Chapter 7 Notes

• Senescence


  Biological aging, aging occurs at cellular level

  
• Normal cells


  • Undergo replicative or cellular senescence
  • Adult somatic cells have a limited number of successive replicative cycles
  • Once that limit is reached, cells stop growing and enter into the state of REPLICATIVE SENESCENCE
• Hayflick limit


  The limit on the number of successive replicative cycles that normal cells can undergo
• Senescent cells


  • Remain metabolically active but have lost the ability to re-enter the active cell cycle
  • Growth factors help sustain the viability of the senescent cells, but are unable to elicit a proliferative response
  • Display growth factor receptors
• In vitro replicative senescence

  1. Serial passaging

  2. Portion of cells that have filled one dish are removed and introduced into the second dish and allowed to proliferate

  3. Cells are then introduced into a third dish and so forth
• In vitro replicative senescent cells

  • Remain metabolically active
  • Cannot re-enter the active cell cycle
  • Acquire a large cytoplasm ("FRIED EGG")
  • Can persist for months
• In vivo replicative senescence

  • Keratinocyte stem cells in the skin lose proliferative capacity with age
  • Thinning of the keratinocyte layer
  • Loss of the ridge architecture
• Immortal cells


  Cells that show unlimited replicative capacity
• Cancer cells are often found to be immortal, which suggests that immortalization is integral to cancer cell transformation to a neoplastic growth state
• Cell culture induction of senescence

  1. Normal cell division

  2. Induction of CDKI inhibitors

  3. Cell cycle arrest

  4. Senescence
• Senescent cells


  • Small size
  • Few focal contacts
  • Few actin fibres
  • Inhibits Cdk complexes
  • Blocks phosphorylation of pRB
  • Cell cycle arrest
  • Large size
  • Many focal contacts
  • Many actin fibres
• Senescence occurs in aging tissues
• Senescence in aging tissues

  • Normal rat kidney tissue: stained for acidic β-galactosidase
  • Increased staining associated with senescence
• Senescence in aging tissues

  • IHC staining for p16 antibody - cells in the tissue undergoing senescence
  • IHC staining for Ki67 - dividing cells in the tissue

  
• Senescence is not only attributed to aging cells, it may reflect stress that the cells have sustained

  
• Senescence


  A halt in cell proliferation with retention of cell viability over extended periods of time
• Crisis (Senescence induced apoptosis)

  Apoptosis associated with cell crisis
• Cancer has to overcome two hurdles before immortality: senescence and crisis (senescence induced apoptosis)
• Cells have a generational clock that measures elapsed cell generations
• Telomeres


  Protective shields for the chromosomal ends
• Telomere dysfunction can contribute to cell physiological stress
• Telomere shortening


  Shortening of telomeres correlates with population doublings, telomere length varies between 7-13 kbp and undergoes 50-100 bp shortening per cell generation
• Mechanism of telomere shortening


  1. Telomeric DNA of normal human cells proliferating in culture progressively shorten during each growth-and-division cycle, until they become so short that they can no longer effectively protect the chromosomes

  2. At this point, crisis occurs, chromosomes fuse, and apoptosis will result
• Telomeric DNA


  • Formed from the repeating hexanucleotide sequence 5' TTAGGG 3' in one strand (G-rich) and the complementary 5'CCCTAA3' in the other strand (C-rich)
  • Telomeric DNA is formed from several thousands of these hexanucleotide sequences (5-10 kb stretches of sequence)
  • G-rich strand is longer resulting in a 3' single-strand overhang
• Telomeric DNA


  • The overhang's molecular configuration is in a T-loop
• Telomeric DNA and proteins

  • Telomeric DNA + telomeric proteins = telomeres
  • Some proteins possess domains that recognize and bind to the hexanucleotide sequence present in the S and SS regions of telomeric DNA
• Telomere shortening due to end-replication problem

  1. DNA synthesis must be initiated at the 3-0 end of an existing DNA strand, this will be the primer that will nucleate DNA strand elongation

  2. In the absence of a DNA primer, the 3-end of an RNA molecule can serve as a primer for DNA synthesis
• Telomere shortening due to end-replication problem

  1. Leading strand - DNA polymerase III

  2. Lagging strand - Okazaki Fragments, RNA primase lays down RNA primers, DNA pol III lays down the DNA, DNA pol I replaces the RNA primers with DNA, DNA ligase links the Okazaki fragments

  3. Loss of end telomeric DNA sequences
• Human telomerase


  Prevents telomere shortening
• How telomerase prevents telomere shortening

  Telomerase recognizes the tip of the repeat sequence in telomerase, uses its own RNA template (hTR) and elongates the parental strand in 5' to 3' direction, DNA polymerase can fill in the complementary strand
• Expression of telomerase prevents crisis
• Proposed mechanism of telomerase preventing senescence


  During replicative senescence telomeres lose their single-stranded overhangs, DNA damage signal induces p53 which induces cell arrest, telomerase expression prevents this from occurring
• Telomerase and generational clock

  Telomerase expression is repressed in postembryonic cell lineages, these cell lineages are granted only limited postembryonic replicative potential before they enter crisis, cancer cells re-express telomerase and turn back the generational clock
• Telomere loss leads to chromosome abnormalities in cancer
• Mechanism of telomere loss leading to chromosome abnormalities

  Telomere loss leads to breakage-fusion-bridge (BFB) cycles
• How BFB cycles promote cancer

  BFB cycles lead to increase in chromosomal rearrangements and the amplification and deletion of chromosomal segments adjacent to breakpoint, resulting in novel oncogenes, amplified oncogenes, loss of TSGs

  
• How cancer cells stabilize their genomes

  Cells with unstable genome grow slowly, cells can die as a result of catastrophic genomic instability, cancer cells express hTERT to stabilize their genomes, hTERT repairs chromosome ends in cancer cells and stops the BFB cycles, cancer cells survive and continue to extensively proliferate, chromosomal rearrangements acquired prior to hTERT expression remain in cancer cells

  
• Pathway to immortality


  Normal cell -> Telomere length shortening -> Transformation -> Cancer cell -> hTERT expression -> Genome stabilization