2. cell cycle Flashcards
1
Q
what are three types of systems that can be used for cancer research?
A
- people
- animal models
- cell based systems
2
Q
how can people be used for cancer research?
A
epidemiology, autopsy, genetics, GWAS
3
Q
what can epidemiology show us about cancer?
A
making connections between the population and causative agents of the disease - most connections have now been made
4
Q
what can GWAS show us about cancer?
A
they can link certain alleles with a pre-disposition to developing cancer
5
Q
what can autopsies show us about cancer?
A
look and see how the disease progressed from early to late stage in corpses
6
Q
how are animal models useful in cancer research?
A
they are good for identifying key players that can be extrapolated back to human cancer progression
7
Q
how are cell based systems a powerful tool?
A
they give us information at the biochemical level of different disease setting and identify what affects this
8
Q
what is required in order to view cancer progression?
A
a full animal system
9
Q
name one process that it is not fully possible to study in a cell systems?
A
metastasis - you can look at migration but you cannot see how it migrates and ends up at a distal location
10
Q
name 4 different types of cells that can be used as in vitro models of cancer
A
- primary cells
- immortalised cells
- transformed cells
- metastatic cells
11
Q
what type of cells are the closet we can get to the WT setting when using cells as in vitro models of cancer?
A
primary cells, these are obtained directly from the organism
12
Q
what are the downsides of using primary cells? (3)
A
- they have limited lifespan
- they don’t divide much
- there is lots of variation in primary cells
13
Q
what three types of cells can grow indefinitely in culture?
A
- immortalised cells
- transformed cells
- metastatic cells
14
Q
give an example of immortalised cells? and what can be done to them?
A
Swiss 3T3 cells - you can transform these cells by the addition of an oncogene
15
Q
what are three properties of immortalised cells?
A
- they are growth factor dependent
- they are anchorage dependent
- they are contact inhibited
16
Q
out of the three cell types that can grow indefinitely in culture, which are the most similar to the WT setting?
A
immortalised cells
17
Q
what type of WT cells are not anchorage dependent?
A
blood cells
18
Q
what is anchorage dependence?
A
when cells need tissue culture plastic as substrate to proliferate
19
Q
what is contact inhibition?
A
once cell to cell contact is obtained and a monolayer has formed, cells stop proliferating
20
Q
if a cell is not contact inhibited what occurs?
A
they proliferate out of the monolayer of the dish
21
Q
give an example of a transformed cell
A
K-Ras 3T3
22
Q
give four properties of transformed cells in virto?
A
- substrate independent
- contact independent
- reduced growth factor dependence
23
Q
when transformed cells are injected into nude mice what are they?
A
tumorigenic
24
Q
give an example of metastatic cells?
A
HeLa cells - from late stage metastatic cervical carcinoma
25
when were HeLa cells isolated?
in the 60s
26
in addition to the properties of transformed cells, what property do metastatic cells also have?
they are able to metastasise - can form colonies at different locations
27
how can you study the effect a gene has on anchorage dependence?
take Swiss T3T cells, insert gene of interest and see what affect this has on anchorage dependence i.e. can it now grow in solution
28
what does a focus formation assay look at?
contact inhibition
29
what can be deduced from the focus formation assay?
cells that grow out of the monolayer are not contact inhibited
30
what does the soft agar assay look at?
substrate dependence
31
what can be deduced from the soft agar assay?
cells that can grow in suspension in slightly solidified agar are not substrate dependent
32
what two things can be assed when cells are injected into mice?
- tumourogenesis
| - metastasis
33
what is the gold standard for being able to determine whether you are looking at a tumour suppresser or oncogene?
being able to define all the players in a system to determine exactly what is happening
34
oncogenes promote and tumour suppressers prevent what 6 things?
- substrate independence
- transformed appearance
- release from contact inhibition
- reduced growth factor requirement
- tumour formation in nude mice
- genetic instability
35
if a single cells is given unlimited ability to proliferate in 40 days, what size will it become? why does this not occur in tumour formation?
cubic metre
| cells are limited by nutrients, space and oxygen availability
36
what has been reported in clinically obese people?
tumour up to a meter in size, that have been hidden by padding and so given lots of time to grow
37
what are the 4 stages of the cell cycle?
G1, S, G2 and M
38
what happens in G1 phase?
the cell decides whether to go through the cell cycle or not
39
why is it bad to only go part the way through the cell cycle?
gene imbalance will arise from partially replicated genome
40
why might a cell decide not to divide?
the conditions are not right or the cell is not fit to do so
41
what is deregulated in cancer?
the cell cycle, cell enter the cell cycle when they should not
42
what can happen to a quiescent cells waiting in G0?
it might receive signals from the environment to enter the cell cycle and start proliferating
43
what is the restriction point?
this is the stage in G1 of the cell cycle where once you have passed you are committed to the cell cycle
44
what sort of genes are de-regulated in lots of cancers?
genes that are involved in stopping/allowing cells to pass the restriction point
45
what are four experimental systems that are used to study factors involved in the restriction point?
- somatic cell hybrids
- budding and fission yeast
- sea urchins
- virology
46
what is it called when two cells fuse together but their nuclei remain separate? and what does this allow us to do?
heterokaryon
| this allows us to see the affect that one nucleus has on another
47
what are carried using somatic hybrids to show that the are diffusible factors that regulate cell cycle?
two cells populations were synchronised in G1 and S phase and cells of each phase are fused together. G1 phase nucleus starts to replicate its DNA i.e. is in S phase
48
what do the somatic hybrid tell us about the S phase nucleus?
the S phase nucleus has told the G1 nucleus to start replicating its DNA earlier than it would have done if it did not receive these signals.
this indicates that there are diffusible factors which drive G1 into S phase
49
if cells in G1 and G2 phase are fused what occurs? and what does this tell?
nuclei remain in G1 and G2 phase
| factors that are expressed in S phase are no longer expressed by the time the cell is in G2 phase
50
if cells in S phase and G2 phase are fused what occurs? and what does this show us?
G2 phase nucleus starts to replicate its DNA, i.e. is driven onto S phase
this shows us that factors released by S phase nucleus are dominant
51
what was concluded from the somatic cell hybrid experiments?
S phase was controlled by a diffusible dominant factor and that the factor was lost in G2 cells
52
what systems were used to identify these diffusible factors?
fission yeast, budding yeast and sea urchins
53
who worked on Fission yeast and who's work on Sea Urchins Oocyetes to determine these diffusible factors?
Paul Nurse
| Tim Hunt
54
how do fission yeast divide?
they elongate their cell body while the replicate their DNA and then they split into two cells of the same size as the original
55
what factors was identified in fission yeast?
cdc2
56
what two phenotypes were observed in fission yeast?
Wee phenotype - where yeast go through division too quickly
| cdc phenotype - cell division cycle mutants - these were key for defining the soluble factor
57
how many gene can be mutated in order to get each of the fission yeast phenotypes?
one gene is mutated in the wee phenotype
| about 60 genes can be mutated to get the cdc phenotype
58
what type of protein was giving rise to the cdc2 mutants?
a 34kDa protein kinase
59
what type of experiment Nurse carry out to identify the genes mutated that give rise to the cdc phenotype?
- he used complementation experiments where he used libraries of plasmid put back into elongated yeast in order to isolate genes causing mutations
- when WT version of the gene mutated put into yeast WT division is restored
60
what did nurse see about cdc2 expression in the cell cycle ?
cdc2 is expressed at high levels throughout the cell cycle
61
what did he see regarding kinase activity in vitro and in vivo?
- in vitro cdc2 is inactive
| - in vivo cdc2 has periodic activity, with peak activity in S phase and low activity in G1 and G2 phase
62
what was discovered in Urchins Oocytes?
cyclins
63
what is useful about working Urchin Oocytes?
- they are 0.1mm in length and so it is possible to inject them with things and observe them under the microscope
- all cells are at same stage in the cell cycle before fertilisation
64
what did Hunt do in order to label proteins in Oocytes?
injected the with radiolabelled 35S methionine which would be incorporated into proteins
65
what did labelling proteins in Oocytes allow hunt to do?
this allowed him to harvest oocytes at defined times, fractionate proteins and see what proteins are expressed at different times in the cell cycle
66
how did Hunt stimulate cell cycle in Oocytes?
mocking fertilisation
67
what did Hunt observe about cyclin expression?
it was expressed for short periods right before mitosis
| as cells go through the cell cycle it is periodically expressed
68
what did Hunt show in relation to Nurse's work? and what did he do to prove this?
he showed that this cyclin expression was mirroring cdc2's activity and so is a regulatory subunit for the kinase
he showed that when the cyclin was added to cdc2 in vitro, there was a high constant kinase activity
69
a similar screen to Nurse was carried out on what type of yeast? and what did it look at?
budding yeast
| this looked at cdc28 that is almost identical at the amino acid level to cdc2
70
how evolutionarily close are budding and fission yeast and what does this suggest?
they are about as evolutionarily close to each other as they are to us and so things that are conserved between then will be conserved between us
71
what is the equivalent human proteins of cdc2?
cdk1
72
how many cyclin dependent kinases are there in human?
15
| 6 of which are important in cell cycle regulation
73
what type of kinases are cyclin dependent kinases?
they are serine threonine kinases
74
what is the conserved sequence that cyclin dependent kinases phosphorylate?
S/TxR/K
75
what are expressed at different stages of the cell cycle? and what does this mean?
cyclins
| this means that different cdks are active at different stages of the cell cycle
76
what are the function of cyclins?
the bind cdks and activate them
77
cyclins interact preferentially with specific cdks, TRUE or FLASE? and what does this mean?
TRUE
| this means that different cdks are active when different cyclins are expressed
78
what type of cyclin is expressed in early G0 phase?
D type
79
what order of the cyclins expressed in the cell cycle?
D, E, A, B
80
describe cyclin expression throughout the cell cycle with reference to cell cycle phases
- cyclin D starts to be expressed in G0 and peaks just before G1 phase, it remains persistent throughout the cell cycle
- cyclin E is expressed in G1 phase
- cyclin A is expressed in S phase and G2 phase
- cyclin B is expressed in M phase
81
how can you synthetically make cdks and cyclins? and how can this be used to assay which cdks cyclins preferentially bind?
- couple transcription and translation in an Eppendorf
- radioactively label cdks
- immunoprecipitate out for each cyclin
- when cdk binds cyclin it will be viewed on the gel
82
how does immunoprecipitation work?
antibody on solid phase specific to protein of interest, add proteins to this to see it they associate with protein of interest, when protein of interest is pulled out run complex on denaturing gel to see what has associated
83
what cdks does cyclin D bind?
cdk4/cdk6
84
what cdks does cyclin E bind?
cdk2
85
what cdk does cyclin A bind?
cdk2/cdk1
86
what cdk does cyclin B bind?
cdk1
87
when mutated, what may a cyclin become?
oncogenic
88
what cyclin is expressed as a cell exits quiescence?
cyclin D
89
what happens to cyclin E when cdc2 swaps it for cyclin A?
cyclin E is degraded
90
what phase does cyclin A persist from and into?
from S phase into G2 phase
91
what does cyclin A swap cdk2 for?
cdk1
92
what does cdk1 swap cyclin A for in M phase?
cyclin B
93
the expression of the correct cyclins activates cdks in the correct order, what does this ensure?
this ensures that cell cycle is driven in the correct direction
94
what do cdks do in the cell cycle?
they phosphorylate their substrate and assist each stage of the cell cycle
95
what are over expressed in many cancers? and what type in particular and what affect does this have?
cyclins
particularly cyclin D and E, they cyclins that are expressed at the start of the cell cycle as once the cell cycle is started it will keep going
this facilitates entry into the cell cycle
96
what might this over expression of cyclins be due to? and how can this be looked at?
the gene may have been amplified in the genome
this can be observed using nucleic acid fluorescent probes to a control locus and a specific cyclin - the control locus will be present once on each chromosome and the cyclin will be amplified throughout the genome
97
what types of mutations may result in an cyclin becoming oncogenic?
- amplification of gene
- translocation in fount of a strong constitutively active promoted
- transcriptional up-regulation due to over expression of transcription factor
98
what is seen in many multiple myeolomas?
translocation of the strong Igg promoter in front of cyclin D which leads to its amplification to kick start the cell cycle
99
the is a strong link between cell cycle deregulation and what?
aberrant proliferate in many cancers
