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BMS379 Cancer Biology > Lecture 6 Cell Cycle > Flashcards

Lecture 6 Cell Cycle Flashcards

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1
Q

What are the two functions of cyclins

A

Activate catalytic subunits of the relevant Cdk and target these catalytic subunits within the cell to specific substrates

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2
Q

How are temperature-senstivite mutations in yeast used to study cell cycle genes

A

Temperature sensitive mutations are used to screen for specific cell cycle genes. These are mutations that only manifest at specific temperatures when the permissive temperature is changed to a restrictive temperature

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3
Q

What was the significance in the similarity between the protein sequence of MPF and the base sequence of cdc2

A

The sequence of the cdc2 gene identified by Paul Nurse was almost identical to that of MPF identified by Masui and Lokha. It was later determined that MPF actually consisted of cdc2 and cyclin B the protein found to oscillate in sea urchin eggs by Tim Hunt

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4
Q

What is useful about female frogs that allows the study of their eggs extremely easy

A

Injection of progesterone into a female frog will cause her to lay eggs

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5
Q

Which region of cdks becomes phosphorylated to increase catalytic activity

A

T loop

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6
Q

Explain the experiments carried out by Rao and Johnson and how this developed our understanding of the cell cycle

A

Rao and Johnson took cells that were in interphase and metaphase and fused them together by infecting both with Sendai virus that elicited fusion. Fusion of the interphase and mitotic cells caused the interphase cells to enter mitosis prematurely regardless of where they were in the cell cycle. These experiments demonstrated that mitosis was somehow the dominant program for a cell

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7
Q

What is meant by the execution point of a particular gene

A

The point in the cell cycle that a mutation in a gene causes arrest

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8
Q

What is significant about yeast cdks

A

Yeast possess only one cdk and this is capable of mediating the entire cell cycle

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9
Q

Explain the phenotype of wee1 temperature sensitive mutant S.pombe relative to the cell cycle

A

Wee1 mutants are shorter than normal because they are dividing prematurely and spending less time in G2

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10
Q

How can you morphologically determine where S. cerevisiae yeast are in the cell cycle

A

Depending on the size of the daughter bud you can determine where the cell is in the cell cycle

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11
Q

Describe an in vivo assay that can be use to determine whether cells are in S phase

A

Incubate cells with halogenated nucleotide derivatives such as bromo-deoxyuridine which will be incorporated into the DNA if the cells are in S phase. These halogenated bases can then be recognised with specific antibodies that recognise individual halogens

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12
Q

Below is a table containing a list of the stages of the cell cycle. Fill in the column with the average time that a cell spends in each phase?

A

See completed table below

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13
Q

Why can’t we investigate the effects of mutating genes involved in the cell cycle and how is this overcome

A

If you mutate a gene that controls the cell cycle in a yeast cell it is likely to kill that cell and hence there will be no cells to study.

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14
Q

How was cdc2 identified

A

Yeast cells were mutagenized and colonies with the elongated phenotype were identified. A library of yeast genes were then introduced into cells from these colonies to see which one rescued the wild type phenotype. This lead to the identification of cdc2

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15
Q

How did Tim Hunt discover cyclins

A

Bathed sea urchin eggs in radioactive methionine and observed the radioactive proteins produced. He observed that a particular protein accumulated for a time but then periodically disappeared just before the cells divided (mitosis). This implied that these proteins were being destroyed and this operated in parallel with egg division. This was later found to be cyclin B whose levels fluctuate during the cell cycle and peak before mitosis

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16
Q

How have Xenopus eggs been used to observe replication and mitosis

A

Centrifugation of Xenopus eggs creates a pellet containing the nuclei and on top of that a cellular extract. This concentrated cell extract retains all of the functionality of the egg containing the proteins and lipids as well as preserving the functional mRNA. If DNA or chromatin is added to this cell extract with will acquire its own nuclear envelope in vitro. This allows you to observe replication and mitosis easily

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17
Q

How does Eukaryotic replicator selection occur

A

Origin replication complex (ORC) binds to the replicator sequence. Helicase loading proteins Cdc6 and Cdt1 then bind to ORC to convert the single stranded replicator sequence into a pair of replication forks. Mcm2-8 then also binds to complete formation of the pre-RC

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18
Q

What is the effect on the cell cycle of introducing dominant negative Akt into cells

A

This increases the activity of p21Cip1 and p27Kip1 subsequently leading to a global inhibition of proliferation

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19
Q

Which component of the pre-replication complex was found to mediate the transition into S phase

A

Removal of cdt1 allows the functional recruitment of mcms and S phase to occur

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20
Q

Describe the role of phosphatases in regulating cdk activity

A

The cdc25 phosphatases antagonise wee1 and dephosphorylate cdks returning them to an active state

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21
Q

What kind of cell cycle arrest is indicated by the two FACS traces below how do they suggest different arrest points

A

The univariate FACS graph on the left shows that all the cells are arrested in G1 phase. However the bivariate FACS trace suggest that these cells are trying to enter S phase indicated by cells in the dashed box however these cells are unable to complete S phase and enter G2. Hence this is a DNA replication problem rather than a growth one.

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22
Q

Which proteins are responsible for the phosphorylation of Cdks that increase their catalytic activity

A

The CDK7–cyclin H complex consisting of the cdk-activating kinase (CAK)

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23
Q

Regulation of cdk activity occurs only at the level of cyclin binding T or F

A

F – additional regulation is going on

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24
Q

What kind of protein is S. pombe cdc2

A

Cyclin-dependent kinase (Cdk1)

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25
Q

Given that the average S phase lasts 7.5 hours and this accounts for 35% of the cell cycle how long is the average cell cycle

A

21.42 hours – 7.5/0.35

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26
Q

How can you determine the amount of time cells spend in M phase

A

Stain the cells for tubulin using fluorescently labelled antibodies for tubulin. Then count the number of cells that have formed the mitotic spindle/metaphase plate. The percentage of the cells can then be used to work out the percentage of the cell cycle this stage must account for

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27
Q

Explain the interaction between cdc2 cdc25 and wee1

A

Cdc25 and wee1 both act upstream of Cdc2. Cdc25 is a positive upstream regulator whereas wee1 is a negative regulator

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28
Q

How can phosphorylation of cdks be used to negatively regulate its activity

A

Phosphorylation of threonine 14 and tyrosine 15 residues by wee1 and myt1 act to inhibit cdk activity

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29
Q

What is the limitation of temperature-sensitive mutations

A

You will always miss some genes due to the permissive and restrictive temperatures you chose

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30
Q

Specifically how does the CKI p15INK4B act

A

p15INK4B binds cdks directly at the kinase active site blocking its activity by interfering with ATP binding

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31
Q

Bivariate FACS analysis allows you to more accurately pinpoint where within the cell cycle that cells are arresting as a result of an intervention. What is its limitation

A

It often can’t distinguish between mitosis and G2 phase

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32
Q

What were the 4 key experiments that laid the foundations of understanding how cell cycle phase transitions are brought about?

A

Yoshio Masui - showed that factor in the cytoplasm of a Xenopus egg cell arrested in M phase was capable of inducing M phase in an ooctyte arrested in G2 phase, this was termed maturation promoting factor

Fred Lohka - purified MPF and incubated it with histone H1 and radioactive ATP, this lead to the identification that MPF had protein kinase activity

Tim Hunt - bathed sea urchin eggs in radioative methionine and incubated cells over a period of time, taking protein lysates at regular intervals and looking for radioactivity. One of the radioacitve proteins was found to accumulate for a time but then periodically disappeared just before the cells divided. This was later found to be cyclin B

Paul Nurse - identified cdc2 by introducing a library of yeast genes into cell cycle mutant to determine which one rescued wild type phenotype. Cdc S. pombe mutants were elongated due to a failure to divide. Cdc2 was found to be highly homologous to MPF and was later found to be a component of the complex along with cyclin B identified by Hunt

33
Q

Which cyclin controls entry into M phase

A

Cyclin B

34
Q

Draw a FACS trace to represent the typical distribtution of cells within the cell cycle

A

See completed diagram below

35
Q

Below is a table with some of the known mammalian cyclins. Determine where within the cell cycle these cyclins are required?

A

See completed table below

36
Q

What is the significance of the pre-replication complex and the cell cycle in cancer testing

A

Antibodies that recognise the pre-replication complex (specifically Mcms) are profoundly useful for detecting malignant cells in cervical samples. It makes it far easier to identify malignant epithelial cells in smear tests

37
Q

Explain what is meant by the licencing factor hypothesis

A

In every cell cycle DNA is licenced to undergo one round of replication only. During replication the licence is destroyed/removed from DNA. Thus DNA inside the nucleus cannot access licensing factor because it resides in the cytoplasm

38
Q

What is the effect on the cell cycle of introducing constitutively active Akt into cells

A

This prevents any activity of p21Cip1 and p27Kip1 subsequently leading to over-proliferation

39
Q

Which two proteins are responsible for mediating the stages of the cell cycle

A

Cyclin-dependant kinases and cyclins

40
Q

Outline the difference in terms of molecular structure of inactive and activated cdks

A

When inactive the active site of Cdks is sterically obstructed by the activation loop

41
Q

How did Fred Lohka’s experiments add to the idea of a maturation promoting factor

A

Lohka purified MPF from a series of biochemical purifications using fractions from the mature Xenopus egg to find the protein response for inducing maturation of immature oocytes. This isolated fraction was incubated with histone H1 and radiolabelled ATP. Histone H1 was used to test for protein kinase activity as it contains a number of phosphorylatable serine residues and allowed Lohka to investigate if the maturation promoting factor was a protein kinase. The proteins were then separated out and exposed to a film in order to pick up radioactivity that would prove that MPF was indeed a kinase. As predicted a band of radioactivity was seen indicating the MPF was capable of inducing phosphorylation of histone H1

42
Q

Once a mature Xenopus oocyte is laid what phase of the cell cycle does it arrest in

A

Meiotic metaphase

43
Q

How do cyclin-dependent kinase inhibitors (CKIs) generally act

A

These proteins bind the kinase and occlude its ability to bind to its substrate

44
Q

Explain the usefulness of schizosaccharomyces pombe in studying the cell cycle

A

S. pombe only grows in one direction and so the length of the organism can tell you which phase of the cell cycle it is in

45
Q

How can mutations that affect the cell cycle be easily studied in S. pombe

A

Mutations in genes that cause changes in the length of the yeast are affecting the various stages of the cell cycle

46
Q

What is the name of the gene involved in the G2 to M phase transition during the cell cycle of S.cerevisiae

A

Cdc28/2

47
Q

Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur

A

G1

48
Q

When do cdk inhibitors usually operate

A

During G1 and S phase

49
Q

Cyclin dependent kinases are important in the temporal control of DNA replication. During which stage of the cell cycle are cdk levels particularly high and particularly low

A

G1 phase – low cdk activity. S phase – high cdk activity

50
Q

What is the S. cerevisiae homologue of cdc2

A

Cdc28

51
Q

How does the anti-proliferative signal TGF-β act to mediate contact inhibition

A

TGF-β promotes the synthesis of p15INK4B that blocks D cyclins involved in transition in G1. This halts the cell cycle preventing proliferation

52
Q

Explain how bivariate FACS is carried out and how it differs from univariate

A

Bivariate FACS uses the additional parameter of the rate of DNA replication per cell. It involves the addition of a halogenated nucleotide derivative which will be incorporated only in cells in S phase. This then becomes the y axis of the FACS trace with the proportion of cells indicated using colour intensities. This allows you to determine what stops the cell cycle and at what point in the cell cycle much more accurately

53
Q

What was licencing factor later identified to be

A

Licencing factor turned out to be components of the pre-replication complex

54
Q

Explain the phenotype of cdc25 temperature sensitive mutant S.pombe relative to the cell cycle

A

Cdc25 mutants at restrictive temperature are elongated too because they aren’t dividing – hence have the same phenotype as cdc2

55
Q

How can phosphorylation of Cdks be used to regulate their catalytic activity

A

Phosphorylation at T172 in CDK4 and T160 in CDK2 residues located in the T loop are viral for proper catalytic activity

56
Q

Specifically how does the CKI p57Kip2 act

A

p57Kip2 binds near the active site and interferes with substrate binding

57
Q

Discuss the clinical implications of mis-regulated CKIs in cancer

A

Mis-regulated CKIs are seen in many cancers such as mammary carcinomas. These are associated with a more severe prognosis in these patients

58
Q

Explain the phenotype of cdc2 temperature sensitive mutant S.pombe relative to the cell cycle

A

Cdc2 mutants at restrictive temperature are elongated because they aren’t dividing

59
Q

Xenopus laevis ooctyes grow without dividing for months before they are laid and fertilised prior to laying what stage of the cell cycle are these cells arrested in

A

G2

60
Q

What can be said about the relationship between the number of cell cycle mutants found in each strain of yeast at a particular point in the cycle and the amount of time they spend in that phase

A

They are proportional the longer a yeast strain spends in a particular phase of the cell cycle the more likely you are to discover mutants arrested in that stage

61
Q

Explain how univariate FACS can be used to determine the proportion of cells in each phase of the cell cycle

A

Firstly the cells within the population have their DNA stained using propidium iodide or DAPI. These cells are then passed through a flow cytometer and the relative DNA content of each cells is measured using the degree of fluorescence given off. Cells in G1 phase have a relative DNA content of 1 whereas cells in G2 and M phase have a relative DNA content of 2 due to DNA replication having taken place. Cells in S phase have a relative DNA content of between 1 and 2 because they are undergoing DNA replication and so will have a DNA content between G1 and G2. You can then determine the proportion of cells in each phase of the cell cycle using the fact that the area under the curve is proportional to the number of cells in that phase.

62
Q

What are the yeast homologues of cyclins

A

Clb and Clns

63
Q

What phenomena is seen during the first 8 cell cycles of the developing Xenopus embryo

A

There is no change in the size of the embryo as the cells aren’t undergoing transcription. The cells themselves get smaller but increase in number so no overall change is seen

64
Q

How was the amount of time a cell is in S phase determined

A

To determine the amount of time a cell spends in the synthesis phase of the cell cycle you first need to establish the number of cells in S phase and synthesising DNA within a colony. To do this 32P containing phosphate is included in the medium on which the cells are cultured. The cells actively synthesising DNA and are hence replicating will incorporate this radioactive phosphate into the genome. These cells can then be visualised by exposing a film to the colony and the radiation emitted from the cells undergoing S phase will leave black spots in the film. These can then be counted. The percentage of colonies stained positive for radioactivity can give an indication of the percentage of cells undergoing S phase

65
Q

What two families of proteins regulate Cdks

A

Activators such as the cyclins and cdk-activating kinases (CAKs) and the inhibitors such as CKIs

66
Q

Below is a table comparing the different phases of the cell cycle in S. pombe and S. cerevisiae. Fill in the blanks to outline how they differ?

A

See completed table below

67
Q

Roughly what percentage of cells are in S phase at any one time

A

35%

68
Q

Temperature sensitive mutations introduced into yeast will cause arrest of all cells at the same point in the cell cycle once switched to the restrictive temperature T or F

A

T

69
Q

How often does each cell division in the early Xenopus embryo occur

A

Every 30 minutes

70
Q

Why does S phase only happen once in most cells

A

The pre-replicative complex marks where the origins of replication are. During S phase the pre-replicative complex is knocked off and is degraded which means that it needs to be re-made before the next cell cycle. This acts to limit the cycle to a single S phase per cycle

71
Q

What are mcms

A

Mcms are helicases that unwind the DNA allowing S phase to begin

72
Q

Outline Masui’s experiments on the effects of injecting cytoplasm from a Xenopus egg into an arrested oocyte and what this showed

A

Masui injected the cytoplasm of a mature Xenopus egg that was arrested in metaphase of mitosis into an oocyte arrested in G2 phase. Injection of this cytoplasm led to that cell maturing and induced early entry into M phase which was visualised by the formation of a white spot at the animal pole of the oocyte. This lead to the idea that a factor in a cell arrested in M phase is capable of inducing M phase in a cell in G2 phase a factor subsequently named maturation promoting factor (MPF)

73
Q

How do growth factors exert their effects on the cell cycle by regulating CKIs

A

Mitogens such as growth factors control cell cycle clock partly through regulating CKI function. This is mediated through the PI3K and Akt pathway whereby there is an inhibition of p21Cip1 and p27Kip1. This inhibition of these CKIs inhibits their inhibition of cell cycle progression so you subsequently get growth

74
Q

How is cyclin-dependant kinase (cdk) activity important in limiting pre-RC formation and activation to specific points in the cell cycle

A

Cdk levels are high during S phase of the cell cycle. High cdk levels leads to the phosphorylation of already formed pre-RC thus activating them and leading to formation of the replication origin. Cdk also acts to phosphorylate the individual components of the pre-RC particularly the Cdc6 Cdt1 and ORC elements. Phosphorylation of these constituent elements leads to their inactivation and hence inhibition of new pre-RC formation during S phase. During G1 cdk levels are low and thus there is little phosphorylation of Cdc6 and Cdt1 and hence more pre-RC formation

75
Q

What DNA replication event occurs in S phase

A

Origin activation – the unwinding of DNA and recruitment of DNA polymerase

76
Q

What feature is indicative of mitosis

A

Chromosome condensation

77
Q

What type of protein kinases are cdks

A

Serine/threonine kinases

78
Q

Schizsaccharomyces pombe spend very little time in M phase T or F

A

F – they spend a long time in M phase

79
Q

If the cells spend roughly 1 hour in M phase what percentage of cells in a colony would show tubulin staining indicative of mitosis

A

5% - 1/20 x 100

BMS379 Cancer Biology (10 decks)

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