Life and Death of Cells and Organisms

Life and Death of Cells and Organisms

BI2BC45 Cells and Immunity, Dr Mike Fry 2024

Learning Outcomes

By the end of this lecture, understanding the following is expected:

• Factors affecting cell lifespan and aging.
• How specific processes impact aging and potential drug targets to extend healthy lifespan:
  • Telomere length
  • DNA damage/repair systems
  • Energy levels and metabolism
  • Signaling pathways (Insulin/IGF, PI3K, Tor, Sirtuins)
  • Autophagy (self-eating)

Why Can't We Live Forever?

Aging Scenarios

• Scenario A:
  • Subclinical aging occurs early.
  • Age-Related Diseases (ARD) lead to death around 65-70 years.
• Scenario B:
  • Subclinical aging occurs early.
  • Medical intervention addresses age-related diseases.
  • Individuals live to approximately 85 years.
• Scenario C:
  • Subclinical aging addressed with anti-aging interventions and medical interventions.
  • Age-related diseases are delayed.
  • Lifespan extended to approximately 100 years.

Cellular Changes Associated with Aging

• Hematopoietic Stem Cells (HSC):
  • Altered location in bone marrow microenvironment.
  • Altered cellular metabolism.
  • Decreased hematopoietic reconstitution potential.
• Innate Immune Cells:
  • DC (Dendritic Cells):
    • Defective homing to secondary lymph node.
    • Decreased antigen uptake.
  • Macrophages:
    • Decreased phagocytosis.
    • Defective bactericidal function.
    • Increased production of inflammatory cytokines.
  • Neutrophils:
    • Decreased ROS (Reactive Oxygen Species) production.
    • Reduced phagocytosis.
    • Defective bactericidal function.
  • NK Cells (Natural Killer Cells): Reduced cytolytic potential.
  • Impaired proliferation in response to cytokine stimulation (IL-2).
• Adaptive Immune Cells:
  • T Cells:
    • Reduced development, leading to reduced numbers of naïve CD4+ T cells.
    • Increased numbers of effector and memory CD4+ T cells.
    • Decreased CD8+ T cell proliferation in response to IL-2.
    • Decreased CD8+ T cell expansion and proliferation in response to antigenic stimulation.
  • B Cells:
    • Reduced development, resulting in fewer naïve B cells.
    • Decreased responsiveness to IL-7 stimulation.
    • Increased numbers of low affinity antibodies; overall decrease in antibody affinity.
    • Increased numbers of memory B cells.

Parabiosis and Immune Factors in Aging

• Parabiosis:
  • Involves the conjoining of two organisms to study the effects of shared circulatory systems.
• Aging:
  • Associated with decreased neurogenesis, impaired synaptic plasticity, and impaired cognition.
• Rejuvenation:
  • Rejuvenating factors can increase neurogenesis.
  • The effect on synaptic plasticity and cognition remains unknown.

Factors Playing a Role in Aging

• Proliferation of cells
• Cell replacement
• DNA damage and repair
• Energy and metabolism

Cells and Proliferation

• Cells exhibit a range in their ability to grow and divide.
• Some cells (e.g., nerve cells and erythrocytes) reach a mature differentiated state and do not divide; these are post-mitotic cells.

Human Cell Replacement Rate

Cells and Proliferation (Stem Cells)

• Stem cells divide continuously throughout life.
• Examples include cells in the epithelium lining the intestine and cells that give rise to various blood cell types.

Cells and Proliferation (Intermediate Cells)

• Many cells are intermediate between post-mitotic and stem cells.
• They remain quiescent but can be triggered to divide by appropriate signals (e.g., liver cells after liver damage).

Properties of Stem Cells

• A stem cell can self-renew and give rise to either cell precursors or cells entering a terminal differentiation pathway.
• Depending on tissue requirements, a stem cell can remain transiently dormant or undergo steady-state cycling.
• A precursor cell can undergo several rounds of cell divisions and acquires distinctive features characteristic of each lineage as it differentiates.
• Differentiated cells are nonmitotic with a finite life span.
• Differentiating cells of a lineage follow a unique maturation sequence.

Cells and Proliferation (Primary Cells)

• Primary cells isolated from a tissue will undergo approximately 30-50 cell divisions before stopping and becoming senescent.
• The number of cell divisions depends on the age of the individual from whom the cells were taken; cells from an embryo grow longer than those from an adult.

Telomerase

• Telomerase is an enzyme that maintains the telomere ends of chromosomes.
• Insufficient telomerase activity limits the number of mitotic divisions and forces the cell into senescence, defining the finite capacity of cell division.

Telomere Shortening and Tumor Suppression

• Telomere shortening and the limited lifespan of a cell are considered potent tumor suppressor mechanisms.
• Most human cancers express human telomerase reverse transcriptase (hTERT).
• Ectopic expression of hTERT in primary human cells confers immortal cell growth in culture.

Telomere Length and Telomerase Activity

• High Telomerase Activity:
  • Germ cells, stem cells, tumor cells, and immortal cell lines have high telomerase activity.
  • This maintains functional telomeres, genomic stability, and high proliferative capacity.
• Low or Absent Telomerase Activity:
  • Somatic tissues and primary cells have low or absent telomerase activity.
  • Telomere exhaustion leads to chromosomal abnormalities, genomic instability, growth arrest, and apoptosis.
• Telomerase activity/telomere length is regulated in a developmentally- and tissue-specific manner.
• It is highly variable among age-matched individuals and determined by both genetic and environmental factors.
• Newborns show no gender-related differences, but adult females generally have greater telomere length than males.

Telomerase Complex

• hTR (Telomerase RNA Component)
• TERT (Telomerase Reverse Transcriptase)
• Accessory Proteins: GAR1, Dyskerin, NHP2, NOP10, Reptin, Pontin, TCAB1

Telomerase Activity and Molecular Pathology

• Telomerase components include hTR, DKC1, NHP2, NOP10, and hTERT.
• Shelterin proteins (Apollo, TPP1, TIN2, POT1, RAP1, RTEL, CST, TRF1, TRF2) protect telomeres.
• Molecular Pathology:
  • Premature telomere loss with DNA damage response can lead to stem cell depletion and cell death, resulting in diagnoses like Dyskeratosis Congenita.
  • Telomere elongation with higher replicative potential, altered gene expression, and chromosomal instability can lead to tumor initiation, such as in Chronic Lymphocytic Leukemia (CLL) or familial melanoma.
  • Altered metabolism can result in metabolic syndrome (e.g., obesity).

Cell Lines

• On rare occasions, cells that would normally stop growing become altered and appear immortal, forming a cell line.
• Cell lines are useful experimentally as they often retain the phenotype and growth characteristics of the original cells.

Cell Transformation

• Transformation is an additional change associated with the potential for malignant growth (cancers).
• Transformed cells lose normal growth control and may exhibit alterations such as anchorage-independent growth.
• Normal cells typically only grow when anchored to a solid substrate.

DNA Damage and Repair

Types of DNA Damage

• Single-strand issues:
  • Base excision
  • Mismatch excision
  • Nucleotide excision
• Double-strand issues

Model Organisms and Genome Sizes

DNA Damage Response

• DNA damage can be caused by:
  • Cellular metabolism
  • Viral infection
  • Radiation
  • Chemical exposure
  • Replication errors
• The DNA damage response involves:
  • Cell cycle checkpoint activation
  • Transcriptional program
  • DNA repair
  • Apoptosis activation

Endogenous and Exogenous DNA Damage

• Endogenous DNA Damage:
  • Depurination: 10,000 lesions/cell/day
  • Cytosine deamination leading to base transition: 100-500 lesions/cell/day
  • SAM-induced methylation: Approximately 600 3-meA and 4,000 7-meG lesions/cell/day
  • Oxidation: 10-30 06-meG and 400-1500 8-oxo-dG lesions/cell/day
• Exogenous DNA Damage:
  • Peak hour sunlight: Generates approximately 100,000 pyrimidine dimers and (6-4) photoproducts per cell/day.
  • Cigarette smoke: Generates 45-1029 aromatic DNA adducts.
  • Various X-ray procedures induce Double-Strand Breaks (DSBs), with the number of lesions/cell varying based on exposure (e.g., Chest X-rays: 0.0008 DSBs, Body CT: 0.28 DSBs).
  • Radiation treatments and accidents also induce DSBs, with the quantity varying based on exposure level (e.g., Tumor PET scan: 0.4 DSBs, Chernobyl accident: 12 DSBs).
  • Airline travel: Approximately 0.0002 DSBs/hour.
  • Space mission (60 days): Approximately 2DSBs.

Timeline of DNA Damage and Repair Understanding

• 1775: Concept of oncogenes introduced.
• 1900-1950: Introduction of the concept of hereditary material.
• 1928-1961: Establishment that hereditary material (DNA) can be damaged by endogenous and exogenous agents.
• 1953: Reporting of the helical structure of DNA.
• Circa 1958: Discovery that DNA damage can be repaired
• 1955-1961: Establishment of the link between mutagenesis and carcinogenesis.
• 1960: Alkylating agents shown to react with and damage DNA.
• 1969: Apoptosis defined.
• 1969-2015: Discovery that the failure to repair DNA contributes to cancer; individuals with disorders resulting from defective DNA repair are cancer-prone.
• 1972: Cell cycle checkpoints proposed.
• 1974: Xeroderma pigmentosum identified as the first DNA repair disorder.
• 1975: Ames test established to identify carcinogens via analysis of mutagens.
• 1975-1985: Viruses proposed as a major cause of cancer.
• 1981: Concept of tumor suppressors introduced.
• 1984: Ataxia telangiectasia reported to be a radiosensitive disorder characterized by cancer predisposition.
• 1989: Microsatellite instability identified in Lynch syndrome tumors and shown to be due to MMR deficiency. MSH2 identified as the first Lynch syndrome locus..
• 1990: Mutator phenotype for cancer cells proposed.
• 1993: p53 mutations identified in cancers; the role of p53-dependent surveillance pathways recognized as cancer suppressive.
• 1997-1999: BRCA1 and BRCA2, which are mutated in hereditary breast cancer, shown to function in homologous recombination.
• 1997: Oncogene expression shown to lead to activation of the p53-p21 pathway, and thus to senescence or apoptosis.
• 1999: Concept of caretaker and gatekeeper genes introduced.
• 2000: Oncogene expression shown to cause replication instability.
• 2002-2003: Oncogene expression shown to cause deregulated metabolism leading to ROS production and DNA damage.
• 2002: DDR reported to be an anticancer barrier in early-stage tumorigenesis; mutations in DDR genes shown to occur in later-stage tumors.
• 2005: Significance of replication stress and replication fork stability appreciated.
• 2014: Multiple mutations in DDR genes identified in cancers; acquired characteristics of cancer cells defined.

DNA Damage and Repair in Bacteria (N. meningitidis and E. coli)

• DNA damage can occur due to oxygen metabolism, replication infidelity, UV light, X-rays, and mutagens.
• This can lead to various types of DNA lesions, including 8-oxo-G (BoxoG), AP sites, mismatches, cyclobutane pyrimidine dimers (CPD), and double-strand breaks.
• N. meningitidis shows several unique features in DNA repair compared to E. coli:
  • Fewer DNA glycosylases, potentially due to habitat adjustment.
  • Lacks MutH or has an unidentified homologue/superfluous function.
  • Fully functional NER despite being less exposed to UV light in the host; the pathway may have redundant functions.
  • No SOS boxes or LexA homologue identified, suggesting a lack of RecA-controlled DNA-damage-inducible response.
  • Fewer TLS polymerases, suggesting unique features of TLS polymerase DinB.
  • More antioxidants but reduced alkylation reversal, also potentially due to habitat adjustment.

Inherited Syndromes with Defects in DNA Repair

DNA Damage During Replication

• Damage can occur during replication.
• Errors can be reduced by proofreading, where DNA polymerase moves backward to degrade the newly synthesized strand and then moves forward again to synthesize the correct sequence.

Importance of DNA Polymerase

• DNA polymerase plays a critical role in survival, as demonstrated by experiments with modified DNA polymerase.

Chemical Damage

• Deamination of 5-methylcytosine to thymine requires base excision repair to prevent permanent changes in the sequence following replication.

Base Excision Repair (BER)

• BER is used to correct chemically altered bases with little helix distortion.
• The process involves:
  • DNA glycosylase cleaving the altered base.
  • An AP endonuclease cleaving the deoxyribosylphosphate.
  • Pol-β and DNA ligase inserting the correct nucleotide and closing the gap.

Mismatch Excision Repair

• Mismatch excision repair corrects errors that occur during DNA replication.
• The process involves:
  • Proteins like MSH2 and MSH6 recognizing the mismatch.
  • MLH1 and PMS2 endonuclease cleaving the DNA.
  • DNA helicase and exonuclease removing the incorrect sequence.
  • DNA polymerase and ligase filling the gap with the correct bases.

Nucleotide Excision Repair (NER)

• NER repairs helix-distorting adducts, such as thymine dimers caused by UV radiation.
• The process involves:
  • Cleavage of the DNA fragment (~24 nt on the 5' side and 5 nt on the 3' side of the adduct).
  • Filling in by pol-δ or -ε, PCNA, and RPA.
  • Closure by ligase.

Xeroderma Pigmentosum (XP) Syndrome

• XP is characterized by a 1000-fold increased risk of cancer.
• Skin cancer appears at a mean age of 10 years instead of 60 years.
• Seven out of eight genes identified for XP function in nucleotide excision repair.

Double-Strand Breaks

• Double-strand breaks (DSBs) in DNA are severe lesions that can lead to gross chromosomal rearrangements if not repaired correctly.
• They are caused by ionizing radiation (e.g., X- and γ-radiation) and some anticancer drugs.
• DSBs are repaired by:
  • Homologous recombination (inefficient in human cells).
  • Non-homologous end-joining (NHEJ), which is inaccurate and error-prone.

Non-Homologous End Joining (NHEJ)

• NHEJ is an error-prone repair mechanism for double-strand breaks.
• It involves:
  • Resection of single strands by exonuclease.
  • Bringing DNA strands together, possibly with limited base pairing.
  • Filling in the strands and joining them by ligation.
  • Reconstruction of the double helix, often with several base pairs missing from the original wild-type sequence.

Homology-Directed Repair

• Homology-directed repair uses a sister chromatid as a template to repair double-strand breaks accurately.
• The process involves:
  • Resection by exonuclease.
  • Base-pairing with unwound DNA of the sister chromatid.
  • Strand extension.
  • Disengagement and pairing.
  • Filling in gaps and restoring the wild-type helix.

NHEJ and Homology-Directed Repair Proteins

• NHEJ involves proteins such as KU70-KU80, DNA-PKcs, Artemis, XRCC4, and LIG4.
• Homologous recombination involves proteins such as ATM, BRCA1, MRN, γH2A.X, CtIP, EXO1, BLM, RPA, RAD51, and BRCA2.

CRISPR/Cas and DNA Repair

• CRISPR/Cas9 makes use of these repair mechanisms.
• It uses a guide RNA to target a specific genomic DNA sequence.
• Cas9 induces a double-strand break.
• Repair can occur via NHEJ (leading to insertions or deletions (InDels) and potential premature stop codons) or homology-directed recombination (for precise gene editing).

DNA Damage Checkpoints

• DNA damage checkpoints are critical for maintaining genomic stability.
• Key checkpoints include:
  • Intra-S-phase checkpoint
  • Spindle-assembly checkpoint
  • Spindle-position checkpoint
  • DNA-damage checkpoint (G1, S, G2, and M phases)
• These checkpoints involve various proteins and enzymes, including ATM/R, ATR, Chk1/2, p53, p21CIP, Cdc25A/C, and cyclin-CDK complexes.

DNA Damage and Repair Signaling

• DNA damage activates kinases such as ATM and ATR, which phosphorylate downstream targets like p53, CHK1, and CHK2.
• p53 activation leads to the transcription of genes involved in cell cycle arrest, DNA repair, and apoptosis.
• BRCA1 and other DNA repair proteins act as scaffolds to assemble DNA repair complexes.

DNA Damage, Cancer, and Aging

• DNA damage accumulates from both exogenous (radiations, chemicals) and endogenous (ROS, replication errors, hydrolysis) sources.
• Accumulation of mutagenic lesions can lead to cancer.
• DNA repair mechanisms counteract DNA damage, but their efficiency and accuracy decline with aging, leading to defects in cellular functions, cell death, and senescence.

Aging

Insulin/IGF-1 and TOR Signaling in Aging

• Insulin/IGF-1 signaling and TOR (Target of Rapamycin) are key pathways involved in aging.
• Reduced insulin/IGF-1 signaling and TOR activity are associated with increased longevity.
• DAF-2 receptor and DAF-16/FOXO transcription factor play crucial roles in this pathway.
• Sirtuins (e.g., SIR-2) are also involved in longevity.

Dietary Restriction and Aging

• Chronic food limitation or dietary restriction extends lifespan.
• This involves:
  • Reduced Insulin/IGF-1 signaling.
  • Reduced TOR activity.
  • Increased SIR-2.1 and AMP kinase signaling.
  • Activation of transcription factors like SKN-1, PHA-4, and DAF-16.

Mechanisms of Lifespan Extension

• Dietary restriction and rapamycin (TOR inhibitor) extend lifespan through different mechanisms:
  • Reduced S6 kinase activity.
  • Increased autophagy.
  • Reduced general translation.
  • Increased respiration.

Fasting and Dietary Restriction in Aging

• Periodic fasting and fasting-mimicking diets have various benefits:
  • Increased stress resistance and lifespan.
  • Multi-system regeneration.
  • Reduced adiposity and cancer risk.
  • Decreased inflammatory diseases.
  • Improved immune and cognitive function.
• These interventions also affect biomarkers and risk factors for diseases like diabetes and cardiovascular disease (CVD).

p53, Caloric Restriction and Autophagy

• Caloric restriction and compounds like resveratrol affect aging through various pathways:
  • Glucose starvation and IGF-1 reduction activate AMPK.
  • Sestrin 1/2 signal is activated by Glucose starvation and IGF-1 reduction activate AMPK.
  • AMPK inhibits mTOR through TSC1/TSC2.
  • Sirt-1 is also activated and promotes longevity
  • p53 is affected by PI-3K, PTEN, and MDM2.
  • mTOR inhibition leads to autophagy and longevity.

Sirtuin 1 (SIRT1)

• SIRT1 is a deacetylase involved in aging.
• Acetylation is a post-translational modification regulated by acetylases and deacetylases.
• SIRT1 deacetylates histones and other proteins, affecting a wide range of cellular processes.

Nutrient Status and Autophagy

• In nutrient-rich conditions, mTORC1 is active, inhibiting autophagy.
• In nutrient starvation conditions and with mTOR inhibition (e.g., by rapamycin), autophagy is induced.
• This involves:
  • Activation of ULK1/2 and mAtg13 complex formation.
  • Inhibition of S6K and 4E-BP1.
  • Activation of Vps34.
• Autophagy promotes recycling and reduces protein synthesis and cell growth.

What is Autophagy?

Autophagy Process Overview

• Autophagy is a cellular process that involves the degradation and recycling of cellular components.
• Nutrients and stress can trigger autophagy via mTOR, AMPK, and HIF pathways.
• The process involves:
  • Formation of an autophagosome.
  • Fusion with a lysosome to form an autophagolysosome.
  • Metabolism of cellular components to produce amino acids, nucleosides, fatty acids, and sugars.
• Autophagy plays a role in organelle and protein quality control, recycling, and biomass production.

Pathological and Physiological Functions of Autophagy

• Autophagy plays a role in various physiological and pathological processes:
  • Neurodegenerative diseases
  • Type II diabetes
  • Fatty liver
  • Infectious diseases
  • Cell death
  • Cancer
  • Innate immune system
  • Cardiomyopathy
  • Aging

Summary: Factors Affecting Cell Lifespan and Aging

• Telomere length
• DNA damage/repair
• Energy levels and metabolism
• Signaling pathways (Insulin/IGF, PI3-kinase, Tor, Sirtuins)
• Autophagy (self-eating)