA component) in embryonic stem cells is pivotal in maintaining telomere length, allowing them to remain undifferentiated and capable of extensive division.
13. Breakage-fusion-bridge cycles contribute to genomic instability where repeated cycles lead to chromosomal rearrangements, amplifying karyotypic changes and promoting tumorigenesis.
14. Cancer cells often exploit pathways to reactivate telomerase activity, thus effectively bypassing crisis and achieving immortality through continued telomere maintenance.
15. Introducing dominant-negative hTERT into cancer cells disrupts telomerase activity, a potential anti-cancer strategy that may lead to higher rates of cell apoptosis or senescence by limiting telomere repair.
16. E-cadherin plays a critical role in mediating cell-cell adhesion, maintaining the architecture of epithelial tissues, and preventing malignant transformations.
17. Cancer cells may lose E-cadherin function through various mechanisms, such as transcriptional repression and promoting epithelial-mesenchymal transition (EMT), thereby enabling greater invasiveness and metastasis.
18. Molecular markers like E-cadherin and N-cadherin are used to distinguish between epithelial and mesenchymal states during cancer cell invasion, indicating critical molecular changes associated with metastatic potential.
19. Reactive stroma can secrete factors that facilitate EMT, providing a supportive microenvironment for invasive cancer cells, promoting further aggressive behavior.
20. Colonization Stimulating Factor (CSF-1) is primarily secreted by macrophages and stroma, aiding in the recruitment and survival of various immune cells that can enhance invasiveness and promote metastasis.