Cancer biology

Define the cell cycle. What are the four main phases, and what key events occur in each?

The cell cycle is the series of events that a cell goes through to grow and divide. It consists of four main phases:

• G1 phase (Gap 1): Cell grows and synthesizes proteins.

• S phase (Synthesis): DNA is replicated.

• G2 phase (Gap 2): Cell continues to grow and prepares for mitosis.

• M phase (Mitosis): Cell divides into two daughter cells. This includes mitosis and cytokinesis.

How do normal and cancerous cell cycles differ in terms of regulation and progression?

In normal cells, the cell cycle is tightly regulated by checkpoints and regulatory proteins. In cancer cells, mutations disrupt these controls, leading to unchecked proliferation, resistance to apoptosis, and genomic instability.

Explain the concept of endoreplication. How does it deviate from the canonical cell cycle, and what are its physiological consequences?

Endoreplication is a process where cells replicate their DNA without undergoing mitosis, resulting in polyploidy. This process uses the same regulatory proteins as the mitotic cycle and contributes to tissue specialization and disease.

What qualifies a protein as a “cell cycle protein”? Give at least three examples from different functional classes (e.g., kinases, inhibitors, transcription factors).

A cell cycle protein is any protein involved in the regulation, progression, or execution of the cell cycle. Examples include:

• Cyclins (e.g., Cyclin B)

• Cyclin-dependent kinases (e.g., CDK1)

• CDK inhibitors (e.g., p21)

• Checkpoint proteins (e.g., ATM/ATR)

How does deregulation of cell cycle proteins contribute to cancer development? Mention specific proteins and mechanisms.

Deregulation of cell cycle proteins can lead to cancer through mechanisms such as increased cell proliferation and evasion of apoptosis. Key proteins include cyclins and cyclin-dependent kinases (CDKs) that promote progression through the cell cycle, and tumor suppressors like p53 and Rb that normally act to inhibit uncontrolled growth. For example, overexpression of Cyclin D can lead to unchecked transition from G1 to S phase, while loss of p53 (a tumor suppressor) disables the DNA damage checkpoint, allowing mutations to accumulate.

Describe the structure and general function of cyclin-dependent kinases (Cdks) in cell cycle progression.

Cyclin-dependent kinases (Cdks) are serine/threonine kinases that, when bound to a cyclin, phosphorylate target proteins to regulate the cell cycle. They control key transitions, such as G1 to S and G2 to M.

What is the role of phosphorylation in Cdk regulation? Explain using Cdc25 and Wee1 as examples.

Cdks are activated by cyclin binding and phosphorylation by CDK-activating kinase (CAK). Inhibitory phosphorylation by Wee1 and activation by dephosphorylation via Cdc25 modulate their activity.

Describe the role of cyclin-B1–Cdk1 in mitosis. How is its activity regulated by subcellular localization?

Cyclin-B1–Cdk1 is essential for the initiation of mitosis, promoting the breakdown of the nuclear envelope and chromatin condensation. Its activity is regulated by localization, being retained in the cytoplasm until the cell is ready for mitosis, where it then translocates to the nucleus. Cyclin-B1–Cdk1 is kept inactive in the cytoplasm through phosphorylation. Upon activation by Cdc25C and dephosphorylation, it translocates into the nucleus during prophase to initiate mitosis.

How did Paul Nurse’s work with Schizosaccharomyces pombe contribute to our understanding of Cdk function?

Paul Nurse's research with Schizosaccharomyces pombe revealed key insights into cell cycle regulation by identifying the cdc2 gene, which encodes a Cdk. His experiments demonstrated that Cdk activity is essential for cell cycle progression and that Cdk function is conserved across species. He identified CDK1 in fission yeast and demonstrated its central role in regulating cell cycle transitions through phosphorylation, fundamentally advancing our understanding of eukaryotic cell cycle control.

Explain the difference between Cdk activation through cyclin binding and its inhibition through CKIs. Use a named Cdk, such as CDK2, to illustrate

Cdk activation occurs when cyclins bind to Cdks like CDK2 ,when bound to Cyclin E, drives the G1/S transition leading to a conformational change that permits phosphorylation and activation. In contrast, cyclin-dependent kinase inhibitors (CKIs) bind to Cdks, preventing their activation by blocking cyclin binding or interfering with necessary phosphorylation, thus regulating the cell cycle progression. It can be inhibited by CKIs like p21, which binds the CDK2-Cyclin E complex, preventing substrate access.

What is a cyclin? Describe its relationship with Cdks.

A cyclin is a regulatory protein that controls the progression of the cell cycle by activating cyclin-dependent kinases (Cdks). Cyclins bind to Cdks at specific phases of the cell cycle, leading to a conformational change that activates the Cdk, allowing it to phosphorylate target substrates and drive cell cycle transitions.

Tim Hunt discovered cyclins in sea urchins. What was significant about their pattern of expression?

Tim Hunt discovered that cyclins exhibit a distinct pattern of synthesis and degradation during the cell cycle, with their levels rising and falling at specific phases. This cyclical expression pattern is crucial for the timely activation and inactivation of cyclin-dependent kinases, thereby controlling cell cycle progression.

Map the expression of cyclins (e.g., Cyclin D, E, A, B) to the cell cycle phases and describe the transitions they regulate.

Cyclin D is expressed in early G1 phase, promoting progression through G1; Cyclin E appears in late G1, facilitating the G1/S transition; Cyclin A is present in S phase, supporting DNA replication; Cyclin B is expressed in G2 and peaks during mitosis, driving the G2/M transition.

How does the degradation of cyclins contribute to unidirectional cell cycle progression?

Proteasome-mediated degradation ensures cyclins are removed after their function is complete, preventing backward progression or re-initiation of earlier phases. This unidirectional progression is crucial for maintaining the integrity of the cell cycle and ensuring that cells do not prematurely re-enter previous phases.

Why are cyclins considered key timing regulators of the cell cycle rather than constant activators?

Cyclins are considered key timing regulators because they are synthesized and degraded in a cyclical manner, leading to transient activation of cyclin-dependent kinases (CDKs) at specific points in the cell cycle. This ensures precise timing of cell cycle events rather than constant activation, which could disrupt normal progression.

Compare and contrast the positive and negative regulators of Cdk activity, including examples of both.

Positive regulators, like cyclins and CAK, bind to Cdk to activate their kinase activity, while negative regulators, such as CKIs (p21 and p27) and Wee1 kinase, inhibit Cdk activity by binding to the cyclin-Cdk complex, preventing progression through the cell cycle.

What is the role of phosphatases such as Cdc25C in cell cycle control? How is their activity modulated?

Phosphatases like Cdc25C activate cyclin-dependent kinases (CDKs) by removing inhibitory phosphate groups, thus promoting cell cycle progression. Their activity is regulated by various signals, including checkpoints and phosphorylation events that can either promote or inhibit their function.

Explain how CKIs such as p21 and p16 inhibit Cdks. What are the differences between the Ink4 and Cip/Kip families?

CKIs like p21 and p16 inhibit Cdks by binding to the cyclin-Cdk complex, preventing its activation and thus halting cell cycle progression. The Ink4 family specifically binds to Cdk4 and Cdk6, inhibiting their interaction with cyclins, while the Cip/Kip family can bind to multiple Cdks and can function as a competitive inhibitor or modulator of cyclin activity.

Discuss the checkpoint mechanisms that monitor DNA damage or incomplete replication. How do these influence cell cycle arrest?

Checkpoint mechanisms, such as the G1/S and G2/M checkpoints, monitor for DNA damage and incomplete replication. When damage is detected, proteins like p53 or ATM activate signalling pathways that lead to cell cycle arrest, allowing for repair before progression. If repair fails, apoptosis may be induced

Illustrate how proteolysis (e.g., through the ubiquitin-proteasome system) controls cell cycle transitions, focusing on the G1/S checkpoint.

Proteolysis, primarily through the ubiquitin-proteasome system, regulates cell cycle transitions by marking specific proteins for degradation. At the G1/S checkpoint, the degradation of inhibitors such as cyclin-dependent kinase inhibitors (CKIs) and the timely removal of cyclins are crucial for progression into S phase. E3 ubiquitin ligases (e.g., SCF, APC/C) target cyclins and CKIs for degradation. For example, degradation of p27 allows CDK2-Cyclin E to drive G1/S progression.

What are some laboratory techniques used to measure cell cycle progression? Describe at least two and how they work.

Laboratory techniques to measure cell cycle progression include flow cytometry and BrdU incorporation assays. Flow cytometry analyzes DNA content in cells to determine the distribution among cell cycle phases, while BrdU incorporation assays involve the use of a thymidine analog to label dividing cells, allowing detection of S phase activity.

How can flow cytometry be used to analyse cell cycle distribution? What markers or dyes are typically used?

Flow cytometry can be used to analyze cell cycle distribution by measuring the DNA content of individual cells. Commonly used markers include propidium iodide and DAPI, which stain DNA, allowing the identification of cells in different phases of the cell cycle based on their fluorescence intensity.

Describe how the expression levels of cyclins or Cdks can be monitored experimentally. Which methods are protein-specific?

The expression levels of cyclins or Cdks can be monitored experimentally using methods such as Western blotting and immunofluorescence. Western blotting detects specific proteins using antibodies, while immunofluorescence allows visualization of protein localization and abundance within cells. ELISA is another method that quantitatively measures protein levels using specific antibodies.

What is the role of immunofluorescence microscopy in visualizing cell cycle protein localization?

Immunofluorescence microscopy is used to visualize the localization of cell cycle proteins within cells by employing specific antibodies labeled with fluorescent dyes. This technique allows researchers to examine the spatial distribution and expression levels of proteins like cyclins and Cdks during different phases of the cell cycle.

How can synchronization techniques help study cell cycle traverse, and what are some common methods of cell synchronization?

Synchronization techniques help to align cells in the same phase of the cell cycle, facilitating the study of specific phases. Common methods of cell synchronization include serum starvation, chemical treatments, and the use of mitotic inhibitors.

What is the primary function of the p53 protein in the cell cycle?

The primary function of the p53 protein in the cell cycle is to act as a tumour suppressor that regulates the cell cycle, DNA repair, senescence, and prevents the proliferation of cells with damaged DNA. It does so by inducing cell cycle arrest, initiating DNA repair mechanisms, or triggering apoptosis if the damage is irreparable.

Explain how p53 functions as a “tumour suppressor.”

p53 functions as a tumor suppressor by controlling the cell cycle and ensuring the integrity of the genome. It activates the expression of genes involved in cell cycle arrest, DNA repair, and apoptosis, thus preventing the proliferation of potentially cancerous cells. It acts like a brake on uncontrolled cell proliferation

What is the structural form (oligomeric state) of functional p53, and why is this important?

Functional p53 exists primarily as a tetramer, which is crucial for its ability to bind DNA and regulate gene expression effectively. This oligomeric state allows p53 to achieve the necessary stability and conformation for its role as a tumor suppressor.

Describe the half-life of p53 and how its levels are regulated under normal conditions.

The half-life of p53 is relatively short, typically around 20 to 30 minutes under normal conditions. Its levels are regulated by MDM2, a protein that promotes the ubiquitination and proteasomal degradation of p53, keeping its concentration low to prevent unnecessary cell cycle arrest or apoptosis.

What role does Mdm2 play in regulating p53 stability and function?

Mdm2 is an E3 ubiquitin ligase that targets p53 for ubiquitination, leading to its degradation by the proteasome. By controlling p53 levels, Mdm2 helps to prevent excessive activation of p53's tumour suppressor functions under normal cellular conditions. inhibition of Mdm2 allows for p53 to accumulate.

How do missense mutations in p53 act as dominant negatives?

Missense mutations in p53 can produce proteins that interfere with the function of the wild-type p53, preventing it from binding to DNA and regulating target genes properly. This dominant negative effect impairs the tumor suppressor activity of p53 and can lead to tumorigenesis. Missense mutations in p53 can produce a defective protein that inhibits the function of the wild-type protein by incorporating into tetramers and rendering them inactive.

Explain the concept and significance of “loss of heterozygosity” in the context of p53 mutations.

Loss of heterozygosity refers to the loss of one allele at a gene locus in which the other allele was already mutated, leading to the complete inactivation of a tumor suppressor gene like p53. This phenomenon is significant in cancer biology as it can eliminate the protective effect of the normal p53 allele, promoting tumorigenesis.

Why is p53 described as the “guardian of the genome”?

p53 is described as the "guardian of the genome" because it plays a critical role in maintaining genomic stability by regulating the cell cycle, promoting DNA repair, and inducing apoptosis in response to cellular stress or DNA damage. Its function ensures that damaged cells do not proliferate, thus preventing tumor development.

What is the mechanism by which p53 activates transcription of target genes?

p53 binds to specific DNA sequences as a tetramer and activates transcription of target genes involved in cell cycle arrest (e.g., p21) and apoptosis (e.g., Bax, PUMA). This binding enhances RNA polymerase recruitment and initiates transcriptional activation, leading to the expression of genes that help restore cellular integrity and prevent tumor formation.

Which domains of p53 are critical for its transcriptional activity?

The transactivation domains, particularly TAD1 and TAD2, along with the DNA-binding domain, are critical for p53's transcriptional activity. These domains facilitate the binding of p53 to target gene promoters and the recruitment of transcriptional co-activators.

What are some examples of genes directly activated by p53, and what are their functions? (e.g., p21, Bax, PUMA)

Examples of genes directly activated by p53 include p21, which inhibits cyclin-dependent kinases to induce cell cycle arrest; Bax, which promotes apoptosis by releasing cytochrome c; and PUMA, which also promotes cell death in response to DNA damage. These genes are essential for maintaining cellular integrity and preventing cancer development.

How does p53 activation lead to either cell cycle arrest or apoptosis, depending on the extent of damage?

p53 activation leads to cell cycle arrest or apoptosis depending on the severity of cellular stress. If damage is mild (low DNA damage), p53 activates genes like p21 to halt the cell cycle for repair; if damage is extensive (high DNA damage), it promotes pro-apoptotic genes such as Bax and PUMA to trigger programmed cell death.

Explain how the balance between cell cycle arrest and apoptosis is influenced by other factors such as Myc or phosphorylation patterns.

The balance between cell cycle arrest and apoptosis is influenced by various factors, including the levels of oncogenes like Myc, which binds to p21 promoter and prevent its activation which can promote proliferation and suppress apoptosis, and phosphorylation patterns of p53 that can affect its stability and transcriptional activity. This interplay determines whether cells will repair themselves or undergo programmed cell death in response to stress.

What types of cellular stress or damage activate p53? (e.g., UV, IR, oxidative stress, oncogene activation)

Cellular stress or damage such as DNA damage from UV radiation, ionizing radiation (IR), oxidative stress, and oncogene activation activate p53. These stresses trigger a response that can lead to cell cycle arrest, DNA repair, or apoptosis.

Describe how phosphorylation activates p53 and blocks Mdm2-mediated degradation.

Phosphorylation of p53, typically by kinases in response to cellular stress, stabilizes the p53 protein and prevents its interaction with Mdm2, which is an E3 ubiquitin ligase that normally targets p53 for degradation. This results in increased p53 levels, enhancing its role in activating genes involved in cell cycle arrest and apoptosis.

What is the role of ATM in the activation of p53?

ATM (Ataxia Telangiectasia Mutated) is a key kinase that detects DNA damage and phosphorylates p53 at specific residues, enhancing its stability and transcriptional activity. This phosphorylation is crucial for p53's role in cell cycle regulation and apoptosis in response to genotoxic stress.

How does p53 activation influence the expression of p21, and what is p21’s role in the cell cycle?

p53 activates the transcription of p21, a CDK inhibitor that halts the cell cycle at G1/S checkpoint, allowing DNA repair and preventing the propagation of damaged DNA. This regulation is critical for maintaining genomic stability.

What are the mitochondrial proteins regulated by p53 that contribute to apoptosis, and how do they function? (e.g., Bax, Bad, Bak, Bcl2, PUMA, NOXA)

p53 regulates several mitochondrial proteins that promote apoptosis, including Bax, Bad, Bak, and PUMA while inhibiting anti-apoptotic proteins like Bcl-2. These proteins contribute to mitochondrial outer membrane permeabilization, leading to the release of cytochrome c and subsequent activation of caspases that initiate the apoptosis cascade.

Describe the formation and role of the apoptosome in p53-mediated apoptosis.

The apoptosome is a large protein complex formed in response to apoptotic signals, including those initiated by p53. It activates initiator caspases, leading to a cascade of events that ultimately result in programmed cell death, effectively eliminating damaged or unnecessary cells. Cytochrome C binds Apaf-1 to form the apoptosome, which activates caspase 9 and initiates a caspase cascade leading to apoptosis.

Why is p53 considered the most commonly mutated gene in human cancers?

p53 is considered the most commonly mutated gene in human cancers due to its critical role in regulating the cell cycle, DNA repair, and apoptosis. Mutations in p53 lead to loss of function, allowing cells with damaged DNA to proliferate, contributing to tumorigenesis. Its central role in controlling cell proliferation and death makes it a prime target for inactivation.

How does loss of p53 function lead to increased genomic instability?

Without p53, cells bypass checkpoints and accumulate mutations, leading to greater malignancy.This loss of function impairs DNA repair mechanisms and allows damaged cells to survive and divide, contributing to increased rates of genomic instability and tumor progression.

What are some early and late roles of p53 mutations in multistage carcinogenesis?

Early roles of p53 mutations in multistage carcinogenesis include disruption of normal cell cycle checkpoint regulation and enhancement of cell survival despite DNA damage. Late roles involve promotion of genomic instability and tumor evolution, contributing to the development of aggressive cancer phenotypes.

How do inherited (germ-line) mutations in p53 contribute to cancer predisposition (e.g., Li-Fraumeni syndrome)?

Inherited (germ-line) mutations in p53 contribute to cancer predisposition, such as in Li-Fraumeni syndrome, by resulting in a loss of p53 function, which is crucial for cell cycle regulation and DNA repair. This predisposition leads to an increased risk of developing various types of cancers at an early age, as these mutations promote uncontrolled cell proliferation and genomic instability.

How does the loss of p53 function relate to prognosis and treatment resistance in cancer patients?

Loss of p53 function is often associated with poor prognosis in cancer patients, as it contributes to treatment resistance by allowing tumors to evade apoptosis and maintain unchecked growth. This results in more aggressive cancer behaviors and complicates therapeutic strategies, making it harder to achieve effective responses to treatment.

List and describe the different mechanisms by which tumour cells can evade p53 regulation.

Interaction with viral proteins

Overexpression of Mdm2

Nuclear exclusion

Inactivation of ARF

Viral protein interaction (e.g., HPV E6 in cervical cancer), nuclear exclusion (neuroblastoma), Mdm2 overexpression (sarcoma), inactivation of ARF and altered feedback loops can all disrupt p53's regulatory functions, leading to compromised cell cycle control and increased tumorigenesis.

What are the consequences of p53 loss on G1/S checkpoint control?

The consequences of p53 loss on G1/S checkpoint control include the failure to halt the cell cycle in response to DNA damage, resulting in unregulated progression through the cell cycle, allowing replication of damaged DNA. This can lead to increased mutation rates, genomic instability, and ultimately contribute to tumorigenesis.

Why do tumour cells under selective pressure evolve to inactivate p53 or its pathway components?

Tumour cells under selective pressure evolve to inactivate p53 or its pathway components to enhance survival and proliferation despite the presence of DNA damage or stress, allowing them to bypass growth restrictions and maintain aggressive behavior. This adaptation facilitates resistance to therapies aimed at inducing cell death.

What are alternative mechanisms (other than direct mutation) that inhibit p53 signalling in tumours?

Alternative mechanisms that inhibit p53 signalling in tumours include the overexpression of inhibitors like Mdm2, the loss of ARF, and aberrant regulation of upstream signalling pathways such as the PI3K/AKT pathway. Additionally, interactions with viral oncoproteins can also compromise p53 function.

How does the mutation of upstream regulators like ATM or CHK2 affect p53 pathway integrity?

The mutation of upstream regulators like ATM or CHK2 disrupts p53 pathway integrity by impairing the activation of p53 in response to DNA damage. This can lead to diminished p53 activity, resulting in insufficient cell cycle arrest and increased risk of genomic instability.

What are current therapeutic strategies targeting p53 in cancer treatment?

Current therapeutic strategies targeting p53 in cancer treatment include reactivating mutant p53 using small molecules, gene therapy to restore p53 function, and utilizing compounds that increase the levels of wild-type p53 or enhance its activity. Other approaches involve the use of drugs that inhibit Mdm2 or MdmX, which are negative regulators of p53.

Describe the concept of “super p53” and its implications in cancer resistance.

"Super p53" refers to genetically engineered forms of the p53 protein designed to enhance its tumor-suppressing functions, making cancer cells more susceptible to therapies. Its implications in cancer resistance include preventing tumor progression and improving patient outcomes by restoring the function of p53 in malignancies where it is mutated or dysfunctional.

What are the mechanisms of p53 gene therapy (e.g., Gendicine), and how is it applied clinically?

Mechanisms of p53 gene therapy, such as Gendicine, involve delivering a functional copy of the p53 gene to tumor cells, leading to restored p53 activity. Clinically, it is applied to treat various cancers by inducing apoptosis in cancer cells and enhancing the effects of other therapies.

How do p53-based oncolytic viruses (e.g., Onyx015) work selectively in cancer cells?

p53-based oncolytic viruses, such as Onyx015, are engineered to selectively infect and replicate in cancer cells with mutated or absent p53 while sparing normal cells. They exploit the defective apoptotic pathways in cancer cells to induce cell death and boost the immune response against tumors.

Why have p53 vaccines not yet progressed to phase III clinical trials?

p53 vaccines have not yet progressed to phase III clinical trials primarily due to challenges in achieving robust and consistent immune responses, along with difficulties in patient selection and defining appropriate endpoints for efficacy. Additionally, there have been concerns about the safety profiles of these vaccines and the variability in p53 mutations among different cancers, which complicates the development of universally effective vaccines.

What are telomeres and where are they found in the genome?

Telomeres are repetitive DNA sequences located at the ends of linear chromosomes, protecting them from degradation and preventing them from fusing with other chromosomes. They play a crucial role in cellular aging and stability.

What DNA sequence is repeated in human telomeres, and what enzyme uses this sequence as a substrate?

Human telomeres consist of repetitive sequences of TTAGGG, and the enzyme telomerase uses this sequence as a substrate to extend telomeres.

Explain the protective roles of telomeres at chromosome ends. Why are these important?

Telomeres protect chromosome ends from degradation and prevent the chromosomes from fusing with each other. This protection is crucial for maintaining genomic stability and preventing cancerous transformations.

Also stops ends from activating genome damage checkpoints and ensures accurate replication during cell division.

Prevents loss of sequence by exonuclease attack and helps maintain chromosome integrity by preventing deterioration from the cell's replication process. This is essential for preserving genetic information and preventing cellular aging.

What is the role of telomere-binding proteins?

Telomere-binding proteins are essential for protecting telomeres, regulating telomere length, and facilitating the assembly of the telomere complex. They help maintain chromosomal integrity and prevent DNA damage response activation at the chromosome ends. These proteins stabilize telomeres and assist in the recruitment of telomerase and other proteins necessary for telomere maintenance.

Describe the T-loop structure and the role of TRF2 in its formation.

The T-loop structure is a protective configuration formed at the end of telomeres, created when the single-stranded overhang of DNA folds back and invaginates into the double-stranded region. TRF2 (Telomeric Repeat Binding Factor 2) is crucial in facilitating the formation of the T-loop, helping to stabilize telomeres and protect them from being recognized as DNA damage. The single-strand G-rich extension is tucked back into the double-stranded region of the telomere to form a protective loop structure.

What is the “end replication problem” and how does it contribute to telomere shortening?

The "end replication problem" refers to the inability of DNA polymerase to fully replicate the ends of linear chromosomes during DNA replication, leading to progressive shortening of telomeres with each cell division. This shortening can eventually trigger cellular aging and limit the number of times a cell can divide.

The end-replication problem arises because leading strand synthesis fails to reproduce the last part of the telomere, leaving a blunt leading-end telomere without it characteristic and crucial 3' overhang. Telomerase solves this problem by adding single-stranded TTAGGG repeats to the telomere end

Why does DNA replication machinery fail to fully replicate chromosome ends?

DNA replication machinery lacks the ability to synthesize the 3' ends of linear chromosomes, resulting in incomplete replication of chromosome ends during cell division.

What happens to telomere length during successive rounds of cell division in the absence of telomerase?

Telomere length progressively shortens with each cell division due to incomplete replication of the chromosome ends. This leads to eventual cellular senescence or apoptosis when telomeres become critically short.

What is telomerase and how does it function to extend telomeres?

Telomerase is an enzyme that adds repetitive nucleotide sequences, specifically TTAGGG, to the ends of telomeres, counteracting telomere shortening that occurs during DNA replication. This extension allows cells to divide more times without undergoing senescence.

What components make up telomerase, and what is the specific role of hTERT?

Telomerase consists of a protein component called hTERT (human telomerase reverse transcriptase) and an RNA component that serves as a template for adding DNA repeats. hTERT is crucial for the enzyme's catalytic activity, allowing it to extend telomeres.

What is the Alternative Lengthening of Telomeres (ALT) pathway and how does it differ from telomerase action?

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent mechanism that some cells use to maintain telomere length through homologous recombination and other DNA repair processes. Unlike telomerase, which directly adds repetitive sequences to telomeres, the ALT pathway relies on the exchange of genetic material between telomeres to preserve their length.

In what types of cells is ALT typically observed?

The ALT pathway is typically observed in certain types of cancer cells, particularly those that are deficient in telomerase activity. Such cells include sarcomas, gliomas, and some breast cancers.

Is telomerase expressed in all human cells? Which cell types typically express telomerase?

Telomerase is not expressed in all human cells; it is typically expressed in stem cells, germ cells, and certain cancer cells, allowing them to maintain telomere length and proliferate.

What happens to telomere length when telomerase is artificially introduced into normal human fibroblasts?

When telomerase is artificially introduced into normal human fibroblasts, telomere length increases, leading to extended cellular lifespan and enhanced proliferation, effectively bypassing normal cellular senescence.

What percentage of human malignancies show upregulated telomerase expression?

Approximately 85-90% of human malignancies show upregulated telomerase expression, which contributes to their immortality and unchecked growth.

What is replicative senescence, and how is it related to telomere length?

Replicative senescence is the process by which normal somatic cells cease to divide after a certain number of divisions, primarily due to telomere shortening. This phenomenon is closely related to telomere length, as critically short telomeres trigger cellular aging and ultimately lead to cell cycle arrest.

How do telomeres signal for a DNA damage response when they become critically short?

Telomeres signal for a DNA damage response by activating pathways such as the p53 and ATM/ATR signaling cascades when they reach a critically short length. This response leads to cellular senescence or apoptosis, thereby preventing the propagation of damaged cells.

What is the Hayflick limit?

The Hayflick limit is the maximum number of times a normal somatic cell can divide before cell division stops, typically around 40 to 60 divisions, due to telomere shortening.

How does telomere shortening limit the proliferative capacity of cells?

Telomere shortening limits the proliferative capacity of cells by progressively reducing the length of telomeres with each cell division. When telomeres become critically short, they activate DNA damage response pathways that induce cellular senescence or apoptosis, thus inhibiting further cell division.

Why is replicative senescence considered a tumour suppressive mechanism?

Replicative senescence is considered a tumour suppressive mechanism because it prevents damaged or dysfunctional cells from dividing further, thereby reducing the potential for tumorigenesis and maintaining genomic stability.

What is the relationship between telomere shortening and the activation of p53?

Telomere shortening leads to the activation of the tumor suppressor protein p53, which responds to DNA damage caused by critically short telomeres. This activation triggers cell cycle arrest and promotes replicative senescence or apoptosis, preventing the proliferation of potentially cancerous cells.

What happens if a cell continues to divide despite having critically short telomeres?

If a cell continues to divide despite having critically short telomeres, it can lead to genomic instability, resulting in chromosomal alterations and potential oncogenic transformations. This uncontrolled division may increase the risk of tumor formation.

How can the loss of checkpoint function (e.g., p53) in the context of telomere shortening lead to genomic instability?

The loss of checkpoint function, such as p53, allows cells to bypass critical cell cycle controls in the presence of short telomeres. This results in continued division despite DNA damage, leading to an accumulation of genomic alterations and increasing the risk of tumorigenesis.

What types of chromosomal abnormalities can arise from dysfunctional telomeres?

Dysfunctional telomeres can lead to chromosomal abnormalities such as chromosome fusions, deletions, translocations, and aneuploidy. These alterations can disrupt normal cellular processes and contribute to oncogenesis.

Why is telomere erosion considered a barrier to tumour development?

Telomere erosion is considered a barrier to tumor development because it limits the number of times a cell can divide, thereby preventing the accumulation of mutations and maintaining genomic stability. When telomeres shorten excessively, cells undergo senescence or apoptosis, blocking the progression of cancer.

How does telomerase enable cancer cells to become immortal?

Telomerase extends the length of telomeres, preventing their erosion during cell division. By maintaining telomere length, cancer cells are able to bypass the normal limits on cell division, allowing them to proliferate indefinitely and contribute to tumor growth.

Why might telomerase be a promising target for cancer therapy?

Telomerase is often upregulated in cancer cells, allowing them to maintain telomere length and avoid senescence. Targeting telomerase could limit cancer cell proliferation and promote their death.

What is the clinical consequence of extensive telomere erosion observed in early tumour progression?

Extensive telomere erosion in early tumor progression can lead to increased genomic instability, promoting the development of malignant cells and contributing to tumorigenesis.

what is senescence?

A cellular state where cells lose the capacity to divide and grow, often as a response to stress or damage, preventing the proliferation of potentially harmful cells.

Describe how growth factors interact with receptor tyrosine kinases (RTKs) to initiate intracellular signalling. Use EGF and EGFR as a model

Growth factors such as EGF (epidermal growth factor) bind to the extracellular domain of RTKs. This induces receptor dimerization (or oligomerization) and activates the intrinsic tyrosine kinase domain. Activation leads to autophosphorylation of specific tyrosine residues on the intracellular domain, creating docking sites for downstream signalling proteins. These phosphorylated tyrosines recruit SH2 domain-containing proteins that initiate downstream signalling cascades such as the Ras-MAPK pathway or PI3K-Akt pathway.

Explain the structural changes that occur upon growth factor binding to its receptor that allow signal transduction

Ligand binding to RTKs causes conformational changes that lead to receptor dimerization and activation of the kinase domain. This results in transphosphorylation of tyrosine residues on adjacent receptors, creating a platform for recruitment of signalling complexes via phosphotyrosine-binding domains.

What is autocrine signalling, and how does it differ from paracrine signalling? Provide examples of how cancer cells exploit autocrine signalling

Autocrine signalling occurs when a cell produces a growth factor that it also responds to. Many cancer cells exploit this mechanism to sustain proliferation independent of external signals. For example, glioblastoma cells secrete PDGF and express PDGF receptors, establishing a self-stimulating growth loop. In contrast, paracrine signalling involves the release of growth factors that affect nearby cells, rather than the secreting cell itself. This difference allows cancer cells to foster an environment promoting tumor growth and survival.

Discuss the enzymatic function of Src and how its tyrosine kinase activity contributes to cellular transformation

Src is a non-receptor tyrosine kinase that plays a crucial role in cellular signaling pathways. Its enzymatic function includes phosphorylation of tyrosine residues on target proteins, which drives processes like proliferation, survival, and migration, often leading to cellular transformation in cancer. It phosphorylates tyrosine residues on multiple substrates, modulating functions like growth, differentiation, and migration. Mutant v-Src lacks regulatory control and is constitutively active, promoting transformation via uncontrolled phosphorylation of signalling proteins.

Explain the cycle of tyrosine phosphorylation and how it regulates signalling dynamics, including the opposing roles of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs)

Tyrosine phosphorylation is a reversible post-translational modification that regulates protein function and cellular signaling dynamics. Protein tyrosine kinases (PTKs) add phosphate groups to tyrosine residues, activating or enhancing the function of target proteins, while protein tyrosine phosphatases (PTPs) remove these phosphates, effectively turning off or inhibiting the signaling pathways, allowing precise control over cellular responses. This cycle ensures that signalling is reversible, tightly controlled, and appropriate in duration and intensity.This dynamic interplay between PTKs and PTPs is essential for maintaining cellular homeostasis and regulating processes such as cell growth, differentiation, and apoptosis.

What is transphosphorylation, and why is it critical for receptor activation and downstream signalling?

Transphosphorylation occurs when one receptor in a dimer phosphorylates its partner on tyrosine residues. This process is essential for activating RTKs and creating binding sites for downstream adaptor proteins such as Grb2.

What is the SH2 domain, and how does it contribute to specificity in signalling cascades?

The SH2 domain is a protein domain that binds specifically to phosphorylated tyrosine residues on target proteins. This interaction enables proteins containing SH2 domains to selectively recognize and interact with activated signaling molecules, thereby enhancing specificity and propagation of cellular signaling cascades. SH2 domains are ~100 amino acid motifs that bind phosphorylated tyrosines within specific amino acid contexts. They are found in proteins like Src, Grb2, and PLCγ, facilitating specific recruitment to activated receptors and ensuring precise signal propagation.

Describe how PH (pleckstrin homology) domains facilitate the localisation of signalling proteins to the membrane. What lipid molecules do they bind?

PH domains are protein domains that bind specifically to phosphoinositides, a subset of lipid molecules found in the cell membrane. This binding enables the localization of signaling proteins to the membrane, where they can interact with other signaling components and propagate cellular signals. Like bringing Akt/PKB into proximity with their activators or substrates, a critical step in propagating membrane-based signals.

How do modular domains like SH2 and PH influence signal propagation and pathway insulation in the cell?

Modular domains like SH2 and PH influence signal propagation by providing specificity and localization to signaling proteins. SH2 domains enable selective interactions with phosphorylated tyrosines, while PH domains anchor proteins to membranes through binding to phosphoinositides, ensuring that signals are correctly transmitted and pathways are insulated from non-specific interactions. Modular domains provide selectivity and spatial organization in signalling pathways. For instance, SH3 domains recognize proline-rich motifs, while SH2 domains recognize phosphotyrosines. This modularity allows rapid, reversible, and specific assembly of signalling complexes.

Describe the Ras activation cycle. How is Ras activated by growth factor receptors?

Ras is inactive when bound to GDP. Upon receptor activation, adaptor proteins like Grb2 recruit SOS (a GEF), which promotes GDP-GTP exchange on Ras. Active Ras-GTP triggers downstream pathways (e.g., MAPK cascade). Inactivation occurs via intrinsic GTPase activity, accelerated by GAPs. This cycle involves the conversion of Ras from its inactive GDP-bound form to the active GTP-bound state, initiated by growth factor receptor signaling.

How does the Grb2-SOS-Ras axis link activated RTKs to downstream signalling pathways?

The Grb2-SOS-Ras axis connects activated receptor tyrosine kinases (RTKs) to downstream signaling pathways by recruiting the guanine nucleotide exchange factor (GEF) SOS to RTKs. This interaction facilitates the activation of Ras by promoting the exchange of GDP for GTP, leading to the initiation of pathways such as the MAPK cascade.

Discuss how mutations in Ras can lead to sustained signalling and contribute to oncogenesis

Mutations in Ras can lead to sustained signaling by impairing its intrinsic GTPase activity, causing Ras to remain in its active GTP-bound form for extended periods. This prolonged activation promotes continuous cell proliferation and survival signals, contributing to oncogenesis by driving uncontrolled cellular growth and tumor formation. These mutations often occur at key residues, such as G12, G13, or Q61, and prevent hydrolysis of GTP, leading to persistent signaling through downstream effectors.