Cancer biology

• Principles of the cell cycle

  • Rounds of protein degradation and protein synthesis of specific proteins help control the cell cycle
  • Regulatory phosphorylation events keep a tight control on the cell cycle
  • Some key regulatory proteins can be tumour suppressors or oncogenes (tumour causing)
• There are 2 landmark phases:

  • S-phase: DNA synthesis phase; genome is replicated.
  • M-phase: mitotic phase; chromosomes are segregated to daughter cells
• How do you monitor cells in S-phase?

  DNA replication can be monitored by incorporating 3H[thymine] and cells can only pick up the thymidine label in S phase. If the length of the cell cycle is known, the length of S phase can be cocirculated from the percentage of cells labelled with 3HT.
• Analysis of DNA content with a flow cytometer (fluorescence activated cell sorter – FACS)


  Cells stained with a dye that fluoresces when it binds DNA

  • the amount of fluorescence is directly proportional to the amount of DNA in each cell
• Which phase are most cells in?

  G1 - cell growth and normal metabolic activities
• Experimental demonstration that cells contain factors that stimulate entry into mitosis, what is that factor?

  Entry into the M phase is triggered by activation of a protein kinase: maturation promoting factor (MPF). MPF only present in M phase cells

  • MPF has two subunits:
  • kinase subunit
  • regulatory subunit: cyclin
  • MPF binds to cyclin which activates the kinase domain. The cyclin is only present in M phase.
• Cyclin regulated by rounds of protein synthesis and protein degradation
• • When cyclin concentration is low, the kinase lacks the cyclin subunit and is inactive
  • When cyclin concentration rises, the kinase is activated and entry into mitosis is initiated
• A model of cell cycle regulation in yeast

  • The cell cycle is controlled primarily at two points, start point of mitosis and the G2-M transition
  • Requires the activation of MPF-like (MPF only in mammals) cyclin-dependent kinases (Cdks) encoded by cdc genes
  • A third major transition occurs at the end of mitosis and is triggered by a drop in miotic cyclins

  A) cdc2 kinase

  B) cdc2 kinase

  
• Control of the Cell Cycle: The Role of Protein Kinases

  • Cdc-activating kinase (CAK) phosphorylates a threonine on CDK (stimulatory)
  • Wee1 phosphorylates a tyrosine on the Cdk (inhibitory). Wee1 holds cells back from progressing prematurely in the cell cycle so they have enough time to grow
  • The double phosphorylation of threonine and tyrosine inactivates the Cdk
  • To activate the Cdk, the phosphataes Cdc25 removes the phosphoryl group from tyrosine and Cdk can stimulate entry into mitosis
  • back in interphase, the phosphoryl group on threonine is removed and Cdk is inactive
• Cyclin D1 controls G1 to S phase transition
• Cdk control of pRB (retinoblastoma)

  pRB is a repressor of S phase genes

  • In G1, the transcription factor E2F is bound to pRB (active) on promoters, holding E2F in a repressive state which promotes gene repression.
  • Activation of Cdk phosphorylates pRB and it releases E2F, turning E2F into an activator and helps transcribe genes for S phase

  Therefore, S phase can only occur when pRB is phosphorylated and E2F is not bound to pRB. Unphosphorylated pRB will lead to repression of S phase genes
• Other inhibitory proteins of the cell cycle

  • Progression through the S phase is inhibited by the action of p27, which is a Cdk inhibitor.
  • p27 forms a trimeric complex with CycA and Cdk2, preventing their activity.
  • Elimination of this cell cycle progression inhibitor causes excess cell proliferation
• relating it back to MAPK

  Growth factors stimulate the MAPK pathway

  • One of the early target genes is Cyclin-D1, a key G1 cyclin to start the cell cycle
• • Normal cells can not proceed through the cell cycle if their DNA is damaged
  • If cells override the cell cycle checkpoints with existing DNA damage, this could lead to cancerous mutations
• Sources of DNA damage in every day life

  • UV exposure from the sun
  • Chemical carcinogens
  • Replication induced damage
  • X-rays
  • Gamma rays
  • Radon gas
• DNA damage response reactions

  1. repair of DNA damage
  2. activation of a DNA damage checkpoint, and arrest of the cell cycle for repair
  3. OR apoptosis - programmed cell death

  cancer is a result of unrepaired or poorly repaired (repaired with low fidelity) DNA damage
• Cell cycle Checkpoints

  Mechanisms to halt the the cell cycle if:

  • Chromosomal DNA is damaged
  • Critical events incomplete
  • DNA replication in S phase
  • Chromosome alignment during M phase
• What protein is activated by a double stranded break?

  The protein kinase, ATM. Ataxia-telangiectasia (AT)-mutated
• What protein is activated by a single stranded break?


  A protein kinase ATR (ATM and Rad3-related).

  • usually activated by UV-induced DNA damage which creates single-stranded breaks
• Progress through the cell cycle can be arrested at a checkpoint by:

  1. Sensors that detect chromosomal abnormalities

  • ATR
  • ATM

  1. Transmitters/transducers that signal the information

  • CHK1 and CHK2 (checkpoint kinases)

  1. Effectors that inhibit cell cycle machinery

  • p53 (guardian of the genome)
  • CDC25 (phosphatase that removed tyrosine phosphoryl group from Wee1)
• ATM and ATR activates which checkpoint kinases?

  ATM is activated by double-stranded breaks and activates CHK2. ATR activated by UV radiation induced DNA damage and activates CHK1.
• What does CHK2 do?

  Active CHK2 stabilizes P53 by phosphorylation. P53 then acts as a transcription factor to transcribe P21 gene which is a Cdk inhibitor that arrests the cell cycle thus giving the cell time to repair its DNA. If P53 cannot arrest the cell cycle, then it can also help transcribe a gene for apoptosis.
• What does CHK1 do?

  Once activated by ATR by phosphorylation, CHK1 phosphorylates Cdc25, Cdc25 has to leave the nucleus and go into the cytoplasm where it can't function. Without its function, Cdk's will remain in an inactive state, thus preventing the cell to go through mitosis (cell cycle arrest)
• The G2/M checkpoint

  • Prevents cells from entering M phase if there is DNA damage
  • Activation of CHK1 by ATR results in phosphorylation and inactivation of Cdc25
  • CDC25 inactivation is required for G2/M (and perhaps G1/S phase) arrest
• The G1S checkpoint with a Double Strand DNA break


  • Double strand DNA breaks lead to ATM activation and phosphorylation of p53
  • p53 is stabilized and transcribes p21 gene
  • The protein p21 inhibits the G1 CDK when DNA is damaged at the G1 to S transition.
  • no pRB (repressor of S phase) phosphorylation
  • no transition to S phase
  • p53 can also transcribe cell death proteins if repair is not possible.
• Cell cycle goes from G1 to S to G2 to M phase
• pRB represses genes required for S phase

  • In G1, E2F is bound to pRB on promoters (repressive)
  • Activation of CDKs phosphorylate pRB and it releases E2F, turning E2F into an activator
  • With DNA damage in G1, production of p21 inhibits the CDKS responsible for phosphorylating pRB
  • Cell cycle arrest at G1-S
• • Cancer cells are normal cells that have acquired genetic mutations that enable them to survive and proliferate independently of normal signals
  • Metastasis is the spread of cancer from where it started (primary site)
• Three main groups of tumours

  • Sarcomas – relatively rare, less than 2%
  • solid tumours of connective tissues eg. bone, cartilage, and fibrous tissues
  • Leukemias and lymphomas - approximately 8% of human cancers
  • arise from blood-forming cells (leukemia) and cells of the immune system (lymphomas)
  • Carcinomas - approximately 90% of human cancers
  • originate from epithelial cells
• Causes of Cancer

  1. Carcinogenic chemicals
  2. Ionizing radiation
  3. Viruses – alter the genome
  4. Ultraviolet light
• Viruses and cancer

  • Viruses picked up cancer causing genes a long time ago and can ‘transform’ cells (cause cancer) because they carry genes whose protein products interfere with the cells growth regulating activities (oncogenes)
• Cancer is a genetic disease, but in most cases, not an inherited one


  • Traced to alterations within specific genes
  • Genetic defects arise mostly from somatic mutations during affected individual's lifetime, not inherited from parents
  • Inherited mutations in the BRCA1 and BRCA2 genes increase the risk of breast and ovarian cancer
• Hallmarks of Cancer

  • sustaining proliferative signaling
  • evading growth suppressors
  • activating invasion and metastasis
  • enabling replicative immortality
  • inducing angiogenesis (formation of new blood vessels to deliver and transport materials)
• Contact inhibition

  • It is the inhibition of cell division in normal cells when they contact a neighbouring cell.
  • motility ceases
  • They cannot move over one another so they form a monolayer on the bottom of the dish
  • Tumour cells have lost contact inhibition allowing them to migrate up on top of each other to form a mass (foci) and openly proliferate on top of each other
• • Cancer cells become growth factor independent
  • Proliferate in the absence of serum (contains growth factors) probably due to mutations in their Ras G proteins
• Anchorage independence

  • Cancer cells can grow when suspended in soft agar (no ECM)
  • normal cells need ECM to proliferate and even survive
• Progression of Cancer

  • Mutations in tumour suppressor genes and proto-oncogenes
  • Loss of control over proliferation
  • Loss of adhesion to neighbouring cells and basement membrane
  • loss or mutation in E-cadherins
  • loss of Integrins or integrins just not needed to sustain survival signal
  • Use of matrix metalloproteinases to digest the basement membrane
  • Invasion of surrounding tissue and blood stream or lymph system
• Need around 5-7 mutations in a single cell for it to become cancerous
• Measuring metastasis

  Use of Boyden chambers to assess the ability of cells to migrate (-ECM) or invade (+ECM)