2- Mechanisms of Disease I: Cell growth and cell differentiation Flashcards
Mechanism that turns zygote into mature multicellular organism?
- Cell growth = bigger organism, more cells
2. Differentiation = cells become complex, (usually an end to growth)
State the 3 main groups of diseases related to cell growth and differentiation
- Developmental conditions
Can be related to cell growth or differentiation (or both)
E.g. Neural tube defects like spina bifida - Neoplasia (and metaplasia)
E.g. cancer, tumours - Others, e.g. cardiac hypertrophy
What are the 2 main forms of cell growth?
Hypertrophy
Hyperplasia
What is cell growth balanced by?
cell death
What is hypertrophy?
Increase in cell size
More proteins, more membrane etc
Elevated protein synthesis is a big driver of increased cell size
What is hyperplasia?
Increase in cell numbers
-caused by cell division/ proliferation
What are 3 examples of when apoptosis happens during development?
→ Separation of digits
→ involution
→Immune and nervous system development
What are growth factors?
→ Proteins that Stimulate proliferation (mitogens) and promote survival
e.g. Growth factors and interleukins (EGF, FGF, NGF, PDGF, IGF1, IL2, IL4)
What is the function of TGFβ ?
→ Stimulates differentiation and inhibits proliferation
What is the function of the TNFα family?
→ Induce apoptosis
What are the three broad classes of growth factors?
→ Paracrine
→ Autocrine
→ Endocrine
What is paracrine?
→ produced locally to stimulate proliferation of a different cell type that has the appropriate surface receptor
What is autocrine?
→ produced by a cell that also expresses the appropriate cell surface receptor
What is endocrine?
→ like conventional hormones and released systematically for distant effects
What is the function of Wnt Ligands?
→ can inhibit or promote cell growth depending on context
What are the characteristics of terminally differentiated cells?
→ post-mitotic
→ exit from cell cycle
→ elicit a specific tissue type gene expression
→ cell morphology and function changes
What is common between cell growth and differentiation?
→ The mechanisms governing them
→ governed by the integration of multiple signals intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
→ Signals converge on the promoters of key genes
Promoters act as “co-incidence detectors”
Express gene YES/NO? How much?
How do extracellular signals induce gene expression?
Growth factor binding to its receptor
Activating signalling transduction pathway via kinase cascade
Activate transcription factors in the nucleus
Transcription factors drive expression of downstream genes
Creating a mRNA – exported back to cytoplasm – where protein synthesis happens
Proteins made can remain in cytoplasm or they can be membrane proteins going back to membrane
Or can be transcription factors or other nuclear proteins that return to nucleus and drive expression of further downstream genes
What happens during the M phase?
→ Separation of chromosomes
What happens to daughter cells after mitosis?
→ undergoes interphase and it grows in size, synthesises macromolecules
→ leave the cell cycle- becomes quiescent and is terminally differentiated (G0)
What happens to quiescent cells?
→ can remain in G0 indefinitely
→ can rejoin cell cycle- some stimulus will make them proliferate again
→ can begin a process towards differentiation- terminally differentiated and will be considered post mitotic cells
How many copies of each chromosome do you have in G1 and S phase?
2
How many copies of each chromosomes do you have in G2 and M phase?
4
How is DNA replicated?
→ Semi conservatively
In what direction is new DNA synthesized in?
→ 5’ to 3’
What is FACs used to measure?
→ If a DNA stain is applied, FACs can measure the DNA content of every cell in a population.
What is the amount of DNA in each phase when rate of division is LOW?
→ G1= 60%
→ S= 20%
→ G2/M= 20%
→ see image
What is the amount of DNA in each phase when rate of division is HIGH?
→ G1= 40%
→ S= 40%
→ G2/M= 20%
→ see image
How can we use fluorescence microscopy to view cells in the cell cycle?
Blue= DNA Red = γ-tubulin Green = CHEK2 Yellow = centrioles(γ-tubulin and CHEK2 colocalised)
What happens during prophase?
→ Nucleus becomes less defined
→ microtubular spindle apparatus assembles
→ centrioles migrate to poles
What happens during prometaphase?
→ Nuclear membrane breaks down
→ Kinetochores attach to the spindle in the nuclear region
What happens in metaphase?
→ Chromosomes line up in the equatorial plane
What happens in anaphase?
→ Chromatids separate and migrate to opposite poles
What happens in telophase?
→ daughter nuclei form
What happens during cytokinesis?
→ Division of cytoplasm
→ Chromosomes decondense
What are cell cycle checkpoints?
→ Controls involving specific protein kinases and phosphatases ensuring the strict alternation of mitosis and DNA replication
How many cell cycle checkpoints are there and when do they occur?
→ 3
→ Before the S phase- G1 checkpoint
→ Before the cells start mitosis- G2 checkpoint
→ At mitosis- M checkpoint
What is the checkpoint before S phase for?
→ Checking if the DNA is not damaged
→ right cell size
→ metabolite and nutrient stores- enough energy for division
What is the checkpoint before mitosis for?
→ Checking if the DNA is completely replicated and checking for damage
What is the checkpoint at mitosis for?
→ Checking if the chromosomes are aligned on the mitotic spindle
When are cells responsive to growth factors?
→ G1 phase
→ main site of control for cell growth
What is the function of CDK?
→ recognising target proteins and phosphorylating them
CDK (catalytic subunit) binds to cyclin (regulatory subunit) to form active CDK-cyclin complex which will be able to bind and phosphorylate specific protein substrates.
What are the 4 ways that CDK is regulated?
→ Cyclical synthesis (inducing gene expression) and destruction (targeting to the proteasome)
→ post translational modification by phosphorylation - resulting in activation, inhibition or destruction
→ dephosphorylation
→ binding of cyclin dependent kinase inhibitors (CDKIs)
How does E2F become inactive?
→ Retinoblastoma is unphosphorylated and it can bind E2F
→prevents E2F from being able to bind to the promoter genes and drive their transcription
→ this inactivates E2F and prevents it from stimulating S phase protein expression
How does E2F become active?
→ Cyclin D forms a complex with CDK4
→ Cyclin E forms a complex with CDK2
→ these phosphorylate retinoblastoma protein which can no longer bind to E2F
What does active E2F cause?
→Released E2F able to bind promoters of its target gene
→stimulates expression of more Cyclin E and S-phase proteins e.g. DNA polymerase, thymidine kinase, PCNA etc.
→DNA replication starts.
By becoming partially active drives expression of more cyclin E- creating positive forward feed loop
What is retinoblastoma protein? (RB)
→tumour suppressor protein that is dysfunctional in several major cancers
→prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide
→RB is a key substrate of G1 and G1/S cyclin-dependent kinases
What does E2F cause the transcription of?
→ DNA polymerase
What are the two families of Cyclin dependent kinase inhibitors?
→ CDK inhibitory protein (CIP/KIP) called CDKN1
→ Inhibitor of kinase 4 family (INK4) CALLED CDKN2
What is the function of the CDKN1s?
→ Inhibit all CDK-cyclin complexes (late G1,G2, and M)
→ Are gradually sequestered by G1 CDKs allowing activation of later CDKs
What are CDKN1s stimulated by?
→ stimulated weakly by TGFβ
→strongly by DNA damage (involving TP53)
What are CDKN2s stimulated by?
→TGFβ
What is the function of CDKN2s?
→ Specifically inhibit G1 CDKs
How is cyclin expression induced?
→ Growth factor binds to growth factor receptor on the membrane
→ a signal transducer is activated
→ a cascade occurs and transcription is initiated
Describe the sequential activities in the cycle?
→ Growth factor signalling activates early gene expression (transcription factors -FOS, JUN and MYC)
→ early gene products stimulate delayed gene expression (cyclin D, CDK4/6, and E2F)
→ E2F sequestered by binding to unphosphorylated retinoblastoma protein RB
→ G1 cyclin-CDK complexes hypophosphorylate RB then G1/S CDk hyperphosphorylate RB releasing E2F (G1/S phase)
→E2F stimulates expression of more (E2F responsive genes) cyclin E and S phase proteins (DNA polymerase, thymidine kinase)
How many genes do humans have encoding for CDK?
→10
How many genes do humans have encoding for cyclin protein?
→over 20
What are the three outcomes if DNA is damaged in the cell cycle?
→ Stopping the cycle (CDK inhibitors, CHEK2)
→ attempting DNA repair (nucleotide or base excision enzymes, mismatch repair)
→ Programmed cell death if repair is impossible (BCL2 family, caspases)
Describe how DNA repair genes are expressed via p53?
→ double strand break or a change in base pair occurs
→ the damage is detected by kinases (ATM and ATR)
→ kinases activate CHEK2
→ one of the substrates of CHEK2 is p53
→ when p53 is phosphorylated it cannot be degraded and becomes stable
→ it can bind promoters which express DNA repair genes
