Notes

• Interleukin-8


  Involved in the attraction of inflammatory leukocytes
• Interleukin-8


  • Promotes responses such as angiogenesis, proliferation and enhances cancer cell migration
• MDA-MB-231


  • Epithelial cell lines from breast tissue carcinomas
• MDA-MB-436


  • Multinucleated cell lines derived from adenocarcinomas
• SYTOX Green


  Able to cross the membrane when compromised, can only stain dead cells
• Lamin B1 protein


  Down-regulated biomarker in senescence
• P21


  Cyclin-dependent inhibitor, regulates the progression of G1 and S phase, by inhibiting CDK1, CDK2 and CDK4/6
• Interleukin-6


  Facilitates the repair and induction of counter-signalling pathways that hide and protect the tumour
• Bleomycin


  Antibacterial chemotherapy drug that induces DNA damage
• β-galactosidase activity


  In senescent cells, there is an upregulation of lysosomal biogenesis and have more lysosomes, resulting in higher activity at pH 6.0 compared to normal cells
• p21


  A CDK inhibitor (CDKI) which stops the progression of the cancer cell in G1, thus halting the cell cycle
• qRT-PCR


  1. mRNA or Total RNA is extracted into cDNA, transcription using primers then converts cDNA into double-stranded DNA, which can be measured using fluorescent detection to a specific strand

  2. One-Step - all this occurs in one tube

  3. Two-Step - cDNA and the final steps are separated
• TB - 37

  • TB-37 - Normal cell lines found in breast tissue and expresses markers of the major cell types
• Oncogene HRas (G12V)


  A member of the RAS family, which are GTPases involved in cell signalling pathways, involved in cellular growth, differentiation and survival
• Immunoblotting


  Molecules are separated using gel electrophoresis, a primary antibody is added, a neutral antibody is added to block the gel, then a secondary fluorescent protein is added
• Tyrosine Kinases


  This activates an intracellular pathway, MAPK, which also activates RAS, which then activates ERKs, which then carry out multiple functions, such as differentiation
• Tissue Homeostasis


  Equilibrium of the tissue environment, including cell differentiation/death, immune regulation, as well as temperature/pH
• Tumour Microenvironment (TME)


  Complex ecosystem consisting of cellular and non-cellular interactions within a tumour
• Senescence


  State of irreversible growth, stopped dividing and replicating
• Senescence-associated secretory phenotype (SASP)


  Senescent cells secrete a high level of inflammatory molecules, such as IL-6 and IL-8
• Epithelial cell types


  • Cuboidal
  • Squamous
  • Columnar
• Epithelial cell arrangements


  • Simple
  • Pseudostratified
  • Columnar
• Epithelial cells


  Regulate chemical movement as well as form the extracellular matrix (ECM)
• Inflammatory Cytokines


  • Interleukin-6
  • Tumour Necrosis Factor α
  • Interleukin-1
• SASP CM (Conditioned Media)


  Medium in which cells have been grown, enriched in secretory products
• Caspases


  Family of proteins capable of inducing apoptosis
• Caspase Structure


  2 large α and 2 β subunits, produced as inactive zymogens where they undergo cleavage to become active, with the key cysteine in the active site being the QACxG
• Caspase-mediated apoptosis


  The cell expresses the Fas ligand, which is brought close to a death receptor by a T lymphocyte, this recruits a Fas adaptor protein consisting of a death domain receptor and a death domain, the death domain activates Caspase 8/10, the receptor and the Fas ligand interacts with the adaptor protein to create the DISC complex. Cas 9/10 then work to cleave proteins with the FAS ligand being recycled
• Senescence Inducers


  • DNA damage
  • Epigenetic changes
  • Oncogene activation
• Senolytic Drugs


  Agents that specifically target and induce apoptosis of senescent cells, two main examples are Dasatinib and Navitoclax
• NLRP3


  An inflammasome that senses apoptosis or infection, mediating Caspase 1, which if activated releases pro-inflammatory molecules, such as IL-1β and IL-8
• Gasdermin


  Responsible for the release of pro-inflammatory molecules, this family also regulates pyroptosis
• Pyroptosis


  A programmed inflammatory cell death, which occurs after the cleavage and activation of a gasdermin, leading to loss of cell membrane integrity and release of pro-inflammatory cytokines and cellular proteins that can act as danger signals
• Aberrant heterotypic communication


  Communication between cells that is aberrant, or away from the normal/mutated signalling
• Cellular components of the Tumour Microenvironment in Breast Cancer

  • Endothelial cells
  • Pericytes
  • Fibroblasts
  • Variety of tumour-infiltrating cells
• Tumorigenesis


  Formation of tumours
• Oncogenes


  Genes that can transform a cell into a tumour cell, an example is oncogenic RAS, which upregulates other genes, this may activate senescence and release of IL-6, but may also act to give some protection by decreasing pro-inflammatory release
• Extrinsic Apoptosis Pathway


  DISC assembly and the CARD domain
• Intrinsic Apoptosis Pathway


  Referred to as the Mitochondrial pathway, death signals associated with physiological functions result in upstream signalling and activation of mitochondrial signalling proteins, this stimulates apoptotic signals, which results in a loss of membrane potential, permeability and release of contents, Cytochrome C binds to APAF1 (apoptotic protease activating factor) creating an exposure of the CARD domain, CARD, APAF1 and Cytochrome C all cluster together to form an apoptosome, this then recruits Caspase 9, which causes a cascade and activation of Caspase 3/7
• HMEC


  Human epithelial cell line