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Quiz 6 Flashcards

1
Q

what are the phases of mitosis

A 
prophase
prometaphase
metaphase
anaphase
telophase
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2
Q

when are the centrosomes duplicated

A

during S phase - everything needed for mitosis is synthesized here

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3
Q

when do the centrosomes move to the poles

A

prophase

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4
Q

what happens during prophase

A

centrosomes move to poles, nuclear membrane breaks down, chromosomes condense, sister chromatids held together by centromere

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5
Q

what happens during prometaphase

A

spindle fibers form.

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6
Q

what types of spindle fibers are there

A
  1. astral microtubules: position mitotic spindle
  2. kinetochore microtubules: attach to the chromosomes
  3. polar microtubules: interdigitate with MTs from opposite pole
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7
Q

what happens during metaphase

A

alignment of chromosomes in the center of the mitotic spindle. THIS is when we do karyotyping

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8
Q

what happens during anaphase

A

sister chromatids separate and move to opposite poles, cell elongates

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9
Q

what happens during telophase and cytokinesis

A

telophase: nuclear membrane reforms, chromosomes recondense, spindle disappears
cytokinesis: actin filaments cause separation of daughter cells

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10
Q

what is the purpose of meiosis 1

A

reductive division - separation of homologous chromosomes

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11
Q

what is the purpose of meiosis 2

A

segregation of sister chromatids to be 1n.

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12
Q

what is the diversity generated from meiosis 1 independent assortment

A

2^23 (2^n)

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13
Q

what else creates genetic diversity (other than independent assortment)

A

crossing over (THIS is why we don’t just have separation of homologues but rather DNA synthesis and then 2 divisions)

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14
Q

what structure facilitates recombination

A

synaptonemal complex

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15
Q

describe the synaptonemal complex

A

highly ordered structure formed during meiosis 1. consists of 2 lateral elements and a central element to facilitate recombination.

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16
Q

what is the term for sites of crossing over

A

chiasmata (remnants of synaptonemal complex)

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17
Q

where does crossing over happen for the sex chromosomes

A

at the small region of homology which allows them to pair and cross over. really the main function is to physically keep them together

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18
Q

what is nondisjunction

A

when homologues fail to separate during meiosis 1 or sister chromatids fail to separate in meiosis 2. get trisomy and monosomy as a result

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19
Q

what percent of gametes have abnormal chromosome numbers if non disjunction occurs in meiosis 1 vs 2

A

meiosis 1: 100% abnormal

meiosis 2: 50% abnormal, 50% normal

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20
Q

mitotic index

A

number of cells undergoing mitosis/total number of cells

*done using light microscopy

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21
Q

ki-67

A

ki-67 is an antigen expressed in cells undergoing active division. can be detected using an antibody tag via immunohistochemistry. ONLY in antigen presenting/proliferating cells, allows us to flag cancerous cells.

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22
Q

flow cytometry

A

measures fluoresence of a tag that has integrated into DNA - measures the fluorescence PER cell (G2 and M fluoresce twice as much as G1, can’t tell difference between them!)

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23
Q

where do CDKs phosphorylate

A

ser or thr residue that immediately precedes a proline residue

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24
Q

upregulation vs downregulation of cyclin

A

upregulation via transcription

downregulation via degradation

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25
Q

CDK at early G1, late G1, S, late G2, M

A 
early G1: CDK 4/6 with Cyclin D
Late G1: CDK2 with Cyclin E
S: CDK2 with Cyclin A 
G2: CDK1 with Cyclin A
M: CDK1 with Cyclin B
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26
Q

CDK cyclin at early G1

A

CDK 4/6 with Cyclin D

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27
Q

CDK cyclin at late G1

A

CDK2 with Cyclin E

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28
Q

CDK cyclin at S

A

CDK2 with Cyclin A

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29
Q

CDK at G2

A

CDK1 with Cyclin A

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30
Q

CDK cyclin at M

A

CDK1 cyclin B

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31
Q

what else is necessary for activation of CDK complex?

A

Phosphorlylation at T160 – cyclin binding is necessary but NOT sufficient

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32
Q

what phosphorylates CDK subunit

A

CAK (CDK activating kinase, at T160) and Wee1 at T14 and Y15 (this is inhibitory - juxtaposes where ATP would bind)

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33
Q

what prevents substrate binding in CDK and CDK/cyclin complex

A

part of the CDK called the T loop is in the substrate binding site and prevents binding. Binding of cyclin causes shift, phosphorylation of t loop on T160 by CAK fully moves it so substrate binding site is completely exposed

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34
Q

why is Wee1 inhibitory

A

it juxtaposes where ATP would bind and repels ATP via attaching negatively charged phosphates

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35
Q

how are the phosphates put on by Wee1 removed

A

abruptly

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36
Q

when does CAK/Wee1 phosphorylation occur

A

simultaneously, waiting for inhibitory phosphates to be removed typically

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37
Q

what removes Wee1 phosphates

A

Cdc25c phosphatases

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38
Q

feedback loops in CDK

A

positive feedback of activated CDK/cyclin/P complex on Wee1 – phosphorylates it which makes it less active

positive feedback of CDK/cyclin/P complex on CDC25 phosphatase which makes it MORE active and better at removing inhibitory phosphates

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39
Q

what regulates CDK/cyclin/P activity even if there is no inhibitory phosphates

A

CIP class (P21, P27, P57, inhibits ALL CDKs)

INK4 class (p15, p16, p18, p19, inhibits only CDK 4/6)

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40
Q

how does CIP class work

A

binds to Cyclin-CDK complex and induces a conformational change that prevents substrate binding.

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41
Q

how does INK4 class work

A

binds only to CDK subunit, preventing it from interacting with cyclin

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42
Q

two types of E3 ub ligases

A

SCF and APC/C

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43
Q

similarities between types of E3 ligases

A

both have catalytic, scaffold, adaptor, and variable parts

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44
Q

SCF target and function

A

Cyclin D, E, A. ALWAYS acts as UB ligase, only on things that are already phosphorylated. ALWAYS active and recognizes phosphorylation.

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45
Q

what is SCF important for

A

cyclins in the G1 to S transition (D, E, A)

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46
Q

APC/C target and function

A

cyclin B. NOT always active - acts on cyclin B regardless of state it is in. APC itself needs to be activated via phosphorylation. (If it was always on we wouldn’t ever get cyclin B)

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47
Q

what is APC important for

A

cyclin B, mitotic progression

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48
Q

what phosphorylates APC

A

cofactor - Cdh1 or Cdc20

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49
Q

3 kinds of signals/feedback in cell cycle

A
  1. previous step responsible for activating next step
  2. negative feedback - when next step is activated, previous inactivated
  3. positive feedback, aplification of signal
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50
Q

what upregulates cyclin D

A

downstream in ERK pathway

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51
Q

what does ERK pathway regulate

A

phosphorylates Cyclin D and RB

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52
Q

what is Rb in natural state

A

normally bound to E2F, a txn factor. Rb acts as a repressor when bound and E2F gene expression is off

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53
Q

what happens when Rb gets phosphorylated

A

E2F is released, can now regulate gene expression

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54
Q

what is E2Fs target

A

cyclin E

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55
Q

what are the two types of E2F targets

A
  1. High affinity such as cyclin E

2. low affinity - other enzymes, only binds if these enzymes are present in large amounts (cyclin A)

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56
Q

what is Rb initially phosphorylated by

A

CDK4 and CDK6

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57
Q

what phosphorylates the rigin

A

Cyclin A/CDK2 (low affinity E2F target) and Cyclin E/CDK2 (high affinity E2F target). But ONLY if complex is on top and localized. Polymerase is ultimately what gets phosphorylated.

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58
Q

why doesn’t origin fire again

A

kinase phosphorylates origin proteins at the same time it phosphorylates polymerase. Phosphorylated origin proteins signal to leave and be degraded, and polymerase won’t fire without them despite being in phosphorylated state.

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59
Q

when is pre replication complex formed

A

G1 phase.

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60
Q

what mediates entry into mitosis (G2/M transition)

A

cyclin B/CDK1 phosphorylation

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61
Q

what is cyclin B/CDK1 targets

A
  1. Lamins: when it becomes phosphorylated, nuclear membrane will break down. Non specific phosphatases will removed it – dynamic process. Phosphorylation only occurs if cyclin B/CDK1 is active
  2. condensin. Condenses chromosomes. When Phosphorylated it becomes active.
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62
Q

how is cyclin B increased

A

txn’l regulation caused by loss of a repressor.

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63
Q

what happens at the completion of DNA synthesis

A

CDC25 becomes phosphorylated, this activates Cyclin B/CDK1.

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64
Q

what regulates exit from mitosis (M to G1)

A

activation of APC

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65
Q

how is APC activated

A

becomes associated with a co-factor, either CDC20 or CDH1.

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66
Q

what are APCs substrates

A
  1. Securin. Releases it from separase which cleaves Cohesin and causes sister chromatid separation.
  2. Cyclin B, breaks it down, reversal. Chromosomes decondense, nuclear membrane reforms.
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67
Q

G1 restriction point

A

if cell does not have GFs, will arrest here. Extracellular signalling and cell cycle control connection. After this restriction point, cell is committed to DNA replication.

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68
Q

what does GF signaling result in

A

upregulation of txn of cyclin D. this triggers phosphorylation of pRb and release of E2F

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69
Q

what is the DNA damage checkpoint mediated by

A

p53. Cell wants to stop cycle and repair itself.

70
Q

where is the DNA damage checkpoint

A
  1. prior to entry into S

2. Prior to entry into M

71
Q

G1 DNA damage checkpoint

A

upregulation of tumor supressor p53. Kinase cascade results in phophorylation of p53 and negative regulator Mdm2

72
Q

Mdm2

A

ub ligase for p53. phosphorylation disrupts binding and p53 levels can rise

73
Q

what is p53

A

txn factor. one of its targets is gene encoding p21, CDK inhibitor. p21 binds to CDK complexes and prevents phophorylation of pRb so cell is arrested in G1. p53 can also cause apoptosis

74
Q

how is entry into mitosis prevented by DNA damage

A

DNA damage triggers a kinase cascade that inhibits phosphorylation of CDC 25 so there is no activation of CyclinB/CDK1. P21 can also inhibit Cyclin B/CDK1 (p53 plays a role)

75
Q

what does unreplicated DNA do

A

prevents entry into mitosis. CDC25 needs to be phosphorylated to be active - phosphorylation doesn’t happen until full 4n content achieved (S phase check point)

76
Q

spindle checkpint

A

triggered because of a disruption of the spindle and a loss of attachment of MTs to the kinetochore. causes delay of activation of APC so separase and securin stay attached.

77
Q

primary cells

A

finite lifespan, directly removed from organism

78
Q

immortalized cells

A

unlimited life span

79
Q

transformed cells

A

immortalized cells that have acquired certain properties - if they have the ability to form tumors they are tumorigenic

80
Q

morphology of cancer cells

A

spindle shaped - thought to be due to actin depolymerization. EMT – epithelial to mesenchymal transition

81
Q

what do cancer cells rely on for ATP

A

glycolysis – this is WEIRD because it is inefficient. Hyptoheses:

  1. Grow in hypoxic environment (glycolysis is anaerobic)
  2. Need a lot of AA, fatty acids, etc to grow. This is also whats used in the TCA cycle (which generates more ATP) so wouldn’t work for this to be method of energy production
82
Q

how does a PET scan work

A

pt given glucose, look at where a lot of glucose is being used - signals that cell is undergoing rapid glycolysis

83
Q

contact inhibition

A

normal cells dont proliferate when in contact with other cells - cancer cells DO

84
Q

senescence

A

loss of proliferation without cell death - arrested

85
Q

how could you experimentally stop senescence and allow cells to grow

A

by removing p53 and pRb (p53 signals to cell to degrade)

86
Q

crisis

A

cells die, lose telomerase activity

87
Q

how could you get cells to not enter crisis

A

reactivate telomerase

88
Q

how can you identify a senescing cell

A

has fried egg appearance when you do a beta galactosidase assay. Senescent cells have more lysosomes and therefore a lower pH which has an associated beta galactosidase enzyme

89
Q

what causes initial senescence

A

activation of p53, which causes p21, CIP, inhibits CDKs

90
Q

what happens at M2 crisis

A

there is no telomerase so reaches crisis when real coding portions of gene start to be lost

91
Q

how do cells maintain telomeres

A

telomerase OR other way (ALT cells)

92
Q

RNA tumor virus mechanism

A
  1. viral genome contains activated form of cellular gene (viral genome contains oncogene)
  2. virus integrates into genomic locus that results in aberrant expression of a cellular gene
93
Q

how does ALV work

A

inserts into host genome and can lead to aberrant expression of a gene involved in oncogenesis (will insert in many places that has no proliferative advantage)

94
Q

how can a missense mutation cause cancer

A

constitutive activation. hyperactive protein made in normal amounts.

  1. Ras is normally a GTP ase. Bound to GTP and is active. - G12V or G13V- loses GTP ase activity so ALWAYS active.
  2. Raf - V600E- gets constitutively activated as a kinase

In both of these cases, Ras-Raf-Mek-Erk pathway gets turned on so you get high levels of cyclin D, Rb always phosphorylated, E2F always expressed. Don’t need GF or integrin - growth factor and anchorage independent!

95
Q

how can we visualize gene amplification on chromosome

A
  1. Gene amplified ON chromosome

2. Gene amplified and shuttled out in double minutes.

96
Q

homogenous staining region

A

large region stains the same due to gene amplification in aberrant DNA replication

97
Q

how can gene amplification cause cancer in terms of result

A
  1. Gene products normally limited in the cells get amplified. Myc, CyclinD1, CDK4 all examples.
  2. GF receptors like EGFR or HER2 which require ligand binding to dimerize. If a lot of receptors are made and they are very close to each other, will dimerize independently without GF ligands (GF independent)
  3. Autocrine loop as in PDGF. GF usually comes from other cells to interact with receptor, PDGF amplification causes SAME cell to express both the growth factor and the receptor (still GF dependent)
98
Q

gene rearrangements involving translocations have what mechanisms in terms of the chromosomes

A
  1. Reciprocal translocation on 2 different chromosomes

2. Inversion on same chromosome

99
Q

what parts of gene can be altered via translocation

A

regulatory region (alters expression of a normal protein_ or coding region (causing expression of a novel fusion protein)

100
Q

what gene rearrangements are common in cancer in terms of cell types

A

b and t cells

101
Q

what happens in bcr/abl fusion

A

Abl is tyrosine kinase, usually has its amino terminus negatively regulated. when it fuses to BCR, loses amino terminus and is always active. This causes the substrate protein to always be phosphorylated and activated, which signals cell proliferation and survival causing CML.

102
Q

why is fusion protein therapy easier than most cancer therapy

A

novel protein can be targeted because it will only be found in cancer cells and you don’t run the risk of harming other ells with treatment.

103
Q

what phenotype dominates - tumor or WT

A

WT - know this from fusion of cells

104
Q

what is different about DNA viruses than RNA viruses

A

DNA viruses contain novel oncogenes.

105
Q

how does simian virus SV40 work

A

expresses a large tumor antigen or large T antigen that binds to both cellular p53 and pRb thereby inactivating their function

106
Q

how do adenoviruses work

A

express two distinct proteins that inactive p53 and cellular Rb

107
Q

how does sporadic form of retinal cancer work

A

two somatic mutations in single retinal cell form the tumor - this would be a rare even

108
Q

how does hereditary retinoblastoma work

A

first required mutation is inherited, not THAT uncommon for second mutation to arise which is why it is mostly bilateral

109
Q

distinction between oncogenes and tumor supressors

A

oncogene - gain of function, can arise from mutating single allele
tumor supressors - loss of function, need to mutate BOTH alleles

110
Q

what usually occurs in p53 mutations

A

missense

111
Q

why does p53 become more stable with mutation

A

inability to interact with Mdm2, a Ub ligase for p53.

112
Q

what does oncogene activation by proteins such as myc, ras, and E1a lead to

A

upregulation of a protein called ARF. ARF inhibits Mdm2, thereby upregulating p53. This upregulates target genes like p21, a CDK inhibitor, and PUMA/NOXA promoters of cell death.

113
Q

how is adenamotous polyposis coli protein caused

A

a truncated protein due to frameshift mutation that doesnt allow APC to bind to beta catenin – beta catenin not being degraded means it can always promote cell proliferation

114
Q

what is loss of heterozeigosity (LOH)

A

when there is a missense or frameshift mutation of one allele of a tumor supressor or deletion, the remaining WT allele is deleted.

115
Q

what is transcriptional silencing of a wild type allele

A

when one allele is inactivated by mutation, the remaining one can be silenced through epigenetic means.

116
Q

what does p16 regulate

A

suppressor of cyclin D-CDK4/6

which phosphorylates Rb

117
Q

what is HPV E7 function

A

suppressor of pRb

118
Q

p16 mutations

A

missense, deletion, promoter methylation – if p16 is mutated, no longer suppresses Cyclin D-CDK 4/6 and get pRb phosphorylation and E2F expression

119
Q

cyclin D1 mutations

A

amplification, chromosomal translocations

120
Q

CDK 4/6 mutations

A

amplification and missense mutations that prevent its binding to p16

121
Q

Rb mutations

A

deletion, mutation

122
Q

what does HPV infection lead to

A

expression of E7 protein

123
Q

p53 pathway

A

ARF inhibits MDM2 which inhibits P53

124
Q

what does p53 cause

A

arrest or apoptosis

125
Q

what is the function of HPV E6

A

targets p53 for degradation

126
Q

APC pathway

A

APC and Axin bind beta catenin and degrade it

127
Q

what does B catenin control

A

txn of myc and cyclin D

128
Q

how does the ERK pathway work

A

GF signaling via cell surface receptors. GF can be overexpressed or receptors can be over expressed and receptor is constitutively active as tyrosine kinase

129
Q

how does PI3K pathway work

A

PI3K and downstream Akt can be mutates such that they are always active. PTEN is phosphatase that inhibits PI3K signaling, often deleted in human tumors.

130
Q

what is PTEN

A

phosphatase that prevents PI3K signaling and PTEN often deleted in human tumors

131
Q

what does AKT promote

A

cell survival

132
Q

what does INK4A gene encode

A

two proteins, p16 and ARF. p16 and RF share second and third exon but has their own first exon and promoter. Therefore, any mutation in the second or third exon will affect BOTh proteins!

133
Q

what will get altered if there is a mutation in exon 2 and 3 of INK4

A

p16 will be mutated: therefore pRb affected

ARF mutated: therefore p53 affected

134
Q

what do mutations in Myc and Ras often trigger

A

upregulation of ARF which inhibits MDM2, which activates p53.

135
Q

what is ARF a txnl target for

A

E2F. Inactivation of pRb pathway will upregulate p53.

136
Q

why do cells lose cell-cell junctions in EMT transition

A

loss of E cadherin expression and cell polarity, increased migratory behaviour

137
Q

tumor groth and metastasis steps

A
  1. Angiogenesis
  2. Invasion
  3. Intravasion
  4. Metastasis
  5. Extravasation
  6. Secondary Growth
138
Q

angiogenesis

A

when tumor reaches 1-2 mm, angiogenesis factors produces that induce blood vessel formation. Inhibitors also reduced.

139
Q

invasion

A

tumor cells become attached to sub endothelial cellular matrices via cell surface receptors. Followed by protease mediated degradation of matrix. Migration via chemotaxis using degradation products or tumor associated autocrine motility factors also occurs

140
Q

intravasation

A

tumor cells invade through vascular endothelial cells and their sub endothelial basement membranes and enter vasculature

141
Q

metastasis

A

tumor cells must survive shear of blood flow and attack by immune system, then adhere to endothelial cells of target organ or exposed sub endothelial ECM basement membranes

142
Q

extravasation

A

tumor cells extravasate out of the vasculature and into perivascular stroma. reverse of invasion

143
Q

secondary growth

A

at these distant sites, there is formation and growth of secondary tumor metastasis

144
Q

oncogene addiction

A

tumor cells become dependent upon activated oncogene pathways for their proliferation. targeted chemo uses this. EX: inhibitor of EGF receptor blocks proliferation in normal cells but causes death in cancer cells.

145
Q

necrosis vs apoptosis

A

necrosis: cells and organelles swell and rupture, leakage induces inflammatory response
apoptosis: cells shrink and condense, organelles and membrane retain integrity, remnants phagocytosed, NO inflammatory response

146
Q

DNA ladder

A

way of measuring size – 80-100 bp is nucleosome, see this when cell is undergoing apoptosis

147
Q

flow cytometer in apoptosis

A

cell will be hypodiploid because DNA is getting degraded

148
Q

TUNEL assay

A

breaks in DNA that occur can be labeled using enzyme terminal deoxynucleotidyl transferase - biotinylated dUTP and can be detected using a strepavidin that has a fluorescent tag

149
Q

annexin and phosphatidyl serine flip

A

phosphatidyl serine normally localize to the cytoplasmic side, during phagocytosis, flips to extracellular side but membrane remains intact. Annexin is dye that binds it

*membrane integrity NEEDS to be maintained, otherwise will interact with internal and assay wont tell you anything!

150
Q

how is apoptosis activated in T cells

A

glucocorticoids and antibody binding to the cell surface

151
Q

caspase

A

cysteine aspase – cleave at aspartic acid and have cysteine at the active site

152
Q

how do caspases become active

A

must be processed – get cleaved and lose their pro domains.

153
Q

what are the two types of caspases

A

initiator caspase (8 and 9) and executioner caspase (2, 3, 6, 7) - executioner is what cleaves particular substrates

NOTE: 1 initiator caspase can cleave multiple executioners to make them active

154
Q

what do executioner caspases cleave

A

cellular substrates that result in apoptosis

155
Q

what are the substrates for executioner caspases

A

Proteins involved in DNA degradation (CAD/ICAD)
Proteins involved in DNA repair ( PARP)
Proteins involved in the cytoskeleton (gelsolin)

156
Q

CAD

A

endonuclease that cleaves nuclear DNA. Normally bound to ICAD which inhibits it. ICAD gets cleaved by executioner caspase which frees CAD and makes it active to degrade DNA

157
Q

how do initiator caspases get activated

A

two main pathways:

1) Extrinsic/cell surface receptors
2) Intrinsic (mitochondrial and Bcl-2)

158
Q

how does extrinsic pathway of caspase activation work

A
  1. killer lymphocyte with Fas ligand attaches to Fas Death Cell receptor on target cell
  2. This triggers formation of the disk (FADD adaptor protein with procaspase 8/10)
  3. Caspase 8 or 10 gets activated, executioner caspase gets activated
  4. Cells undergo apoptosis
159
Q

why does the cluster occur with assembly of disk

A

pro molecule has low level of proteolytic activity. if you cluster enough together, get more proteolysis – this triggers cleavage of caspase 8 - THIS can cleave executioner caspase

160
Q

what is the hallmark of instrinsic apoptosis

A

loss of mitochondrial transmembrane potential - diagnostic of the opening of permeability transition pores

161
Q

what is the intrinsic pathway

A
  1. Apoptotic stimulus triggers release of cyt c from mitochondria
  2. Cyt C activates Apaf1 which hydrolyzes dATP to dADP
  3. Apoptosome formed by release of dADP in exchange for dATP or ATP
  4. Apoptosome recruits and activates pro-caspase 9
  5. Caspase 9 cleaves and activates executioner caspases, leading to apoptosis
162
Q

what kind of gene is Bcl2

A

oncogene involved in B cell lymphoma but has nothing to do with proliferation – it is an anti apoptotic gene! (BH 1234)

163
Q

what kind of protein is BH123

A

pro apoptotic - Bax and Bak

164
Q

what kind of protein is BH3 only protein

A

pro apoptotic - Bad, im, Bid, Puma, Noxa

165
Q

what are BH 1-2-3-4 referred to as

A

homology domains

166
Q

what is the basic function of bax and Bak

A

they form pores in the mitochondrial membrane

167
Q

how do bax and bak work

A

an apoptotic stimulus causes the inactive BH123 protein to aggregate and form pores. Cyt C gets released through the pores, or the formation of pores may just disrupt the entire membrane. Bax and Bak are normally cytosolic and monomeric - death signal leads to their insertion.

168
Q

what is the function of Bcl-2

A

binds to bax or bak and inhibits their activity so pores cant be formed and apoptosis doesnt occur

169
Q

what is the function of Bid

A

BIH3 only protein that directly activates Bax and Bak (pro - apoptotic).

170
Q

PUMA and Noxa function

A

block Bcl-2 inhibition of Bax and Bak – this means that pores can be formed and apoptosis can occur

171
Q

what does p53 target in terms of txn’l regulation of apoptosis

A

Bax, Puma, Noxa

McG (11 decks)

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