Molecular biology 2

Question 1

describe what occurs in the G1/DNA damage checkpoint

Answer 1

1. The G1 checkpoint is mediated by a transcription factor- p53
2. in response to DNA damage, levels of p53 increase
3. many cancers have mutations in p53 to bypass this checkpoint

Question 2

what are the roles of p53

Answer 2

1. causes the transcription of p21, a CDK inhibitor
   - CDK inhibition by p21 prevents cell cycle progression
   - p21 also binds to and inhibits PCNA, a component of the DNA replication machinery, preventing its activity
2. stops cell division to allow for DNA repair
   - where DNA damage is irreparable, p53 initials signalling pathways that lead to apoptosis

Question 3

describe what occurs in the G2 checkpoint

Answer 3

1. mediated by the Chk1 kinase as well as p53
2. in response to unreplicated or damaged DNA, Chk1 inactivates the Cdc25 protein
   - without cDC25, CDK1/Cdc2 remains inactive and cells arrest
   - some cancers overexposes CDC25

Question 4

what are the stages of mitosis

Answer 4

• prophase
• prometaphase
• metaphase
• anaphase
• telophase

Question 5

explain what happens in prophase

Answer 5

1. chromosomal material condenses to form chromosomes composed of 2 chromatids attached together at the centromere
2. cytoskeleton is dissembled and mitotic spindle assembled
3. nuclear envelope disperses

Question 6

describe what happens in prometaphase

Answer 6

1. chromosomal microtubules attach to kinetochores of chromosomes
2. chromosomes are moved to spindle equator

Question 7

describe what happens in metaphase

Answer 7

1. chromosomes are aligned along the metaphase plate, attached by microtubules to both poles

Question 8

describe what happens in anaphase

Answer 8

1. centromeres split and chromatids separate
2. chromosomes move to opposite spindle poles
3. spindle poles move further apart

Question 9

describe what happens in telophase

Answer 9

1. chromosomes cluster at opposite spindle poles
2. chromosomes become dispersed
3. nuclear envelope assembles around chromosome clusters
4. daughter cells formed by cytokinesis

Question 10

describe what happens in the M phase checkpoint

Answer 10

1. also known as spindle assembly checkpoint (SAC)
2. unattached kinetochores inhibit APC/C and therefore anaphase initiation

Question 11

describe what occurs in the control of chromatid seperation

Answer 11

1. a cohesion complex binds sister chromatids together
2. cohesins need to be cleaved by separase before chromatids separate in anaphase
3. separase is kept inactive by securing until its degraded by proteolysis via APC/C
4. when cell is ready to proceed, M-CDK phosphorylates APC/C facilitating cdc20 binding, which activates the complex

Question 12

describe the current state of cancer genomics

Answer 12

1. cancer gene census currently lists 578 genes where mutations have been implicated in cancer
   - many relate to cell cycle control and apoptosis

Question 13

what happens when cell cycle control is lost

Answer 13

1. alterations in cell proliferation
2. alterations in DNA damage response
3. alterations in cell growth

Question 14

what are oncogenes

Answer 14

1. drive abnormal cell proliferation
   - may represent the overactive form of normal cellular genes (protooncogenes) or may enter cell as part of a virus

Question 15

what are tumour suppressors

Answer 15

genes that normally inhibit cell proliferation and tumour development
- in tumours, these are often lost or inactivated
- this usually requires 2 mutational events

Question 16

what type of mutation are oncogenes

Answer 16

1. dominant
2. a single mutation event in proto oncogene creates oncogene
   - activating mutation enables oncogene to stimulate cell survival and proliferation

Question 17

what are the 3 ways a proto oncogene can be made overactive and converted into an oncogene

Answer 17

1. deletion or point mutation
2. gene amplification
3. chromosome rearrangement (translocation)

Question 18

what does a normal human male karyotype consist of

Answer 18

22 pairs of autosomes and 2 sex chromosomes- X and Y
- Cancer genes often show karyotypic abnormalities

Question 19

what is the Philadelphia chromosome and what is its role

Answer 19

1. reciprocal chromosomal translocation event
2. found in most chronic myeloid leukaemia patients
3. the ABL1 gene from chromosome 9 and the BCR gene from chromosome 22 fuse
4. codes a hybrid constitutively active tyrosine kinase

Question 20

what are the functions of proto oncogenes

Answer 20

1. growth factors
2. growth factor receptors- eg receptor tyrosine kinases
3. elements of intracellular signalling pathways- regulatory GTPases (Ras)
4. transcription factors
5. genes associated with angiogenesis, invasiveness and metastasis- VEGF and VEGFR

Question 21

describe the effect of the G:C to T:A mutation in the genetic code

Answer 21

1. Glycine >T transversion mutation in codon 12 of Ras protein causes impaired GTPase function, leaving Ras constitutively switched on
2. cell division irrespective of growth factor signalling

Question 22

what type of mutations do tumour suppressor genes cause

Answer 22

1. recessive
   2.mutation event inactivates tumour supressor gene
   - no effect of mutation in one gene copy
2. second mutation event inactivates second gene copy
3. 2 inactivating mutations functionally eliminate the tumour suppressor gene, stimulating cell survival and proliferation

Question 23

what are the 3 types of tumour suppressor genes

Answer 23

1. gatekeepers- monitor cell division and induce apoptosis
   - eg Rb, CKIs, apoptosis genes
2. caretakers- promote genome stability and oppose mutation rates via checkpoints and DNA repair
   - p53
3. landscapers- control the cellular microenvironment
   - genes relating to extracellular matrix proteins, integrins

Question 24

describe the characteristics of Rb mutations

Answer 24

1. can be inherited or non hereditary
2. both copies of Rb need to be mutated for tumour formation
3. sometimes one mutation can be inherited and the second acquired

Question 25

describe the mutator phenotype hypothesis

Answer 25

1. mutation inactivates tumour supressor gene
2. cells proliferate
3. if mutation inactivates DNA repair gene, the cells have acquired a mutation that encourages additional mutations so process accelerates
4. mutation of proto oncogene can then create an oncogene
5. mutation inactivates more tumour suppressor genes
6. leading to cancer

Question 26

what are BRCA 1/2 genes

Answer 26

• caretaker tumour suppressor genes
• increases risk of developing breast cancer

Question 27

what are BRCA mutations

Answer 27

1. double strand DNA breaks can form from replication stress, exogenous stress
2. BRCA can help to repair these breaks so the cell cycle can continue
3. when lost, DNA is repaired through less accurate pathways

Question 28

describe the role of cadherins

Answer 28

1. cadherins mediate mechanical attachment of neighbouring cells
2. within adheren junctions, cadherins link to actin filaments, facilitated by B-catenin linkers

Question 29

describe the role of B-catenin as a landscaper

Answer 29

1. landscaper tumour suppressor gene
2. bound to cytoplasmic domain
3. roles in cell to cell adhesion but also in coordinating proliferation with the Wet signalling pathway
4. B catenin binding to cadherins antagonises Wnt

Question 30

describe the role of B catenin as a proto oncogene

Answer 30

1. when B catenin is not bound to cadherin, forms part of a cytosolic pool
2. free B catenin acts as a transcription factor, up regulating proliferation
3. Apc protein forms a destruction complex
4. Apc inactivated by Wnt signalling at Frizzled

Question 31

when does cancer arise

Answer 31

when suppressive pathway components are overpowered by proliferative pathway components

Question 32

describe the stepwise evolution of cancer

Answer 32

1. genetic alteration and loss of APC leads normal epithelium to become hyper proliferative epithelium
2. increased genetic instability and loss of P53 leads to early adenoma
3. activation of K-Ras leads to intermediate adenoma
4. loss of tumour suppressors leads to late adenoma
5. further loss of p53 leads to carcinoma
6. other unknown alterations leads to metastasis