2024_CancerBiolIntro_2_Penengo

Page 1: Introduction

• Introduction to Cancer Biology

• Lorenza Penengo, contact: penengo@imcr.uzh.ch

• Institute of Molecular Cancer Research, University of Zurich

• Course: BME236, Biomedicine I – Fall 2024

Slide Title: The Updated Hallmarks of Cancer 2011 (Slide 2)

Key Points:

1. Cancer cells exhibit six core hallmarks:

   • Sustaining proliferative signaling: Continuous growth signals.

   • Evading growth suppressors: Ignoring signals that inhibit growth.

   • Resisting cell death: Avoiding programmed cell death (apoptosis).

   • Enabling replicative immortality: Unlimited cell division potential.

   • Inducing angiogenesis: Forming new blood vessels for oxygen and nutrients.

   • Activating invasion and metastasis: Spreading to other tissues.

2. Enabling characteristics facilitate these hallmarks:

   • Genomic instability and mutation: Accumulation of mutations drives cancer progression.

   • Tumor-promoting inflammation: Chronic inflammation supports tumor growth.

Explanation of Visuals:

• The central diagram highlights the hallmarks and enabling characteristics, showing how they interact to sustain cancer development.

Glossary:

• Angiogenesis: Formation of new blood vessels to supply growing tumors.

• Metastasis: Spread of cancer cells to distant tissues.

• Genomic instability: Increased mutation rate in cancer cells.

Key Takeaway: Cancer cells acquire specific traits (hallmarks) and enabling characteristics that allow them to grow, survive, and spread aggressively.

Slide Title: Is the Cancer Phenotype Dominant or Recessive? (Slide 3)

Key Points:

1. Experiment: Normal cells fused with tumor cells:

   • Hybrid cells were non-tumorigenic, indicating that the normal phenotype is dominant over the tumor phenotype.

   • Inactivation of tumor suppressor genes (TSGs) results in tumor formation.

2. Tumor suppressor genes must be inactive in both copies for cancer to develop, as one functioning copy prevents tumorigenesis.

Explanation of Visuals:

• Diagrams illustrate the cell fusion experiment and the role of TSGs. Hybrid cells derived from tumor and normal cells lost tumorigenic potential.

• Microscopic images show hybrid cells with no tumor growth, reinforcing the dominance of normal cell characteristics.

Glossary:

• Tumor Suppressor Genes (TSGs): Genes that inhibit cell growth and prevent tumors.

• Dominant phenotype: The trait that prevails when two cell types are combined.

Key Takeaway: The normal cell phenotype is dominant over the tumor phenotype, as tumor suppressor genes must be inactivated in both alleles for cancer to occur.

Slide Title: Is the Cancer Phenotype Dominant or Recessive? (Slide 4)

Key Points:

1. Recessive behavior of tumor suppressor genes (TSGs):

   • TSGs are recessive, meaning that both alleles must be mutated or inactivated to drive cancer.

   • The presence of one normal allele prevents cancer.

2. Practical implications:

   • Easier to inactivate TSGs than to overactivate oncogenes (e.g., Ras).

   • Diploid genomes (two copies of each gene) reduce the likelihood of complete TSG inactivation, making cancer less probable.

Explanation of Visuals:

• The diagram compares normal and cancerous cells. It explains how one functional TSG allele can suppress tumor development.

Glossary:

• Diploid genome: Cells containing two copies of each chromosome, one from each parent.

• Oncogenes: Genes that promote cell division and can lead to cancer when overactive.

Key Takeaway: Tumor suppressor genes exhibit a recessive phenotype, requiring both copies to be mutated for cancer to occur.

Slide Title: pRb – The First Discovered Tumor Suppressor (Slide 5)

Key Points:

1. pRb (Retinoblastoma protein):

   • The first identified tumor suppressor gene.

   • Mutations in the RB1 gene lead to retinoblastoma, a rare childhood eye cancer.

2. Incidence:

   • Retinoblastoma occurs in 1 in 20,000 children.

   • In hereditary cases, one allele is inherited as mutated, increasing susceptibility to "second hits."

Explanation of Visuals:

• Images of retinoblastoma highlight the clinical manifestation.

• A graph shows the likelihood of cancer development in hereditary vs. sporadic cases.

Glossary:

• pRb (Retinoblastoma protein): A protein that regulates cell cycle progression and prevents uncontrolled cell division.

• Second hit: A second mutation that inactivates the remaining functional allele, leading to cancer.

Key Takeaway: pRb, the first tumor suppressor discovered, demonstrates the "two-hit hypothesis," where both gene copies must be inactivated for cancer to occur.

Slide Title: pRb – The First Discovered Tumor Suppressor (Slide 6)

Key Points:

1. Mutation events:

   • The probability of two independent mutations ("two hits") in the same cell is extremely low, making hereditary predisposition (first hit inherited) significant.

2. Hereditary retinoblastoma:

   • Inherited mutations in one RB1 allele dramatically increase the likelihood of the "second hit" and cancer development.

   • Sporadic cases require mutations in both alleles, which is less common.

Explanation of Visuals:

• Detailed diagrams depict the "two-hit hypothesis," explaining the difference between hereditary and sporadic retinoblastoma cases.

Glossary:

• Hereditary predisposition: A genetic mutation inherited from a parent that increases cancer risk.

• Two-hit hypothesis: Both copies of a tumor suppressor gene must be inactivated for cancer to develop.

Key Takeaway: Hereditary mutations in the RB1 gene significantly increase the risk of retinoblastoma by predisposing individuals to a "second hit," supporting the two-hit hypothesis of tumorigenesis.

Slide 7: Loss of Heterozygosity (LOH) in Tumor Suppressors

Key Points:

• Loss of heterozygosity (LOH) refers to the loss of the normal (wild-type) allele of a tumor suppressor gene, which can lead to cancer progression.

• LOH can occur through three major mechanisms:

  A. Mitotic Recombination:

  • During the S phase of chromosome replication, homologous recombination can occur.

  • In the G2 and M phases of the cell cycle, recombination between homologous chromosomes can result in one daughter cell losing the wild-type allele entirely.

  • This leads to either retention or loss of heterozygosity in daughter cells.

  B. Gene Conversion:

  • DNA polymerase can switch templates between homologous chromosomes while copying DNA.

  • This can result in one chromosome acquiring genetic information from the homologous chromosome, replacing the wild-type allele with a mutated version, leading to loss of heterozygosity.

  C. Defects in Chromosomal Segregation:

  • Errors such as non-disjunction during mitosis can result in an unequal distribution of chromosomes.

  • The extra chromosome with the wild-type allele may later be lost, leaving only the mutant allele and resulting in LOH.

• Historical Significance:

  • LOH was first identified with the retinoblastoma (RB) gene, and its discovery helped in mapping other tumor suppressor genes.

Glossary:

• Loss of Heterozygosity (LOH): The loss of the normal copy of a gene in a heterozygous cell, leaving only the mutated copy, which can contribute to cancer development.

• Mitotic Recombination: A process during cell division where sister chromatids exchange genetic material, potentially leading to LOH.

• Gene Conversion: A mechanism where DNA polymerase copies genetic information from one chromosome to another, potentially replacing a functional gene with a mutated one.

• Non-Disjunction: A chromosomal segregation error during cell division that results in an uneven distribution of chromosomes to daughter cells.

• Tumor Suppressor Genes: Genes that regulate cell division and prevent uncontrolled growth; their inactivation can lead to cancer.

Key Takeaway:

Loss of heterozygosity is a critical event in cancer development and can arise through recombination, gene conversion, or chromosomal segregation defects, leading to the inactivation of tumor suppressor genes.

Slide 8: The Cell Division Cycle

Key Points:

• The cell cycle is a series of events that cells go through to grow and divide into two daughter cells. It consists of distinct phases:

  • G0 (Resting Phase):

    • Cells are in a quiescent state and do not actively divide.

    • Cells can re-enter the cycle if needed.

  • G1 (Gap 1 Phase):

    • The cell grows and synthesizes proteins necessary for DNA replication.

    • A decision window allows the cell to either continue the cycle or exit to G0.

  • S (Synthesis Phase):

    • DNA is replicated, ensuring that each daughter cell will have an identical set of chromosomes.

    • Centriole duplication occurs to prepare for cell division.

    • The S phase entry commitment point determines progression to this phase.

  • G2 (Gap 2 Phase):

    • The cell continues to grow and prepares for mitosis by producing organelles and proteins.

    • The mitotic entry commitment point ensures readiness for mitosis.

  • M (Mitosis Phase):

    • The cell divides its duplicated DNA into two identical sets through stages:

      • Prophase: Chromosomes condense, and the mitotic spindle forms.

      • Metaphase: Chromosomes align at the center of the cell.

      • Anaphase: Sister chromatids separate.

      • Cytokinesis: Division of the cytoplasm, resulting in two separate daughter cells.

    • The mitotic exit commitment point ensures proper segregation of cellular content before completing division.

Glossary:

• Cell Cycle: The ordered sequence of events that leads to cell division and replication.

• G0 Phase: A resting phase where the cell is metabolically active but not dividing.

• G1 Phase: The first gap phase where the cell grows and prepares for DNA synthesis.

• S Phase: The synthesis phase where the cell duplicates its DNA.

• G2 Phase: The second gap phase where the cell prepares for mitosis.

• M Phase: The mitotic phase where the cell divides its genetic material into two daughter cells.

• Cytokinesis: The physical process of cell division that follows mitosis.

• Commitment Points: Critical checkpoints in the cell cycle that ensure conditions are favorable to proceed to the next phase.

• Mitotic Spindle: A structure composed of microtubules that ensures the proper distribution of chromosomes during mitosis.

Key Takeaway:

The cell cycle is a highly regulated process ensuring accurate DNA replication and division into two identical daughter cells. Each phase has critical checkpoints that maintain genomic integrity and prevent uncontrolled proliferation.

Slide 9: The Cell Cycle Clock – Cyclins and CDKs

Key Points:

1. Cyclins and CDKs:

   • Cyclins are proteins that control cell cycle progression by binding to cyclin-dependent kinases (CDKs).

   • CDKs regulate specific phases of the cell cycle via phosphorylation of target proteins.

2. Cyclin-CDK regulation:

   • Cyclins are synthesized and degraded in a tightly controlled manner to ensure proper timing of cell cycle events.

   • Checkpoint signals can inhibit cyclin-CDK activity if errors are detected.

Explanation of Visuals:

• Flow diagram: Depicts cyclin-CDK interactions at different phases of the cell cycle.

• Graphs: Show fluctuations in cyclin levels during the cell cycle, correlating with their activity.

Glossary:

• Cyclin: Regulatory protein that activates CDKs to progress through the cell cycle.

• CDK (Cyclin-dependent kinase): Enzyme that phosphorylates target proteins to drive cell cycle transitions.

Key Takeaway:
Cyclins and CDKs orchestrate cell cycle progression, with their activity tightly regulated to ensure proper division and prevent errors.

Slide 10: The Cell Cycle Clock – Cyclins and CDKs (Detailed View)

Key Points:

• Cyclins and Cyclin-Dependent Kinases (CDKs):

  • The cell cycle is regulated by cyclins and their associated cyclin-dependent kinases (CDKs), which ensure the correct progression through different phases of the cell cycle.

  • Specific cyclins pair with CDKs to regulate key checkpoints in the cycle:

    • Cyclin D with CDK4/6 - regulates the transition from G1 to S phase.

    • Cyclin E with CDK2 - facilitates the S phase entry.

    • Cyclin A with CDK2 - drives the S phase and transition to G2.

    • Cyclin A and Cyclin B with CDK1 - control G2 to M phase transition.

• Cell Cycle Phases and Cyclin Levels:

  • Cyclin levels fluctuate throughout the cycle, controlling transitions between phases:

    • Cyclin D accumulates during G1 phase and prepares cells for DNA replication.

    • Cyclin E peaks at the G1/S transition to initiate DNA synthesis.

    • Cyclin A regulates the S phase and early G2 phase.

    • Cyclin B accumulates during G2 phase and controls mitosis initiation.

• Restriction (R) Point:

  • The R point in G1 phase represents a critical checkpoint where the cell commits to division based on favorable internal and external conditions.

Explanation of Visuals:

• Upper Diagram (Pairing of Cyclins with CDKs):

  • Shows the specific pairings of cyclins (D, E, A, B) with their respective CDKs and how they regulate different cell cycle phases.

• Lower Graph (Fluctuation of Cyclin Levels):

  • Depicts how the levels of cyclins fluctuate throughout the cell cycle, showing their peak activity in specific phases.

Glossary:

• Cyclins: Proteins that regulate the cell cycle by binding to CDKs and activating them at specific phases.

• CDKs (Cyclin-Dependent Kinases): Enzymes that, when activated by cyclins, phosphorylate target proteins to drive cell cycle progression.

• Restriction (R) Point: A critical checkpoint in G1 where cells decide to continue the cycle or enter a resting state (G0).

• Mitosis (M phase): The phase where the cell divides its genetic material into two daughter cells.

• G1 Phase: The first gap phase, where the cell grows and prepares for DNA synthesis.

• S Phase: The synthesis phase, during which DNA is replicated.

• G2 Phase: The second gap phase, where the cell prepares for mitosis.

Key Takeaway:

The cell cycle is tightly regulated by specific cyclin-CDK complexes that control phase transitions, ensuring accurate cell division and genomic integrity.

Slide 11: The Control of Cell Cycle and Proliferation: pRb

Key Points:

• Role of pRb in Cell Cycle Regulation:

  • The retinoblastoma protein (pRb) is a crucial regulator that controls cell cycle progression by inhibiting the transcription factor E2F.

  • In its unphosphorylated state, pRb binds to E2F, preventing it from activating genes required for DNA replication.

  • Upon receiving growth signals, pRb becomes phosphorylated by CDKs (cyclin-dependent kinases), which releases E2F and allows transcription of genes necessary for cell cycle progression.

• Phosphorylation States of pRb:

  • Unphosphorylated pRb (early G1 phase): Binds E2F, blocking transcription.

  • Hypophosphorylated pRb (late G1 phase): CDK4/cyclin D initiates partial phosphorylation, weakening E2F binding.

  • Hyperphosphorylated pRb (G1/S transition): CDK2/cyclin E completes phosphorylation, fully releasing E2F to drive S phase entry.

• The R Point (Restriction Point):

  • A critical checkpoint in the late G1 phase where the cell decides to proceed with division or exit into G0 (quiescence).

  • If conditions are favorable, pRb is inactivated, and the cell progresses to the S phase.

Explanation of Visuals:

• Top Left Diagram:

  • Depicts the interaction between pRb and E2F, showing how phosphorylation of pRb releases E2F to promote gene transcription.

• Top Right Diagram:

  • Illustrates the stepwise phosphorylation of pRb by CDK4/cyclin D and CDK2/cyclin E, leading to the release of E2F.

• Bottom Diagram:

  • Shows the timeline of pRb phosphorylation across the G1/S phases, emphasizing the R point and cell cycle progression.

Glossary:

• pRb (Retinoblastoma Protein): A tumor suppressor protein that regulates the cell cycle by inhibiting E2F transcription factors.

• E2F: A family of transcription factors responsible for activating genes required for DNA synthesis.

• Cyclin-Dependent Kinases (CDKs): Enzymes that regulate the cell cycle through phosphorylation of target proteins.

• Cyclin D/CDK4: Complex that initiates pRb phosphorylation in early G1 phase.

• Cyclin E/CDK2: Complex that completes pRb phosphorylation at the G1/S transition.

• Restriction (R) Point: A key checkpoint in G1 where the cell commits to division based on external signals.

Key Takeaway:

pRb acts as a gatekeeper of the cell cycle by controlling the activity of E2F transcription factors. Its inactivation via phosphorylation is crucial for cell cycle progression, and defects in this regulation can lead to uncontrolled cell proliferation and cancer development.

Slide 12: CDC25 Phosphatases Promote Cell-Cycle Progression

Key Points:

1. Role of CDC25 Phosphatases:

   • CDC25 phosphatases activate cyclin-dependent kinases (CDKs) by removing inhibitory phosphate groups, enabling cell cycle progression.

   • They are essential for transitions between different cell cycle phases, especially the G2/M checkpoint.

2. Mechanism:

   • CDC25 activates CDK1, which drives cells into mitosis (M phase).

   • These phosphatases are regulated by signals ensuring the timing and accuracy of cell division.

3. Clinical relevance:

   • Overexpression or dysregulation of CDC25 phosphatases is linked to cancer, as it can lead to uncontrolled cell proliferation.

Explanation of Visuals:

• Circular diagram: Shows the role of CDC25 in activating CDK1 and other CDKs, highlighting its impact on specific checkpoints (e.g., G2/M).

• Pathway flowchart: Explains the molecular interactions where CDC25 removes inhibitory phosphates to activate CDKs.

Glossary:

• Phosphatase: An enzyme that removes phosphate groups from proteins.

• CDKs (Cyclin-dependent kinases): Enzymes that regulate cell cycle transitions.

• G2/M checkpoint: Ensures cells are ready to enter mitosis.

Key Takeaway:
CDC25 phosphatases are crucial regulators of cell cycle transitions, particularly the G2/M phase, and their dysregulation is associated with cancer progression.

Slide 13: The Cell Cycle: CDK Inhibitors (CKIs)

Key Points:

1. CDK inhibitors:

   • CKIs are proteins that bind to CDKs, inhibiting their activity and halting cell cycle progression.

   • They ensure cells do not divide under unfavorable conditions, such as DNA damage or lack of growth signals.

2. Two classes of CKIs:

   • INK4 family: Inhibits CDK4/6, preventing progression through the G1 phase.

   • CIP/KIP family: Inhibits multiple CDKs (e.g., CDK2, CDK1), blocking G1/S and G2/M transitions.

Explanation of Visuals:

• Flowchart: Shows how CKIs act on specific CDKs to stop the cell cycle at key checkpoints.

• Stimuli chart: Highlights how DNA damage, growth factors, and stress signals regulate CKI expression.

Glossary:

• CKIs (CDK inhibitors): Proteins that suppress CDK activity to regulate the cell cycle.

• DNA damage response: A cellular mechanism that halts the cell cycle to repair DNA.

Key Takeaway:
CDK inhibitors are essential for halting the cell cycle under stress or damage, ensuring genomic integrity and proper cell regulation.

📌 Think of it this way:

• Cyclins = The ON switch for CDKs (they "tell" CDKs when to work).

• CDC25 = The final "unlocking key" that removes the brakes from CDKs so they can function fully.

Slide 14: The Cell Cycle: CDK Inhibitors (Nobel Prize Context)

Key Points:

1. Scientific achievement:

   • The discovery of the regulatory role of CDKs and CKIs in cell cycle control led to a Nobel Prize in Medicine in 2001.

   • This work highlighted the intricate control of cell proliferation.

2. Biological relevance:

   • CKIs act as "brakes" to ensure that cells do not proliferate uncontrollably, playing a critical role in preventing cancer.

Explanation of Visuals:

• Nobel Prize winners: Showcases the scientists who uncovered the mechanisms of CDK and CKI regulation.

Glossary:

• Nobel Prize in Medicine: Awarded for groundbreaking research in biology and medicine.

• CDK regulation: The control of cyclin-CDK activity to ensure orderly cell cycle progression.

Key Takeaway:
The discovery of CDKs and CKIs revolutionized our understanding of the cell cycle, highlighting their importance in regulating cell division and preventing cancer.

Slide 15: Alteration of Cell Cycle Clock in Human Tumors

Key Points:

• Cell Cycle Dysregulation in Various Tumors:

  • Different tumor types exhibit alterations in key cell cycle regulators, which contribute to uncontrolled proliferation and cancer progression.

  • The table summarizes the involvement of critical cell cycle genes/products in various cancers.

• Commonly Affected Cell Cycle Regulators:

  • Rb (Retinoblastoma Protein): A tumor suppressor protein frequently lost or inactivated in most tumor types.

  • Cyclin E1 and Cyclin D1: These cyclins drive the cell through the G1 to S phase transition and are often overexpressed.

  • p16^INK4A: A CDK inhibitor that negatively regulates cyclin/CDK complexes; its inactivation is common in tumors.

  • p27^Kip1: Another CDK inhibitor that prevents cell cycle progression; its downregulation is often observed.

  • CDK4/6: Cyclin-dependent kinases frequently overactivated in cancers to bypass growth control checkpoints.

• Prevalence of Cell Cycle Alterations Across Tumor Types:

  • The percentage of tumors exhibiting alterations in at least one of these genes is remarkably high, often exceeding 80-90%, indicating the pivotal role of cell cycle deregulation in cancer.

  • Tumor types such as lung carcinoma, leukemia, head and neck carcinomas, and lymphoma exhibit alterations in multiple cell cycle genes, leading to high mutation frequencies.

  • Melanoma shows relatively lower rates of alterations compared to other tumor types.

Explanation of Visuals:

• The table lists various tumor types and indicates which cell cycle regulators are altered (‘+’ means altered presence or activity, and ‘+/-’ indicates partial alteration).

• The rightmost column quantifies the percentage of tumors with at least one of these alterations, showing their widespread prevalence in human cancers.

Glossary:

• Cyclins (E1, D1): Proteins that regulate cell cycle progression by activating CDKs.

• CDK (Cyclin-Dependent Kinase): Enzymes that work with cyclins to push the cell through different phases of the cell cycle.

• p16^INK4A and p27^Kip1: Tumor suppressor proteins that inhibit CDK activity and act as brakes to prevent uncontrolled cell division.

• Rb (Retinoblastoma Protein): A tumor suppressor that controls the G1/S transition by inhibiting E2F transcription factors.

Key Takeaway:

The alteration of cell cycle regulators, including cyclins, CDKs, and tumor suppressors such as Rb and p16, is a common feature of various human tumors, highlighting the importance of targeting the cell cycle machinery in cancer therapies.

Slide 16: Take-Home Message

Key Points:

• Precise Control of the Cell Cycle:

  • The cell cycle is tightly regulated to ensure proper duplication and division of a cell into two daughter cells.

• Role of Cyclins/CDKs Complexes:

  • The progression of the cell cycle is controlled by cyclins and cyclin-dependent kinases (CDKs).

• Regulatory Levels of the Cell Cycle:

  1. Levels and Availability of Cyclins:

     • Cyclins accumulate gradually and are rapidly degraded to maintain cell cycle directionality.

     • The anaphase-promoting complex/cyclosome (APC/C) plays a crucial role in ensuring this process.

  2. CDK Inhibitors:

     • These proteins inhibit cyclin-CDK complexes to prevent uncontrolled cell cycle progression.

• Restriction Point (R Point):

  • A critical checkpoint where the cell commits to completing the cell cycle irreversibly.

• Cyclin D/CDK Complex:

  • This complex primes the cell for entry into the cell cycle, specifically transitioning through the G1 phase.

• pRB (Retinoblastoma Protein) and E2F Transcription Factors:

  • Hyper-phosphorylation of pRB by Cyclin E/CDK:

    • Allows E2F-dependent transcription to drive cells past the restriction point.

  • Mechanism of pRB Action:

    1. pRB regulates cell cycle entry by binding or releasing E2F transcription factors.

    2. Hypophosphorylated pRB blocks progression, whereas hyperphosphorylation allows cell cycle continuation.

• CDC25 Phosphatases:

  • Key regulators that activate CDKs by removing inhibitory phosphate groups.

Answers to Key Points for University Exam Preparation:

1. How is the cell cycle controlled to ensure proper cell division?

   • The cell cycle is precisely regulated through cyclin/CDK complexes, regulatory checkpoints, and inhibitors to ensure accurate duplication and division of the cell.

2. What are the key regulatory levels of the cell cycle?

   • The two main regulatory levels include:

     • Cyclin availability and degradation: Cyclins accumulate and degrade to ensure directional progression of the cycle.

     • CDK inhibitors: These proteins regulate CDK activity to prevent unregulated cell cycle progression.

3. What is the significance of the restriction point (R point)?

   • It is a critical checkpoint where the cell commits irreversibly to cell division, ensuring all necessary conditions are met before progressing.

4. What role does Cyclin D/CDK play in cell cycle entry?

   • Cyclin D/CDK primes the cell for progression through the G1 phase, preparing it for DNA replication.

5. How does pRB regulate the cell cycle?

   • pRB binds E2F transcription factors to prevent cell cycle progression; its hyperphosphorylation releases E2F, allowing the transition from G1 to S phase.

6. What is the function of CDC25 phosphatases?

   • CDC25 phosphatases activate CDKs by removing inhibitory phosphate groups, promoting cell cycle progression.

Glossary:

• Cyclins: Proteins that regulate different phases of the cell cycle by activating CDKs.

• CDKs (Cyclin-Dependent Kinases): Enzymes that drive cell cycle progression in association with cyclins.

• Restriction Point (R point): A checkpoint in G1 where the cell commits to completing the cycle.

• pRB (Retinoblastoma Protein): A tumor suppressor protein that regulates cell cycle progression by controlling E2F transcription factors.

• E2F Transcription Factors: Proteins that drive the expression of genes required for DNA replication.

• CDC25 Phosphatases: Enzymes that activate CDKs by dephosphorylation.

Key Takeaway:
The cell cycle is regulated through a balance of cyclin/CDK activity, inhibitory checkpoints, and phosphatase activation to ensure controlled cell division and prevent unregulated proliferation.

Slide 17: The Cell Cycle Is Controlled at Checkpoints

Key Points:

• Purpose of Checkpoints:

  • Checkpoints serve as quality control mechanisms ensuring the cell completes all necessary steps in one phase before advancing to the next.

  • They help prevent errors such as DNA damage, replication stress, or improper spindle formation that could lead to genomic instability or cancer.

• Types of Cell Cycle Checkpoints:

  1. DNA Damage Checkpoint (Interphase):

     • Monitors for DNA damage before entering the next phase.

     • The ATM (Ataxia-Telangiectasia Mutated) and CHK2 (Checkpoint Kinase 2) pathway activates p53, which then promotes p21 to inhibit CDK2 and prevent cycle progression.

  2. Replication Stress Checkpoint (S Phase):

     • Ensures proper DNA replication and integrity of replication forks.

     • The ATR (Ataxia-Telangiectasia and Rad3-Related) and CHK1 pathway activate WEE1, which inhibits CDK2, delaying the cell cycle until replication issues are resolved.

  3. Spindle Assembly Checkpoint (M Phase):

     • Ensures chromosomes are correctly aligned before cell division.

     • Aurora B kinase and the Mitotic Checkpoint Complex (MCC) inhibit the Anaphase-Promoting Complex (APC/C) until proper spindle assembly is confirmed.

• Commitment Points in the Cell Cycle:

  • S Phase Entry Commitment Point: Ensures DNA is ready for replication.

  • Mitotic Entry Commitment Point: Checks for DNA integrity before mitosis.

  • Mitotic Exit Commitment Point: Confirms spindle formation before proceeding to anaphase.

Exam-Ready Answers:

1. What are the main functions of cell cycle checkpoints?

   • They prevent errors by ensuring proper DNA replication, repair, and spindle assembly before the cell advances to the next phase.

2. Which key molecules are involved in DNA damage checkpoints?

   • ATM, CHK2, p53, p21, and CDK2 work together to halt the cycle if DNA damage is detected.

3. How is replication stress managed in the cell cycle?

   • ATR and CHK1 detect stalled replication forks and activate WEE1 to delay progression until the replication issue is resolved.

4. What happens during the spindle assembly checkpoint?

   • Aurora B and MCC prevent APC/C activation, delaying anaphase until chromosomes are properly aligned.

Glossary:

• ATM (Ataxia-Telangiectasia Mutated): A protein that detects DNA damage and activates cell cycle arrest.

• CHK2 (Checkpoint Kinase 2): A kinase that transmits DNA damage signals to halt cell cycle progression.

• p53: A tumor suppressor protein that regulates DNA repair and apoptosis.

• p21: A CDK inhibitor that blocks the cell cycle in response to DNA damage.

• ATR (Ataxia-Telangiectasia and Rad3-Related): A protein that detects replication stress and activates repair pathways.

• WEE1: A kinase that inhibits CDK activity to delay cell cycle progression.

• Aurora B Kinase: A regulator of chromosome alignment during mitosis.

• MCC (Mitotic Checkpoint Complex): A protein complex that prevents premature separation of sister chromatids.

• APC/C (Anaphase-Promoting Complex/Cyclosome): A key regulator of mitosis that initiates chromosome separation.

Key Takeaway:
Checkpoints are crucial for maintaining genomic integrity by monitoring and halting the cell cycle in response to DNA damage, replication errors, and spindle misalignment, ensuring accurate cell division.

Slide 18: Signaling Pathways Involved in Cell Cycle Control and Cancer

Key Points:

1. Signaling pathways in cell cycle control:

   • Multiple pathways regulate cell proliferation and division.

   • Dysregulation of these pathways (e.g., mutations in proteins) contributes to cancer development.

2. Cancer relevance:

   • Certain pathways are more frequently altered in cancers, such as:

     • p53 pathway: Tumor suppressor that halts the cell cycle during DNA damage.

     • RAS-MAPK pathway: Oncogenic pathway promoting proliferation.

     • PI3K-AKT pathway: Regulates cell survival and metabolism.

3. Therapeutic opportunities:

   • Targeting these pathways can help restore normal cell cycle control and prevent tumor growth.

Explanation of Visuals:

• Pathway diagrams: Highlight key signaling pathways and their connections to cell cycle checkpoints.

• Cancer focus: Red sections identify frequently mutated components in cancer.

  • Even Cancer need certain pathways to work so that they can function.

Glossary:

• p53: A tumor suppressor protein that induces cell cycle arrest or apoptosis in response to stress.

• RAS-MAPK pathway: A signaling cascade that promotes cell proliferation and survival.

Key Takeaway:
Cancer often arises from dysregulation of key signaling pathways controlling the cell cycle, making these pathways important targets for therapy.

Slide 19: Cancer Is Also Driven by Epigenetic Changes

Key Points:

1. Epigenetics in cancer:

   • Epigenetic modifications alter gene expression without changing the DNA sequence.

   • These changes include:

     • DNA methylation: Adds methyl groups to DNA, silencing genes.

     • Histone modification: Alters chromatin structure, affecting gene accessibility.

2. Cancer progression:

   • Epigenetic changes can silence tumor suppressor genes or activate oncogenes.

   • They are proven contributors to tumorigenesis and potential targets for cancer therapy.

Explanation of Visuals:

• Diagram of DNA methylation: Shows how methyl groups are added to DNA, repressing gene expression.

• Histone modification image: Illustrates how modifications to histones alter chromatin structure, affecting accessibility for transcription.

Glossary:

• Epigenetics: Study of heritable changes in gene expression not involving changes to the DNA sequence.

• DNA methylation: Epigenetic process that suppresses gene activity.

• Histone modification: Chemical changes to histone proteins that regulate DNA accessibility.

Key Takeaway:
Epigenetic changes, such as DNA methylation and histone modifications, play a significant role in cancer development and represent promising targets for therapeutic intervention.

Slide 20: Cancer Is Also Driven by Epigenetic Changes

Key Points:

1. Epigenetics and cancer:

   • Epigenetic modifications such as DNA methylation and histone modification alter gene expression without changing the DNA sequence.

   • These changes can silence tumor suppressor genes (TSGs) or activate oncogenes, driving cancer progression.

2. Educational context:

   • Courses like BIO 243 and BME 312 emphasize the role of epigenetics in human diseases, including cancer.

3. Impact on therapy:

   • Epigenetic alterations are reversible, making them promising therapeutic targets.

Explanation of Visuals:

• DNA methylation and histone modification diagrams:

  • Show how these changes regulate gene expression by altering chromatin structure.

• Course titles: Highlight the importance of epigenetics in understanding cancer biology.

Glossary:

• Epigenetics: Changes in gene expression that do not involve alterations to the DNA sequence.

• DNA methylation: Addition of methyl groups to DNA, often silencing gene expression.

• Histone modification: Chemical changes to histones that influence DNA accessibility.

Key Takeaway:
Epigenetic changes, including DNA methylation and histone modification, play a crucial role in cancer progression and are potential targets for innovative cancer therapies.

Slide 21: The Hallmarks of Cancer 2022 – New Dimensions

Key Points:

1. Updated hallmarks of cancer:

   • Adds emerging capabilities to the classic hallmarks:

     • Unlocking phenotypic plasticity: Cancer cells adapt to various environments.

     • Non-mutational epigenetic reprogramming: Epigenetic changes that drive cancer progression without mutations.

     • Polymorphic microbiomes: Microbial communities influencing cancer development and immune responses.

2. Core hallmarks remain:

   • Key features like sustained proliferative signaling, evading growth suppressors, and resisting cell death remain central to cancer biology.

Explanation of Visuals:

• Circular diagram: Integrates the new dimensions into the classic hallmarks, emphasizing their interconnected nature.

Glossary:

• Phenotypic plasticity: Ability of cancer cells to alter their behavior and characteristics in response to the environment.

• Polymorphic microbiomes: Diverse microbial populations that influence cancer progression and treatment responses.

Key Takeaway:
The 2022 update expands the hallmarks of cancer to include epigenetic reprogramming, phenotypic plasticity, and the role of microbiomes, highlighting their relevance in cancer research and therapy.

Slide 22: Inactivation of TSGs by Promoter Methylation

Key Points:

• DNA Methylation and CpG Sites:

  • DNA can undergo methylation at cytosine bases specifically in CpG sequences (cytosine-phosphate-guanine).

  • When CpG methylation occurs near a gene promoter, it represses transcription, leading to gene silencing.

  • Tumor suppressor genes (TSGs) can be inactivated through promoter methylation, contributing to cancer development.

• Bisulfite Sequencing Technique:

  • A method used to determine the methylation status of CpG islands.

  • Unmethylated cytosines are converted to uracil, while methylated cytosines remain unchanged, allowing methylation pattern analysis.

• Expression of DNMT3B (DNA Methyltransferase 3B):

  • The images show DNMT3B expression in:

    1. Normal colonic tissue – Low or absent expression.

    2. Differentiated, less aggressive adenocarcinoma – Moderate expression.

    3. Less differentiated, aggressive adenocarcinoma – High expression, indicating a role in cancer progression.

Explanation of Visuals:

1. Methylation Pattern Diagram (left):

   • Shows the methylation status of the RASSF1A gene in different tissue types:

     • Tumor tissue – Extensive methylation (blue dots), indicating gene silencing.

     • Adjacent normal tissue – Partial methylation.

     • Control individual – Unmethylated CpG islands (white dots), indicating normal gene expression.

2. Histological Images (right):

   • Brown staining represents DNMT3B expression levels in different stages of colonic tissue differentiation.

   • Increased staining correlates with tumor aggressiveness.

Glossary:

• Tumor Suppressor Genes (TSGs): Genes that regulate cell growth and prevent uncontrolled proliferation; their loss can lead to cancer.

• CpG Island: A region of DNA with a high frequency of CpG sites, often found near gene promoters.

• Methylation: A chemical modification involving the addition of a methyl group (-CH3) to cytosine, influencing gene expression.

• DNMT3B: An enzyme responsible for adding methyl groups to DNA, playing a role in gene silencing.

• Bisulfite Sequencing: A technique used to determine DNA methylation by converting unmethylated cytosines to uracil.

Key Takeaway:

Promoter methylation of tumor suppressor genes, detected through bisulfite sequencing, plays a crucial role in cancer by silencing genes that prevent tumor growth. The increased expression of DNMT3B is associated with more aggressive tumor behavior.

Slide 23: Other TSGs

Key Points:

1. Examples of tumor suppressor genes:

   • NF1: Mutation in NF1 leads to neurofibrosarcoma, a type of nerve tumor.

   • VHL: Mutation in VHL, associated with hypoxia regulation, contributes to renal cancer.

2. Broader impact:

   • Loss of TSG function across various pathways promotes uncontrolled cell division, invasion, and metastasis.

Explanation of Visuals:

• Lists the roles of NF1 and VHL in preventing cancer and how their mutations contribute to specific cancers.

Glossary:

• NF1 (Neurofibromin 1): Tumor suppressor gene involved in cell growth regulation, often mutated in nerve-related cancers.

• VHL (Von Hippel-Lindau): Tumor suppressor gene that regulates responses to low oxygen (hypoxia).

Key Takeaway:
Tumor suppressor genes like NF1 and VHL are crucial for controlling cell growth and preventing cancer, with their loss leading to specific cancer types.

Slide 24: The Neurofibromatosis (NF1): Familial Cancer Syndrome

Key Points:

1. What is Neurofibromatosis?

   • Neurofibromatosis (NF1) is a genetic disorder characterized by benign tumors of nerve sheaths (neurofibromas).

   • These tumors can sometimes progress to malignant peripheral nerve sheath tumors (MPNSTs).

2. Additional Risks:

   • NF1 patients have a higher risk of developing other tumors, including glioblastomas, pheochromocytomas, and leukemia.

3. Genetic influence:

   • NF1 is a familial cancer syndrome, with a frequency of 1 in 3500 individuals.

   • The genetic background of patients influences the severity of the disease.

Explanation of Visuals:

• Image of neurofibroma: Shows the characteristic benign tumors of NF1.

• Key features: Café au lait spots and subcutaneous nodules are hallmark signs of the condition.

Glossary:

• Neurofibroma: A benign tumor formed on nerves, often associated with NF1.

• Familial cancer syndrome: A hereditary condition that increases the risk of developing specific cancers.

• Café au lait spots: Flat, pigmented skin lesions commonly seen in NF1.

Key Takeaway:
Neurofibromatosis is a familial cancer syndrome that predisposes individuals to benign and malignant tumors, influenced by genetic factors.

Slide 25: Neurofibromin, Encoded by the NF1 Gene

Key Points:

1. Function of Neurofibromin:

   • Neurofibromin is a GTPase-activating protein (GAP) for Ras, a protein that regulates cell growth.

   • Neurofibromin inactivates Ras by converting it from the active (Ras-GTP) to the inactive state (Ras-GDP).

2. Loss of NF1:

   • Loss of neurofibromin function leads to constitutive activation of Ras, promoting uncontrolled cell growth and tumorigenesis.

3. NF1 and signaling pathways:

   • The NF1 gene plays a critical role in suppressing the Ras/MAPK signaling pathway, which is often overactive in cancers.

Explanation of Visuals:

• Diagram: Illustrates how neurofibromin acts as a GAP, hydrolyzing GTP to GDP to inactivate Ras.

• Inactive vs. active Ras states: Shows the switch mechanism regulated by neurofibromin.

Glossary:

• Neurofibromin: A tumor suppressor protein encoded by the NF1 gene.

• Ras: A GTPase involved in cell growth and differentiation.

• GTPase-activating protein (GAP): A protein that enhances the GTPase activity of Ras, turning it off.

Key Takeaway:
Neurofibromin suppresses tumorigenesis by inactivating Ras signaling. Loss of NF1 function leads to uncontrolled cell growth.

Slide 26: Neurofibromin and the Ras/MAPK Pathway

Key Points:

1. Regulation of Ras/MAPK signaling:

   • Neurofibromin suppresses the Ras/MAPK pathway by inactivating Ras, preventing excessive cell proliferation.

2. Ras pathway components:

   • Ras-GTP activates downstream proteins like RAF, MEK, and ERK, promoting cell growth and division.

   • Neurofibromin ensures this pathway is tightly regulated to avoid overactivation.

3. Impact of NF1 mutations:

   • Loss of neurofibromin leads to hyperactivation of the Ras/MAPK pathway, contributing to tumor growth in NF1 patients.

Explanation of Visuals:

• Pathway diagram: Shows the Ras/MAPK signaling cascade, emphasizing neurofibromin's role in turning off Ras.

• Highlighted components: Demonstrate how mutations in NF1 disrupt normal regulation.

Glossary:

• Ras/MAPK pathway: A signaling cascade controlling cell proliferation and survival.

• RAF, MEK, ERK: Key proteins activated in sequence by Ras-GTP to drive cell division.

Key Takeaway:
Neurofibromin ensures controlled cell growth by regulating the Ras/MAPK pathway. Mutations in NF1 cause hyperactive signaling, driving tumor development.

Slide 27: Neurofibromin, Encoded by the NF1 Gene (Clinical Implications)

Key Points:

• NF1 Function:

  • Neurofibromin (encoded by the NF1 gene) is a GTPase-activating protein (GAP) that regulates Ras activity.

  • NF1 converts Ras from its active (GTP-bound) to its inactive (GDP-bound) state.

  • Without NF1, Ras remains active longer, leading to excessive cell signaling and potential tumor formation.

• Mechanism of Ras Regulation:

  • GTP-bound Ras (active): Promotes cell proliferation in response to stimulatory signals.

  • GAPs (such as NF1): Accelerate GTP hydrolysis, converting Ras to its inactive GDP-bound state.

  • Oncogenic mutations in Ras: Prevent proper GTP hydrolysis, leading to persistent Ras activation.

• Experimental Data (Graph):

  • The graph shows Ras activity over time in NF1-deficient (Nf1⁻/⁻) vs. normal cells (Nf1⁺/⁺).

  • In Nf1⁻/⁻ cells, Ras remains active for a prolonged period compared to Nf1⁺/⁺ cells, indicating NF1's crucial role in Ras inactivation.

Explanation of Visuals:

• Diagram (right):

  • Shows the cycle of Ras activation and inactivation.

  • Ras is activated by guanine nucleotide exchange factors (GEFs), which exchange GDP for GTP.

  • NF1 (as a GAP) promotes GTP hydrolysis, inactivating Ras.

  • Oncogenic mutations prevent this inactivation, leading to uncontrolled cell signaling.

• Graph (left):

  • The red line (Nf1⁻/⁻) shows prolonged Ras activity, while the blue line (Nf1⁺/⁺) indicates normal regulation by NF1.

Glossary:

• Neurofibromin (NF1): A tumor suppressor protein that helps regulate Ras signaling by promoting GTP hydrolysis.

• Ras Protein: A molecular switch that controls cell growth and differentiation by cycling between active (GTP-bound) and inactive (GDP-bound) states.

• GTPase-Activating Protein (GAP): Proteins that accelerate the conversion of active Ras-GTP to inactive Ras-GDP.

• Guanine Nucleotide Exchange Factor (GEF): Proteins that activate Ras by exchanging GDP for GTP.

• Oncogenic Mutation: A genetic alteration that contributes to the development of cancer by promoting uncontrolled cell growth.

Key Takeaway:
NF1 acts as a tumor suppressor by regulating Ras activity. Loss of NF1 function leads to prolonged Ras activation, which can contribute to uncontrolled cell proliferation and tumorigenesis.

Slide 28: Von Hippel-Lindau Disease: pVHL Modulates the Hypoxic Response

Key Points:

1. Role of pVHL:

   • pVHL (Von Hippel-Lindau protein) regulates the hypoxic response by targeting HIF-1α (Hypoxia-Inducible Factor-1α) for degradation under normal oxygen levels (normoxia).

   • In hypoxic conditions, HIF-1α escapes degradation, leading to activation of genes involved in angiogenesis, cell survival, and metabolism.

2. Associated cancers:

   • Mutations in the VHL gene predispose individuals to a variety of cancers, particularly renal cell carcinoma (75% of sporadic kidney tumors).

3. Familial context:

   • VHL disease is a familial cancer syndrome with a frequency of 1 in 35,000 individuals.

Explanation of Visuals:

• Pathway diagram: Illustrates the role of pVHL in regulating HIF-1α under normoxic and hypoxic conditions.

• Gene targets: Highlights downstream genes activated by HIF-1α, including VEGF, PDGF, and TGF-α, which promote tumor growth.

Glossary:

• pVHL: A tumor suppressor protein involved in degrading HIF-1α under normal oxygen levels.

• HIF-1α (Hypoxia-Inducible Factor-1α): A transcription factor that promotes survival and angiogenesis under low oxygen conditions.

• Angiogenesis: Formation of new blood vessels to support tumor growth.

Key Takeaway: pVHL plays a crucial role in regulating the hypoxic response by degrading HIF-1α. Mutations in VHL lead to uncontrolled activation of hypoxia pathways, promoting cancer progression.

Slide 29: Von Hippel-Lindau Disease: pVHL Modulates the Hypoxic Response (Visual Context)

Key Points:

• Von Hippel–Lindau (VHL) disease:

  • A genetic condition that predisposes individuals to various cancers, particularly kidney carcinomas, which account for approximately 70% of sporadic kidney tumors.

  • The VHL protein (pVHL) plays a critical role in the cellular response to hypoxia by regulating the degradation of hypoxia-inducible factors (HIFs).

  • Loss of pVHL function leads to uncontrolled angiogenesis, tumor growth, and adaptation to low oxygen conditions.

• VHL and Hypoxia Mechanism:

  • Under normal oxygen levels, pVHL promotes degradation of HIF-2, preventing excessive expression of hypoxia-related genes.

  • When pVHL is mutated or lost, HIF-2 accumulates, leading to increased production of vascular endothelial growth factor (VEGF), which promotes angiogenesis and tumor progression.

• Clinical Manifestations of VHL Disease:

  • Renal cell carcinoma:

    • Increased HIF-2 expression in tumor tissue compared to normal kidney tissue (as shown in staining images).

  • In situ breast cancer:

    • Hypoxia-induced necrotic regions surrounded by VEGF mRNA expression (indicated in white in the histological images).

  • Uncontrolled vascularization in the retina:

    • Leads to abnormal blood vessel growth, a hallmark of VHL disease.

Explanation of Visuals:

• Panel A (left):

  • Histological staining showing HIF-2 expression in renal cell carcinoma compared to normal kidney tissue. The carcinoma shows increased HIF-2 staining, indicating hypoxia adaptation.

• Panel B (middle):

  • Breast cancer tissue showing necrotic cells and their hypoxic regions marked by VEGF expression.

• Panel C (bottom left):

  • Retinal imaging displaying abnormal vascularization due to VHL loss, demonstrating the impact of uncontrolled angiogenesis.

Glossary:

• VHL (Von Hippel–Lindau) Protein: A tumor suppressor protein that regulates HIF degradation and prevents excessive angiogenesis.

• Hypoxia-Inducible Factor (HIF): A transcription factor that mediates the cellular response to low oxygen conditions, promoting angiogenesis and survival.

• VEGF (Vascular Endothelial Growth Factor): A protein that stimulates blood vessel formation, often upregulated in tumors to improve oxygen supply.

• Angiogenesis: The formation of new blood vessels, crucial for tumor growth and metastasis.

• Necrotic Cells: Dead cells resulting from insufficient oxygen or nutrients, commonly found in tumor cores.

Key Takeaway:
Loss of pVHL leads to the accumulation of HIF-2 and overexpression of VEGF, resulting in uncontrolled angiogenesis and tumor progression, particularly in the kidneys and retina.

Slide 30: p53: A Frequently Mutated Tumor Suppressor

Key Points:

1. Prevalence of p53 mutations:

   • p53 is mutated in over 50% of cancers, making it one of the most frequently altered tumor suppressor genes.

   • Mutation rates vary by tumor type, with the highest prevalence in cancers such as ovary, colon, and esophagus.

2. Role of p53 in cancer:

   • Even when p53 mutations do not initiate tumorigenesis, they significantly aid in cancer progression by disabling cell cycle checkpoints and apoptosis.

Explanation of Visuals:

• Bar chart:

  • Illustrates the prevalence of p53 mutations across various tumor types, emphasizing its widespread role in cancer.

Glossary:

• p53: A tumor suppressor protein that regulates the cell cycle and apoptosis.

• Tumorigenesis: The process of tumor formation.

Key Takeaway: p53 mutations are common across cancers and contribute significantly to tumor progression by disrupting critical regulatory pathways.

Slide 31: p53: "The Guardian of the Genome"

Key Points:

• Role of p53:

  • p53 is a critical tumor suppressor protein that helps cells resist various cytotoxic stresses such as:

    • Lack of nucleotides, UV radiation, ionizing radiation, oncogene signaling, hypoxia, and transcription blockage.

  • p53 activation leads to cellular responses such as:

    • Cell cycle arrest, allowing time for DNA repair or leading to senescence.

    • Apoptosis, if damage is irreparable, to prevent the spread of damaged cells.

    • Blockage of angiogenesis, preventing tumor growth.

  • Graph interpretation:

    • Cells with full p53 function (p53+/+) show higher survival rates after X-ray irradiation compared to heterozygous (p53+/-) and knockout cells (p53-/-), demonstrating the importance of p53 in cellular survival under stress.

  • Western blot analysis:

    • p53 expression increases following radiation exposure, along with its downstream effector, p21, indicating activation of the DNA damage response.

Explanation of Visuals:

• A flowchart depicting p53's role in responding to cellular stress by inducing either repair or apoptosis.

• A graph showing cell viability over time after radiation exposure, comparing different p53 genotypes.

• A Western blot showing increased p53 and p21 expression post-radiation exposure.

Glossary:

• p53: A tumor suppressor protein that regulates cell cycle arrest and apoptosis in response to DNA damage.

• Apoptosis: Programmed cell death to eliminate damaged or unnecessary cells.

• Senescence: A state of permanent cell cycle arrest that prevents the proliferation of damaged cells.

• p21: A cell cycle regulator that enforces arrest in response to p53 activation.

Key Takeaway:
p53 is essential for maintaining genomic integrity by responding to stress signals and controlling cell fate through repair or cell elimination.

Slide 32: p53 – The Tight Control of the Guardian

Key Points:

• MDM2 as a Key Regulator:

  • MDM2 ubiquitin ligase is the major negative regulator of p53, controlling its levels through degradation.

  • Under normal conditions:

    • MDM2 binds to p53, leading to its ubiquitination and subsequent degradation by the proteasome.

  • Under stress conditions:

    • p53 escapes MDM2-mediated degradation, accumulates in the nucleus, and activates target genes involved in DNA repair and apoptosis.

  • Regulatory feedback loop:

    • p53 induces the expression of MDM2, creating a negative feedback loop to control its own activity.

Explanation of Visuals:

• The diagram illustrates how p53 activates target genes while being regulated by MDM2, which mediates its degradation via the ubiquitin-proteasome system.

• The process highlights the balance between p53 activation and degradation.

Glossary:

• MDM2: A protein that tags p53 for degradation, preventing excessive p53 activity under normal conditions.

• Ubiquitination: A process where proteins are marked for degradation by attaching ubiquitin molecules.

• Proteasome: A protein complex that degrades unneeded or damaged proteins in cells.

Key Takeaway:
The p53 pathway is tightly regulated by MDM2 to prevent unnecessary cell cycle arrest or apoptosis, ensuring proper cellular function under normal conditions.

Slide 33: p53 – The Tight Control of the Guardian (Multiple Control Levels)

Key Points:

• p53 regulation is highly complex and occurs at multiple levels:

  • Oligomerization: p53 forms tetramers to function as a transcription factor.

  • Phosphorylation: Post-translational modifications control its activity and stability.

  • Inhibitors: Proteins like MDM2 regulate p53 by promoting its degradation.

  • Ubiquitination: Marks p53 for degradation via the proteasome system.

  • Protein levels: p53 stability and function are tightly regulated based on cellular conditions.

• p53 activation triggers transcription of genes involved in:

  • DNA repair.

  • Cell cycle arrest.

  • Apoptosis.

• Feedback mechanisms:

  • Activated p53 induces MDM2 expression, forming a negative feedback loop to regulate its own activity.

Explanation of Visuals:

• The diagram shows the signaling pathways that regulate p53, highlighting its interactions with kinases, DNA damage sensors (ATM/ATR), and cell survival signals.

• It also illustrates how activated MDM2 mediates p53 degradation, ensuring proper cellular balance.

Glossary:

• MDM2: A negative regulator of p53 that mediates its degradation.

• ATM/ATR: DNA damage response proteins that activate p53 upon detecting genomic damage.

• Proteasome: A cellular complex that degrades unneeded or damaged proteins.

• Ubiquitination: A process by which proteins are marked for degradation.

Key Takeaway:
The activity of p53 is tightly regulated through multiple mechanisms to maintain cellular balance and prevent unnecessary cell death or growth arrest.

Slide 34: p53 – Control of Apoptosis Underlies Its Tumor Suppression

Key Points:

• p53 promotes apoptosis, which is a genetically controlled self-destruction process.

• Apoptosis prevents the survival of damaged cells that could lead to cancer development.

• Key functions of apoptosis in tumor suppression:

  • Prevents further malignant transformation of genetically unstable cells.

  • Eliminates cells with severe DNA damage that cannot be repaired.

• p53 can trigger apoptosis through:

  • Activation of pro-apoptotic genes (e.g., BAX, PUMA).

  • Inhibition of survival signals.

  • Induction of DNA fragmentation and cell membrane changes.

Explanation of Visuals:

• Images depict DNA fragmentation during apoptosis, highlighting nuclear condensation and fragmentation as hallmarks of programmed cell death.

• A histological section showing apoptotic cells in pre-cancerous lesions, marked by arrows.

• Morphological analysis demonstrates apoptotic cells being cleared from tissues.

Glossary:

• Apoptosis: A regulated process of programmed cell death that removes damaged or unnecessary cells.

• DNA fragmentation: The breakdown of nuclear DNA, a characteristic feature of apoptosis.

• Senescence: A permanent state of cell cycle arrest that prevents proliferation of damaged cells.

Key Takeaway:
p53 plays a crucial role in preventing cancer by eliminating genetically unstable cells through apoptosis, thereby acting as a safeguard against tumor development.

Slide 35: Cell Immortalization and Tumorigenesis

Key Points:

• Normal cells have a finite replicative potential, meaning they can only divide a limited number of times before they enter senescence or apoptosis.

• Tumor cells evade this limitation by acquiring mechanisms that allow continuous and uncontrolled proliferation, promoting tumor growth.

• Mechanisms ensuring a limited number of cell duplications include:

  • Telomere shortening: Each cell division results in progressively shorter telomeres, eventually leading to cellular aging and growth arrest.

  • Tumor suppressor genes (e.g., p53, Rb): These genes monitor DNA integrity and halt the cell cycle if damage is detected, preventing unlimited replication.

  • Contact inhibition: Normal cells stop dividing when they come into contact with neighboring cells, preventing overgrowth.

  • Cellular stress responses: DNA damage, oxidative stress, and metabolic changes can activate pathways leading to senescence or apoptosis.

• How cancer cells escape these regulatory mechanisms:

  • Telomerase activation: Cancer cells often reactivate telomerase, an enzyme that extends telomeres, allowing indefinite replication.

  • Loss of tumor suppressor function: Mutations in genes like p53 or Rb disable regulatory checkpoints, enabling unchecked growth.

  • Evasion of apoptosis: Cancer cells develop resistance to programmed cell death signals, continuing to proliferate despite damage.

  • Oncogene activation: Genes that promote growth, such as MYC or RAS, are often overactivated in cancer cells, driving proliferation beyond normal limits.

Explanation of Visuals:

• The slide includes a microscopic image of cancer cells, highlighting their irregular structure and invasive nature.

• The diagram explains the concept that while normal cells have limited replication potential, tumor cells acquire mechanisms to bypass these limits.

Glossary:

• Senescence: A permanent state of cell cycle arrest that prevents further replication.

• Telomeres: Protective DNA-protein structures at chromosome ends that shorten with each cell division.

• p53: A tumor suppressor protein that prevents damaged cells from dividing uncontrollably.

• Telomerase: An enzyme that extends the length of telomeres, enabling unlimited replication.

• Oncogene: A mutated gene that promotes cancerous growth.

Key Takeaway:
Normal cells have built-in mechanisms to limit their replication and prevent tumor formation. However, cancer cells develop strategies such as telomerase activation and suppression of tumor suppressor pathways to achieve uncontrolled growth and evade regulatory mechanisms.

Slide 36: Cellular Senescence

Key Points:

1. Barriers to indefinite replication:

   • Senescence and apoptosis are the two main barriers preventing cells from replicating indefinitely.

   • Cellular senescence is a state of permanent cell cycle arrest.

2. Triggers of senescence:

   • Triggered by factors such as telomere shortening, DNA damage, or oncogene activation.

   • Protects against tumorigenesis by preventing the proliferation of damaged cells.

3. Association with aging:

   • Senescent cells accumulate in tissues over time, contributing to aging and age-related diseases.

Explanation of Visuals:

• Graphs: Show the relationship between population doubling and time, highlighting the senescence phase.

• Microscopic images: Compare normal aging kidney tissue and senescent cells with abnormal morphology and chromosomal instability.

Glossary:

• Senescence: A permanent cell cycle arrest preventing damaged cells from dividing.

• Telomere shortening: Loss of protective ends of chromosomes during replication, triggering senescence.

Key Takeaway:
Cellular senescence is a protective mechanism against tumorigenesis but contributes to aging by accumulating non-dividing cells in tissues.

Slide 37: Cellular Senescence (Molecular Evidence)

Key Points:

1. Markers of senescence:

   • Expression of p16 and p21 is elevated in senescent cells, acting as inhibitors of the cell cycle.

   • Increased activity of senescence-associated β-galactosidase (SA-β-gal).

2. Mechanisms:

   • Senescence prevents the propagation of damaged cells with genomic instability.

   • High levels of chromatin markers like H3K9me3 are associated with the senescent state.

Explanation of Visuals:

• Fluorescence microscopy: Shows high expression of chromatin markers in senescent cells.

• Images of senescent cells: Highlight altered morphology and marker expression.

Glossary:

• p16/p21: Cyclin-dependent kinase inhibitors that regulate cell cycle arrest.

• H3K9me3: A histone modification associated with transcriptional repression in senescence.

Key Takeaway:
Cellular senescence is characterized by molecular markers like p16 and H3K9me3, providing evidence of its role in preventing the propagation of damaged cells.

Slide 38: Cell Immortalization and Tumorigenesis

Key Points:

1. Normal cell replication limits:

   • Normal cells can only replicate a limited number of times due to mechanisms like telomere shortening.

2. Tumor cell immortality:

   • Tumor cells acquire the ability to bypass replication limits, often through activation of telomerase or alternative lengthening of telomeres (ALT).

3. Questions to consider:

   • What mechanisms enforce replication limits?

   • How do cancer cells bypass these limits to achieve immortality?

Explanation of Visuals:

• Microscopic image: Depicts tumor cells with abnormal morphology, emphasizing their capacity for unlimited replication.

Glossary:

• Telomerase: An enzyme that elongates telomeres, allowing cells to evade senescence.

• Alternative lengthening of telomeres (ALT): A telomere maintenance mechanism independent of telomerase.

Key Takeaway:
Tumor cells achieve immortality by bypassing replication limits, a key step in cancer progression that allows for indefinite proliferation.

Slide 39: Inactivation of Gatekeepers, Such as p53, Prolongs Life

Key Points:

1. Role of p53 in senescence:

   • p53 acts as a "gatekeeper," enforcing senescence and apoptosis to prevent tumorigenesis.

   • Loss of p53 function circumvents senescence, enabling continued cell division.

2. Experimental example:

   • Simian virus 40 (SV40) large T antigen inactivates p53, allowing cells to bypass senescence and achieve extended proliferation.

3. Clinical implications:

   • Inactivation of p53 in cancer cells promotes immortality and resistance to stress, aiding tumor progression.

Explanation of Visuals:

• Graph: Shows extended population doubling in cells with inactivated p53 due to SV40 large T antigen.

• Diagram: Highlights the role of p53 in enforcing senescence and how its inactivation disrupts this barrier.

Glossary:

• Gatekeeper genes: Genes like p53 that regulate cell cycle checkpoints and apoptosis.

• SV40 large T antigen: A viral protein that inactivates p53, promoting cell immortalization.

Key Takeaway:
The inactivation of gatekeeper genes like p53 removes critical barriers to cell proliferation, enabling immortalization and tumorigenesis.

Slide 41: Telomeres – Introduction

Key Points:

1. What are telomeres?

   • Telomeres are repetitive DNA sequences located at the ends of chromosomes.

   • They protect chromosomes from degradation and prevent end-to-end fusion.

2. Telomere function:

   • Act as protective caps that maintain chromosomal stability during replication.

   • Loss of telomeres leads to chromosomal instability and triggers cellular crises, such as apoptosis or senescence.

3. Structure:

   • Composed of repetitive TTAGGG sequences and associated proteins that form a protective structure called the shelterin complex.

Explanation of Visuals:

• Microscope image: Displays chromosomes with intact telomeres, emphasizing their protective role.

• Schematic: Highlights the repetitive sequence and location of telomeres at chromosome ends.

Glossary:

• Telomeres: Repetitive DNA sequences at the ends of chromosomes that prevent DNA damage.

• Shelterin complex: A protein complex that protects telomeres from being recognized as DNA damage.

Key Takeaway: Telomeres are essential for protecting chromosome ends and ensuring genomic stability. Their loss leads to catastrophic chromosomal events.

Slide 42: Telomeres – End Protection

Key Points:

• Function of Telomeres:

  • Telomeres are protective caps at the ends of chromosomes that prevent DNA degradation and fusion between chromosomes.

  • They shorten with each cell division, leading to cellular aging and eventual cell death when critically shortened.

• Telomere Erosion:

  • Successive cell divisions lead to progressive shortening of telomeres.

  • Once telomeres reach a critically short length, cells enter a crisis state and may stop dividing or undergo apoptosis.

• Chromosome Instability:

  • Loss of telomere protection leads to unprotected chromatid ends that can fuse, resulting in chromosomal rearrangements and mutations.

Explanation of Visuals:

• The top left diagram shows the process of telomere erosion leading to chromosome end-to-end fusions.

• The fluorescence image depicts stained chromosomes in red, highlighting telomere structures.

• The right diagram shows the progressive shortening of telomeres over successive cell generations, eventually leading to a critical crisis point.

Glossary:

• Telomeres: DNA-protein structures that protect the ends of chromosomes from damage.

• Erosion: The gradual shortening of telomeres over time with cell divisions.

• Chromatid Ends: The ends of replicated chromosomes that can fuse if not protected.

• Crisis: The stage where telomeres become critically short, leading to cell death or mutations.

Key Takeaway:
Telomeres shorten over successive cell divisions, leading to chromosome instability and contributing to aging or cancer development.

Slide 43: Telomeres – Structural Dynamics

Key Points:

• Consequences of Telomere Shortening:

  • When telomeres erode, chromosomal ends become unprotected, leading to end-to-end fusions.

  • These fusions result in abnormal chromosome segregation during cell division, causing genomic instability.

• Anaphase Bridge Formation:

  • During mitosis, fused sister chromatids can create anaphase bridges, where chromosomes are stretched and can break.

  • This process leads to further chromosomal rearrangements and potential tumorigenesis.

• Cycle of Chromosome Fusion and Breakage:

  • The slide illustrates how chromosome fusions persist through multiple cell cycles, leading to further mutations and instability.

Explanation of Visuals:

• The top section shows the progression from telomere erosion to chromosome fusion.

• The lower section details the mitotic process, showing normal chromosome segregation versus fused chromosome misalignment and breakage.

• The sequence of diagrams demonstrates the cycle of breakage and re-fusion, contributing to genomic instability.

Glossary:

• Chromosomal Fusion: The joining of two chromosomes due to telomere loss, leading to genomic instability.

• Anaphase Bridge: A structure formed when fused chromosomes fail to separate properly during mitosis.

• Genomic Instability: A state where mutations accumulate due to defective chromosome segregation.

• Mitosis: The process of cell division where duplicated chromosomes are separated into daughter cells.

Key Takeaway:
Telomere shortening leads to chromosomal fusion and breakage cycles, driving genomic instability and potentially contributing to cancer progression.

Slide 44: Telomeres – Comparison of Wild-Type vs. TRF2 Loss

Key Points:

1. Role of TRF2 in telomere protection:

   • TRF2 (Telomeric Repeat Binding Factor 2) is a key component of the shelterin complex.

   • It maintains the T-loop structure, preventing telomere exposure.

2. Impact of TRF2 loss:

   • Loss of TRF2 leads to unprotected telomeres, activating DDR and resulting in end-to-end chromosome fusion.

   • Cells with TRF2 loss display severe chromosomal abnormalities.

Explanation of Visuals:

• Microscopy comparison:

  • Wild-type chromosomes show intact telomeres.

  • TRF2-deficient cells exhibit widespread telomere fusions and genomic instability.

Glossary:

• TRF2: A protein in the shelterin complex that protects telomeres and maintains the T-loop structure.

• Genomic instability: High rates of mutations and chromosomal rearrangements within cells.

Key Takeaway: TRF2 is critical for maintaining telomere integrity. Its loss leads to unprotected telomeres, chromosomal fusion, and severe genomic instability.

Slide 45: Telomeres and Telomerase

Key Points:

1. Function of telomerase:

   • Telomerase is a ribonucleoprotein enzyme that adds repetitive DNA sequences (TTAGGG) to the ends of telomeres.

   • Composed of two key components:

     • TERT (Telomerase Reverse Transcriptase): Catalytic subunit responsible for DNA synthesis.

     • TERC (Telomerase RNA Component): Provides the RNA template for telomere elongation.

2. Role in cell division:

   • Telomerase counteracts telomere shortening, allowing cells to divide beyond their normal limit.

   • It is active in germ cells, stem cells, and most cancer cells but inactive in most somatic cells.

3. Graph insight:

   • HEK cells expressing hTERT (telomerase) maintain stable telomeres and exhibit extended proliferation, whereas cells lacking hTERT undergo senescence.

Explanation of Visuals:

• Diagram: Illustrates the mechanism of telomerase adding repetitive sequences to telomeres, maintaining chromosomal integrity.

• Graph: Compares proliferation capacity of HEK cells with and without hTERT, showing telomerase's role in preventing senescence.

Glossary:

• Telomerase: Enzyme that extends telomeres, ensuring unlimited cell division in specific cell types.

• hTERT: The catalytic protein component of telomerase responsible for synthesizing telomere DNA.

• HEK cells: Human embryonic kidney cells commonly used in research.

Key Takeaway:
Telomerase prevents telomere shortening, enabling extended proliferation in cells such as germ cells, stem cells, and cancer cells.

Slide 46: Telomerase in Cancer

Key Points:

• Telomerase activity is crucial for cancer cell proliferation.

  • When telomerase activity is suppressed, cancer cells lose their ability to sustain neoplastic (abnormal, uncontrolled) growth.

• Experiment findings:

  • Cells with wild-type human telomerase reverse transcriptase (hTERT) maintain consistent growth over time.

  • Cells with a dominant-negative hTERT, which inhibits telomerase function, show a significant reduction in growth.

  • Control vector cells behave similarly to cells with wild-type hTERT, confirming that active telomerase promotes prolonged cell division.

Explanation of Visuals:

• Graphs (left panel):

  • Show the growth of different cancer cell lines (HA-1, SW613, 36M) with wild-type hTERT (green), dominant-negative hTERT (red), and control vector (blue).

  • Cells with dominant-negative hTERT exhibit growth arrest early on, indicating telomerase inhibition hampers proliferation.

• Graph (right panel):

  • HEK (human embryonic kidney) cells with telomerase (blue line) show continuous growth.

  • HEK cells without telomerase (red line) experience growth arrest after around 20 days.

Glossary:

• Telomerase: An enzyme that adds protective DNA sequences to the ends of chromosomes, preventing their shortening and allowing prolonged cell division.

• hTERT (Human Telomerase Reverse Transcriptase): The catalytic subunit of telomerase responsible for extending telomeres.

• Dominant-negative hTERT: A mutated version of hTERT that inhibits normal telomerase function, leading to reduced cell division potential.

• Neoplastic growth: Uncontrolled cell growth that can lead to tumor formation.

• PD (Population Doublings): A measure of the number of times a cell population has doubled in culture.

Key Takeaway:

Suppressing telomerase activity in cancer cells leads to a loss of proliferative capacity, highlighting telomerase as a critical target for cancer therapy.