cell cycle growth and regulation

Question 1

what stage are most cells in the cell cycle *

Answer 1

not constantly dividing - in absence fo grwoth signals they enter G0 or quiescent phase

Question 2

describe cell cycle entry *

Answer 2

requires external stimulus this is translated into transcription of cMyc gene which stimulates the expression of the genes and proteins that regulate the cell cycle - these are transiently stimulated

if you stimulate the receptor with GF - cause increase in transcription of cMyc - this is fast transcription and occurs just before the synthesis phase - cells then enter s phase

the GF receptor is stimulated by a ligand - these are mitogenic factors that activate the signalling cascade

Question 3

describe GF stimulation of signalling pathways *

Answer 3

mitogenic GFs ie growth factor signals from other cells eg hepatocyte GF released after damage

this activates signalling cascades involving receptor protein tyrosine kinase and small G (GTP-binding) protein (Ras)

these are master regulators that go into nucleus and activate a variety of factors (ie regulatory gene proteins, early response genes (C-Myc), delayed response genes) - cascade will activate cell cycle control genes

intermediate early genes that are activated include c-Jun, c-Fos, c-Myc - they are TFs and activate other genes - they are upregulated in cancer

Question 4

what are the key components of signalling pathways *

Answer 4

regulation of enzyme activity by kinases

adapter proteins

regulation by GTP-binding proteins

Question 5

mechanism of signalling by peptide GF *

Answer 5

ligand binds to receptor = receptor dimerisation = cross phosphorylation of tyrosine kinases - activating themselves

the phosphorylation provides docking site for adapter proteins

adaptor proteins bring together kinase and substrate for protein

different phosphorylation sites = different adaptor proteins - this scaffolds cascade and results in signal being relayed

Question 6

describe how cancer treatment targets signalling by peptide GF *

Answer 6

block cascade at beginning

herceptin binds to extracellular domain - prevent ligand binding - prevents all cell signalling

herceptin is anti-Her2 Ab

Question 7

describe the function and mechanism of adaptor proteins *

Answer 7

tyrosine phosphorylation provides docking sites for adaptor proteins

this mediates protein-protein interactions ie protein binding, brings proteins closer together

proteins are modular and contain domains - functional and structural units that are copied in proteins

some domains are important in molecular recognition but have no enzymatic function of their own - bring other proteins together - proteins can swap domains and so swap function

Question 8

describe Grb2 *

Answer 8

it is an adaptor protein - small protein with 2 SH3 regions and 1SH2 region

SH = Src homology regions - these are onchogenes that contain a conserved domain

SH3 recognises proline rich domain - any protein with certain proline sequence will allow SH3 binding - this is consitiuitive ie always bound

SH2 binds to tyrosine - this is inducible depending on the specific sequence context - recognition domain is tyrosine and the 4AA before and after

Grb2 binds to EGF and HER2 receptors at tyrosine phosphorylated domain

this binding activates the cascade

Question 9

describe GTP binding (G) proteins ie Ras *

Answer 9

characterised by binding to GTP which makes it activated - this is normally transient - signal in causes GDP to be removed and bring in GTP

GTP is removed by converting it to GDP by GTPase Activating Proteins (GAP) - this hydrolysis of GTP releases inorganic phosphorus

RAS is then inactive and associated with GDP

exhange factors eg Sos activate Ras by exchanging GTP for GDP

Question 10

descrive how receptor protein tyrosine kinases are linked to Ras *

Answer 10

ligand bind = dimerised GF receptor = phosphorylation = adaptor protein coming to tyrosine kinase at C terminus

Grb domain (adaptor protein) bind to Sos constituitively (ie always)

this occurs close to membrane = activation of Ras - Ras has to be bound to the membrane to remain activated

this results in signal transmission downstream

Question 11

describe how Ras can be oncogenically activated by mutations *

Answer 11

V12Ras - constituitively active because glycine₁₂ is replaced by valine - prevents GAP binding ie inactivation

L61Ras - constituitively active because glutamine₆₁ is converted to leucine which prevents GTP hydrolysis

both mechanisms mean Ras cannot be inactivated

Question 12

describe how Ras transmits downstream signals *

Answer 12

it activates kinases that then phosphorylate each other

the downstream path of Ras is extracellular signal-regulkated kinase (ERK) cascade generically called - Mitogen-activated protein kinase (MAPK) cascades - mitogen refers to the GF

Ras activates Raf which phosphorylates MEK, which phosphrylates ERK

Question 13

significance of B-Raf *

Answer 13

it is an onchogene - mutationally activated in melanomas

Question 14

describe the effect of the Erk pathway *

Answer 14

causes change in protein activity - alter cytoskeleton proteins and transcription factors

also changes gene expression - phosphorylates gene regulatory proteins a and b eg phosphorylation of c-Myc which enters the cell cycle

Question 15

what are cyclin dependant kinases (Cdks) *

Answer 15

they are present in proliferating cells throighout the cell cycle

activity is regulated by interaction with cyclins and phosphorylation

they are cyclically activated protein kinases and they control the cell cycle

Question 16

describe cyclins *

Answer 16

they are transiently expressed at specific points in the cell cycle

regulated at level of expression

sythesised and then degraded

up and down regulation is important for activity

Question 17

describe cyclin-cdk complexes and their roles in the cell cycle *

Answer 17

the start kinase phsophylates cdk which inactivates the cyclin

cyclin is degraded

at the start of G1 - cdk binds to a different cyclin - forms complex that starts dna replication machinery - cyclin is then chopped up

cdk binds to another subset of cyclin - these are mitotic cyclins - cdk1 binds to mitotic cyclin B

Question 18

describe the regulated expression of cyclins *

Answer 18

there is a gradual increase in cyclin then a sudden drop in relative concentration as they are degraded at the end of mitosis

this illustrates the transient down regulation of cyclins

Question 19

describe the regulation of cdks by phosphorylation *

Answer 19

cdk1 binds to cyclin B - this is inactive - there needs to be another level of regulation

cdk is phosphorylated by cdk activating kinase (CAK) and inhibitory kinase (Wee1)

even though Cdk1 is bound to cyclin - it needs inhibitory kinase to be removed before it is active

phosphtase (Cdc25) clips the inhibitory site (removes the phosphate that was added by Wee1) = actuvated complex at end of interphase

Question 20

describe how phosphtase activates cdk1 and mitotic cyclin B *

Answer 20

cd25 activates phosphtase

positive feedback reinforces activation of MPF and drives mitosis

Question 21

why is cdk activatioin complicated *

Answer 21

to check that everything is ready for progression of the cell cycle

Question 22

describe how cdk1-cycB activation fits into the cycle *

Answer 22

in early mitosis - cdk1/cycB is active - mitosis is put on hold - key substrates are phosphorylated

at the anaphase checkpoint the signal from the fully attached kinetochores cause cyclin B to be degraded - cdk1 is inactivated, key substrates are dephsophorylated - mitosis progresses

Question 23

describe teh variation in cyclin cdk complexes *

Answer 23

different cyclins and cdks are required at different stages of cell cycle

in M - cdk1 bind cyc B

between g1 and S - cdk2 binds cycE

in S - cdk2 binds cyc A

cyclins target cdk to specific substrates depending on the cell cycle phase because of the substrate availability at that phase

Question 24

describe how GF stimulation links to cyclins *

Answer 24

GF stim = activation of Ras = production of early immediate gene TFs eg c-jun, c-fos, c-myc

c-myc stimulates cyclin D1 to enter the cell cycle

cdk4/6 binds to cycD and causes cell to enter G1 - this stimulates the synthesis of cyclin E

Question 25

significance of cyclin D1 *

Answer 25

it is an onchogene that is overexpressed in 50% of breast cancers

Question 26

describe the regulated expression of cyclins/cdks *

Answer 26

cdks become sequentially active and stimulate synthesis of genes for next phase - eg cyclin D/cdk4/6 stimulates expression for cyclin E - this gives direction and timing to the cycle

cyclins are suseptible to degredation - hence cyclical activation

cMyc activates cyclinD4/6 - causing cell to go from G0 - G1, this degrades which causes activation of cyclin E which activates A which activates B

B sharply degrades at the anaphase checkpoint

Question 27

what do activated cdk’s do *

Answer 27

they are phosphorylated and bind to cyclins - activated

then they phosphorylate proteins on serine or threonine to drive cell cycle progression

cdk1/cyclin B is the M-phase promoting factor and phosphorylates nuclear lamins - they are the structural component of the nuclear envelope - break down of these causes dissembly of the membrane

cdk2/cyclinE is the start kinase for synthesis - posphorylates retinoblastoma protein which os a tumour suppressor that is inactivated in many cancers

Question 28

describe regulation by Rb *

Answer 28

pRb acts as a break on the cell cycle

cdks phosphorylate at multiple sites, and progressively inactivate pRb

Rb is a tumour suppressor

active rb is found in G0 - it binds to E2F keeping E2F inactivated

cdks phosphorylate rb = inactivate rb = activate E2F which is a TF that allows cyclin E transcription

Question 29

what genes are regulated by TF E2F *

Answer 29

proto-oncogenes - c-myc, n-myc

cell cycle - E2f-1,2,3, pRb, cyclin a and e, cdk2 and 4

dna synth - thymidine kinase, thymidine synthetatse, dihydrofolate reductase, dna polymerase

Question 30

effect when pRb is mutated*

Answer 30

E2f is always activated = cell cycle on full drive

transcribe and increase levels of all cyclin and cdks

Question 31

summarise the regulated control of the production of cyclins *

Answer 31

c-myc promotes cyclinD transcription which promotes kinases that phosphorylate pRb - pRb releases E2f = transcription of cyclin E which binds to cdk2 = kinase phosphorylate pRb = transcription of A which binds to cdk2 = kinase phos prb = increase TF = transcription of cyclin b which binds to cdk1

Question 32

what is deregulated in the cell cycle process in tumours *

Answer 32

more than 1 step - cause deregulation

Question 33

what are the 2 things that regulate cyclin/cdk

Answer 33

phosphtases that deactivate them

inhibitors that bind to them so they cant exert their function - these are cdk inhibitors (CKI)

Question 34

describe the families of CKI *

Answer 34

2 families separated by timing of cell cycle

INK4 family - p15^INK4b p16^INK4a, p18^INK4c, p19^INK4d

CIP/KIP family - p21^CIP1/WAF1, p27^KIP1 p57^KIP2

INK familiy are G1 phase CKIs - they inhibit cdk4/6 by displacing cyclin d

CIP/KIP family - s phase cki - inhibit all cdks by binding to the cdk/cyclin complex (no displacement)

Question 35

describe the control of CKIs *

Answer 35

must be degraded to allow progression of the cell cycle

INK degrade at G1 phase to allow progression of cell cycle

CIP degrade at S so can get activation of cyclin a and b

Question 36

what type of cancer has a large proportion of loss of p16 *

Answer 36

pancreatic

Question 37

what cancer has a large overproduction of cdk4 or cyclin d *

Answer 37

mantle cell cancer

Question 38

what cancer has a loss of RB *

Answer 38

small lung cell cancer

and non-small, but less so

Question 39

what are proto-onchogenes *

Answer 39

normal proteins in cell that become changed

Question 40

what are the onchogenes *

Answer 40

EGFR/HER2 - mutationally activated/overexpressed in breast cancer - herceptin Ab is treatment for metastatic breast cancer

Ras - mutationally activated in many cancers - treated by inhibitors of membrane attachment = reduction in downstream pathways

cyclin D1 overexpressed in 50% breast cancer

B-raf mutationally activated in melanomas - treatment is kinase inhibitors in trials

c-myc is over expressed in many tumours

Question 41

what are tumour suppressors &

Answer 41

rb inactivated in many cancers

p27^{kip1 underexpression - relates to poor prognosis in many malignacies}

Question 42

what makes pathway blocking more difficult *

Answer 42

blocking higher up in the pathway

Question 43

what are motility circles *

Answer 43

when cells have metostatic potential