Molecular biology of cancer 1

Question 1

what is the cell cycle

Answer 1

the process by which a single mother cell gives rise to 2 identical daughter cells

Question 2

why is cell cycle control studied

Answer 2

1. to understand development
2. stem cells
3. differentiation
4. opportunities for therapeutic interventions

Question 3

how do we study cell cycle

Answer 3

1. in whole organisms- yeast
2. in cell free extracts- Xenopus oocytes
3. in cell culture- mammalian cells

Question 4

what are the events of the cell cycle which can be seen under a microscope

Answer 4

1. mitosis and cytokines (M phase) can be easily observed but only occupy a small fraction of the cell cycle
2. the remaining, longer part of the cycle is the interphase

Question 5

describe the process of genetic dissection of the cell cycle using yeast models

Answer 5

involves spindle formation and nuclear envelopes divide
-can see the different cell cycles through microscope

Question 6

what are the advantages in using yeast models for genetic dissection of the cell cycle

Answer 6

1. rapid reproduction
2. genome size <1% of mammals
3. amenable to genetic manipulation- gene deletions, replacement or alteration
4. can proliferate in haploid state- single copy of each gene present
5. can make temperature sensitive mutants

Question 7

how can cell cycle stages be identified

Answer 7

1. by using radiolabelled nucleotides and x ray photography
2. using artificial analogues and antibody staining
3. flow cytometry

Question 8

outline the cell cycle phases

Answer 8

1. G1- first gap phase
   - cell growth
2. S- synthesis phase
   - all DNA in the cells genome is replicated
   - cell growth continues
3. G2- second gap phase
   - cell growth and checking for replication errors
4. M- mitosis phase, usually followed by cytokinesis
   - chromosomes are partitioned into 2 daughter cells which finally divide

Question 9

what is involved in flow cytometry

Answer 9

1. cells stained with DNA dyes to allow discrimination of cells with variable DNA content
2. cells in G1 phase contain half the DNA of cells after DNA replication in G2 and M
3. cells in this process of replication (S phase) contain an intermediate quantity

Question 10

what are the main 2 factors that the cell cycle is controlled by

Answer 10

1. the point of entry into the cell cycle
2. at several critical checkpoints

Question 11

what is the entry into cell cycle controlled by

Answer 11

availability of growth factors

Question 12

describe the effect of availability of growth factors

Answer 12

1. growth factor availability controls the cell cycle at the restriction point in late G1, acting as mitogens
2. induce synthesis of D type cyclins via the MAPK signalling pathway
   - CDK4/6 with cyclin D then drive passage through restriction point
3. if growth factors aren’t available, cells enter a quiescent stage of the cycle called G0
4. once the cell passes the restriction point, its irreversibly committed to dividing, even if the growth factor signal is removed
5. cells have a limited proliferation capacity
   - eventually enter G0 permanently
   - known as replicative senescence

Question 13

outline the cell cycle checkpoints

Answer 13

1. ensure complete genomes are transmitted faithfully to daughter cells
2. a checkpoint in G2 phase arrests cells in response to damaged or unreplicated DNA
3. A checkpoint in M phase arrests mitosis if daughter chromosomes are not properly aligned on the mitotic spindle
4. the G1 checkpoint detects presence of damaged DNA and leads to cell cycle arrest

Question 14

what is cell cycle progression controlled by

Answer 14

cyclin dependent kinases
- CDKs

Question 15

what are CDKs and how do they work

Answer 15

1. protein kinases whose activity rises and falls during cell cycle
2. phosphorylation of intracellular proteins initiate or regulate the major events of the cell cycle
3. partially activated by cyclins and regulated by multiple processes
4. cyclins undergo synthesis and degradation in each cycle
   - CDK levels are constant
   - cyclins accumulate throughout interphase and are rapidly degraded towards the end of mitosis

Question 16

what is the key to cell cycle control

Answer 16

cyclin availability
- controls the activity of CDKs and promotes cycle progression
- when complexed with an appropriate M phase cyclin, the CDK triggers the mitosis machinery
- when complexed with an appropriate S phase cyclin, CDK triggers DNA replication
- there are specific CDK/cyclin complexes for different cell cycle phases

Question 17

which complexes are essential for normal cell cycle

Answer 17

CDK1, cyclin B1 and cyclin A2

Question 18

describe the mechanisms of CDK regulation

Answer 18

1. association with cyclins
2. phosphorylation events
   - activating
   - inhibitory
3. association with CDK inhibitors
4. degradation of cyclins/CKIs

Question 19

what occurs in positive CDK regulation by phosphorylation

Answer 19

CDK activation by cyclin and CAK at threonine 160

Question 20

what occurs in negative CDK regulation by phosphorylation

Answer 20

1. additional regulation of CDKs by inhibitory phosphorylation by Wee1 at threonine 14 and at tyrosine 15
2. opposed by Cdc25 phosphatase

Question 21

describe the CDK regulation by association with CDK inhibitors (CKIs)

Answer 21

1. CKIs are inhibitory proteins
   - bind to CDK and cyclin complexes
   - distort active site
   - insert into ATP binding site
2. p21 and P16 thought to be the dominant inhibitors of cell proliferation in senescent cells

Question 22

what are the 2 families of CKIs in mammalian cells

Answer 22

1. Ink4 family- p15, p16, p18, p19
   - inhibit CDK4/CDK6 (G1 phase)
2. Cip/Kip family- p21, p27, p57
   - inhibit CDK1/CDK2 (G1, S, G2 and M phase)

Question 23

what are the cell cycle control processes

Answer 23

1. control of cell cycle entry
2. once per cycle DNA replication in S phase
3. DNA damage and replication checkpoints
4. the events of mitosis

Question 24

what does MAPK stand for

Answer 24

mitogen activated protein kinase

Question 25

what is the role of RTK

Answer 25

most RTKs activate Ras- a monomeric small GTP binding protein

Question 26

describe the properties of Ras

Answer 26

1. Ras feeds into many intracellular signalling pathways
   - one of these is the MAPK, acting as phosphorylation cascade
   - cascade eventually alters activity of target proteins and causes changes in gene expression

Question 27

how does the MAPK pathway initiate cell division

Answer 27

1. receptor tyrosine kinase interacts with growth factor
2. MAPK phosphorylation cascade leads to expression of transcription factors
3. this causes transcription of cyclin D, which activates CDK4/6

Question 28

describe the properties of the retinoblastoma protein (pRb)

Answer 28

1. a tumour suppressor protein
2. usually binds to and inactivates E2F proteins
3. when cell is ready to divide (G1 restriction point), phosphorylation of pRb frees E2F to act as a transcription factor
4. E2F acts on many S phase genes, leading to cell cycle entry and DNA synthesis

Question 29

what is the main target of CDK4/6 with cyclin D

Answer 29

Rb protein

Question 30

when does non mutagenic replication of every nucleotide occur

Answer 30

occurs only once per cycle

Question 31

describe how cancer chemotherapeutic agents work

Answer 31

1. stop cell division by blocking DNA replication, causing DNA damage or blocking mitosis specifically
2. this causes growth arrest by triggering cell cycle checkpoints
3. cancer treatments can also act via inhibition of growth factor signals, reducing entry into cell cycle (herceptin)