T&H9 Cell proliferation and death Flashcards
______ are protein complexes that contain DNA-binding proteins that assemble on origins at the end of the previous mitosis.
pre-replicative complex (pre-RC)
Once bound by the ____, an origin is licensed for replication. It remains inactive throughout ___ phase of the cell cycle and begins to fire in the ____ phase leading to the DNA synthesis at the origin.
- pre-RC
- G1
- S
Chromosome segregation is powered and organized by _________.
microtubules
Formation of the mitotic spindle is facilitated by _________, these are organelles that sit at either spindle pole and generally act to organize microtubules.
centrosomes
Centrosomes are centered on 2 compact, barrel-shaped cylinders of microtubules called ______.
centrioles
Centrosome replication begins in early ______ phase.
S
Describe the phases of mitosis.
- prophase: begins with chromosome condensation in nucleus, 2 centrosomes separate from one another and microtuble spindle forms between them
- prometaphase: nuclear envelope breaks down, which allows microtubule spindle to interact with chromosomes, sister chromatids lose almost all of their length-wise cohesion but remain tightly juxtaposed at centromeres
- metaphase: sister chromatids become bi-oriented in center of spindle aligned in a plane called the metaphase plate
- anaphase: abrupt/total loss of sister chromatid cohesion occurs which allows opposing microtubules to pull sister chromatids apart and drag them to opposite poles
- telophase: events of early mitosis are reversed, spindle is taken apart, and cytokinesis occurs
_______ is the term used for spitting of mother cell into 2 daughter cells.
cytokinesis
What are the two molecular mechanisms central to cell-cycle regulation.
CDKs and E3 ubiquitin ligases
CDK activity is almost entirely dependent on binding of a ______.
cyclin
_______ stimulates enzymatic activity of CDK and influences substrate selection by the CDK.
cyclin
In normal cells, CDK1 forms complexes with _____ and ____ type cyclins, CDK2 forms complexes with _____ and ____ type cyclins, and CDK4 and CDK6 form complexes with _____ cyclins.
- CDK1: A- and B-type cyclins
- CDK2: A- and E-type cyclins
- CDK4/6: D-type cyclins
In contrast to the other cyclins, the abundance of ____ cyclins does not change during cell cycle.
D-type
When a cyclin binds a CDK, a ______ in activation loop of CDK is phosphorylated by the _______.
- threonine
- CDK-activating kinase (CAK)
In humans, CAK is another cyclin-CDK complex composed of _______.
cyclin H/CDK7
_____ and ______ kinases inhibit cyclin B/CDK1 complexes by phosphorylating amino acids of CDK1 on residue Y15. _______, a positive regulator of cell cycle, remove phosphates from these amino acids, thus promoting their activity.
- MYT1 and WEE1
- CDC25 phosphatases
The primary mechanism for targeted protein degradation is __________ in which a small protein ubiquitin is covalently attached to a target protein.
ubiquitin-mediated proteolysis
APC/C is an E3 ligase that binds to substrate-binding proteins, ______ or _____, in a cell-cycle dependent manner.
CDC20 or CDH1
How do the E3 ligases, SCF and APC/C, differ?
- SCF is constitutively active but cannot target substrate until it is phosphorylated
- APC/C ligases are only active during late M and G1 phases of cell cycle
The cell cycle oscillates between 2 states. The first state lasts from anaphase to end of next G1 phase. The second state lasts from beginning of S phase to metaphase. Describe the activity of these states.
- 1st state (anaphase to end of G1): APC/C activity is high, CDK1 and CDK2 activity are low, and pre-RC assembly on replication origins is permitted
- 2nd state (early S phase to metaphase): CDK2 activity is high, APC/C is low, pre-RC assembly on replication origins is not allowed.
Replication origins can become capable of initiating DNA replication during _____ phase when pre-RCs assemble on them.
G1 phase
Absence of CDK1 and CDK2 kinase activity and high activity of APC/C during G1 phase combine to create a biochemical environment that allows _______.
pre-RC assembly
When phosphorylated by cyclin A2-CDK2 or cyclin B-CDK1, _____ dissociates from the APC/C complex, abolishing its activity.
CDH1
Low activity of ___ and ______ in G1 phase prevents CDH1 from becoming phosphorylated, so APC/C complex remains high which keeps ____ and ____ levels low.
- CDK1 and CDK2
- cyclin A2 and cyclin B
To reinforce low CDK1 and CDK2 activity, APC/C targets SKP2 for degradation, whose function is to degrade ________.
CKI p27
Like G1, G0-phase cells have low _______ activity and high ___ activity. Unlike G1, G0-phase cells have little to no ________ activity due to sharply repressed ______ transcription.
- low CDK1 and CDK2 activity
- high APC/C activity
- cyclin D-CDK4/6 activity
- cyclin D
At G1/S transition, positive feedback loops that suppress CDK2 activity and keep APC/C activity active are overturned. Cyclin D-CDK4/6 complexes are thought to have 2 key roles in this process. Cyclin D-CDK4,6 binds to ____ which prevents it from inhibiting cyclin E-CDK2/ A-CDK2. Cyclin D-CDK4/6 phosphorylates 3 related pocket proteins, _______, ____, and _____ which leads to their release from E2F transcription factors. E2Fs complex with DP protein and bind to sequences found in the promoters of genes that encode proteins important for ______ or ______ (ie, cyclin E, cyclin A2, and EM2).
- p27
- Rb, p107, and p130
- S-phase entry or DNA synthesis
The rise of cyclin A2-CDK2 activity at G1/S transition triggers _______.
DNA replication
Late G2-phase mammalian cells experience surge of _______ activity that initiates events of early mitosis.
cyclin B-CDK1
Cyclin B concentration rises throughout S- and G2 phase and is induced by ________.
FOXM1 transcription factors
Cyclin B-CDK1 phosphorylates and activates ________, which remove inhibitory phosphate groups.
CDC25 phosphatases
During mitosis, fully active APC/C ubiquinates what 3 crucial substrates?
- cyclin B1
- cyclin B2
- Securin
c-myc overexpression can stimulate cell growth by inducing genes involved in ________.
ribosome synthesis
______ and ____ signaling network is central regulator of animal cell growth and proliferation.
PI3K and mTOR
Glycolysis has important role in supplying cellular building blocks, like _______.
NADPH
Anabolic state is programmed by growth factor signaling pathways and transcription factors, particularly _____ network and ____, respectively.
- PI3K/mTOR
- c-myc
The vast majority of cancers contain genetic or epigenetic alterations that loosen control over G1/S transition. Typical alterations include amplification of genes that encode proteins that drive G1/S transition (_______) as well as genomic deletions, point mutations, and promoter methylation that remove G1/S inhibitors, like _____ and _____.
- cyclin D1
- Rb and p16
Mechanisms that correct common chemical changes to DNA bases, such as _________ and _______ operate constitutively.
BER and NER
In G1 phase, cells primarily repair DSBs through what repair pathway?
NHEJ
In S and G2 phase, cells repair DSBs by ___ and _____ repair pathways.
NHEJ and HR
In S and G2 phases, DNA damage kinases inactivate ________ and activate _____. This arrests cells before G2/M transition point by preventing ____________.
- inactivate CDC25 phosphatases
- WEE1 kinase
- surge of Cyclin B-CDK1 activity
The spindle assembly checkpoint serves to delay _____ until all replicated chromosomes are bi-orientated and under tensions.
anaphase
The spindle assembly checkpoint blocks anaphase by preventing full activation of ______.
APC/C CDC20 complex
The primary defect detected by the spindle assembly checkpoint is ________ that are not attached to microtubules.
kinetochores
Misregulation of mitotic proteins can cause aneuploidy by at least 3 interconnected mechanisms:
- chromosomal instability
- Extra centrosomes
- Tetraploidy
________ arises when anaphase of cytokinesis fail.
tetraploidy
Name the anti-apoptotic proteins.
- BCL-2
- BCL- Xl
- BCL-W
- A1
- Boo
- MCL-1
Overexpression of BCL-2 in mammalian cells inhibits release of ______ as well as other factors important for apoptosis, including ____, ____, and _______, from the mitochondria.
- cytochrome C
- apoptosis inducing factor (AIF)
- SMAC
- OMI
Overexpression of _______ prolongs survival of cells and increases their resistance to apoptotic stimuli.
BCL-2
Caspases are present in the ____ in their inactive forms (procaspases or zymogens) and require proteolytic cleavage at ___ residues to be activated.
aspartate
What is the domain and subunits of caspase 9?
- CARD
- p20 and p10
What is the domain and subunits of caspase 8?
DEDs
p10 and p20
What is the domain and subunits of caspase 3?
does not have domain
p10 and p20
Apoptosis protease activating factor-1 (APAF-1) has a critical role for the ______ apoptotic pathway.
intrinsic
______, _____, and ______ are all required for activation of downstream effectors, CASP3, -6, and -7.
Cytochrome C, APAF-1, and CASP9
In the mitochondrial (intrinsic) apoptotic pathway, oncogene activation, irradiation, or DNA damage activates ____, which activates proapoptotic proteins. Activation of _____ and _____ leads to mitochondrial permeabilization which releases ______ into the cytoplasm. This clusters with _____ and _____ forming the apoptosome. The apoptosome activates ______, which then activates effector caspases _______, _____, and ___, which lead to apoptosis.
- p53
- BAK and BAX
- Cytochrome C
- APAF-1 and pro-CASP9
- CASP9
- CASP 3, CASP6 and CASP7
______ plays a crucial role in the mitochondrial pathway through transactivation of essential BCL-2 family members, including ____, _____ ____, and ______.
- p53
- PUMA, BAX, NOXA, FAS
What are the death receptors and ligands for the death receptor (extrinsic) apoptotic pathway?
Receptors:
- TNFR-1
- FAS
- DR5
- DR6
Ligands:
- TNF and LT-alpha
- FASL
- TL1A
- APO2L/TRAIL
In the death receptor (extrinsic) apoptotic pathway, FAS/FASL binding leads to aggregation of FAS receptor proteins to form a trimer and recruit FADD, which then recruits pro-caspase _____ and forms the ______. This leads to dimerization of pro-caspase _____ within the ______ leading to its activation and subsequent activation of effector caspases, _____, ______, and ______.
- 8
- death-inducing signaling complex (DISC)
- 8
- DISC
- CASP 3, 6 and 7
p53 affects the death receptor apoptotic pathway by transactivating _______ in response to DNA damage.
FAS/DR5
________ (inhibitor of CASP8 and CASP10, is transcriptionally upregulated by _____ and is also regulated by _______.
-c-FLIP
- NF-KB
- ubiquitylation
Communication between death receptor and mitochondrial apoptotic pathways is best demonstrated by CASP8 cleavage of ___, which generates a truncated form (____) that cooperates with _______ to form openings in outer mitochondrial membrane, leading to release of proteins, such as cytochrome C.
- BID
- tBID
- BAX
Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited human disorder that is characterized by lymphoproliferation, accumulation of CD4/CD8- T lymphocytes, and autoantibody production. It is associated with heterozygous mutations of ______ and ____. These patients are at risk to develop lymphomas.
- FAS and FASL
Necroptosis is induced if ______ and ___ are not inactivated by CASP8. Occurs if death receptor signaling pathway is impaired or CASP8 is lost.
RIPK1 and RIPK2
Active RIPK1 and RIPK2 trigger ____.
necroptosis
______ was identified initially in studies of human follicular B-cell LSA carrying the translocation t(14:18).
BCL-2 gene
Most cells in normal adult tissues are in what phase of the cell cycle?
G0
Levels of ___ vary during cell cycle while levels of ______ remain constant.
cyclins
CDKs
What 2 canine malignancies are associated with loss of function of p16/INKA4?
mammary carcinoma
melanoma cell lines
What 2 canine malignancies are associated with loss of function of CIP/KIP?
Mammary carcinoma
gastric carcinoma
Which method and DSB repair results in greater genomic instability and why?
- NHEJ due to loss of base pairs
For caspase enzymes, how is the name related to its function?
cysteine-dependent aspartate specific proteases
By what pathway do the most anti-cancer treatments work?
radiation and most chemo agents kill through activation of the mitochondrial apoptotic pathway
Name laboratory techniques to identify apoptosis.
- electron microscopy
- TUNEL assay
- Flow cytometry
- Western blot
_______ assay identifies DNA strand breaks that occur when DNA fragmentation occurs in the last phase of apoptosis. It does this by labeling the breaks with _________, which are then detected via immunoperoxidase techniques.
- TUNEL
- deoxyuridine triphosphates
What flow cytometry stains are used to identify apoptotic cells?
Annexin V / Pyridinium iodine
_____ can detect, localize, and quantify proteins involved in apoptotic signaling.
Western blot
Active caspases are ___________ complexes composed of what?
heterotetrameric
2 large subunits and 2 small subunits
All caspases contain an active site _________.
pentapeptide
Which caspases has pro-inflammatory functions?
CASP1, -4, -5, -11, and -12 a
What are the non-apoptotic functions of CASP8?
- Blood vessel formation during embryogenesis
- proliferation of T and B cells
Name the initiator caspases.
CAP8, CASP9, and CASP10
Name the executioner caspases.
CASP3, CASP6, and CASP7
Name the caspase substrates for the following:
- cytoskeletal proteins (n=2)
- nuclear proteins (n=2)
- proteins involved in DNA repair (n=3)
- cell-cycle proteins (n=4)
- cytokines (n=2)
- apoptotic proteins (n=6)
cytoskeletal proteins
- actin, gelsolin
nuclear proteins
- Lamin A, B
Proteins involved in DNA repair
- PARP1, RAD51, DNA-PKcs
cell cycle proteins
- p21, p27, cdc27, Rb
cytokines
- IL-1b, IL-18
Apoptotic proteins
- caspases, BCL-2, BCL-XL, BID, BAX, ICAD
The caspase-activated DNase (CAD) is inactive when associated with its inhibitor ICAD. In response to apoptotic stimuli, ______ is cleaved by caspases allowing CAD to cleave DNA of apoptotic cells to produce ___________, a characteristic of apoptosis.
- ICAD
- internucleosomal DNA
The BCL-2 family member and anti-apoptotic protein ______ interacts with APAF-1 and CASP9, and forms a tertiary complex, which negatively regulates CASP9 activation.
BCL-XL
Caspase activity is modulated by various members of the family of inhibitors of apoptosis (IAPs). Several IAPs bind directly to caspases, such as _____, ____, and ____ and inhibit their function. This IAP-mediated inhibition can be antagonized by the proteins ___ and _____, which bind directly to XIAP and suppress its inhibition of caspases.
- CASP3, -7, and -9
- SMAC and OMI
Stimulation of the mitochondrial pathway leads to activation of effector caspases, such as CASP3, and CASP6, that can subsequently process and activate CASP _____.
8
What chemotherapeutic has been reported to induce expression of death receptors (DR4, DR5, FAS) in many cancer types, thereby enhancing TRAIL and FAS induced apoptosis?
Doxorubicin
BCL-2 family proteins share several conserved domains known as ____ regions, including ______, ____, _____, and ______. These domains allow formation of dimers between BCL-2 family members and are essential for their function.
- BH regions
- BH1, BH2, BH3, and BH4
