BIOL 472 Exam II

What is the cause of breakage-fusion-bridge (BFB) cycles?

C. chromosomes sticking end-to-end due to loss of telomeres

What are the main four checkpoints in the cell cycle?

1. End of G1 2. In S 3. End of G2 4. in M (anaphase blocked if chromatids are not properly assembled on mitotic spindle)

What are the steps of apoptosis?

1. loss of cell membrane integrity leading to blebs 2. Condensation of chromosomes 3. Enzymatic digestion of DNA 4. Fragmentation of nuclei 5. Disintegration into apoptotic bodies consumed by macrophages

Which of the following events drive(s) the cell cycle past the Restriction Point in late G1? A. Hyperphosphorylation of RB by cyclin E-CDK2 activity B. Increase in p21/Cip1 levels inhibiting cyclin activity C. PTEN activity leading to PIP3 conversion to PIP2 D. All of the above

A. Hyperphosphorylation of RB by cyclin E-CDK2 activity

Which of the following statements about NF1 is FALSE? A. It regulates Ras signaling through stabilizing the Ras-GTP state B. cells lacking NF1 have higher levels of GTP-bound Ras C. NF1 is an example of Ras-GAP D. Both B and C are both false

A. It regulates Ras signaling through stabilizing the Ras-GTP state

Some cancer cells escape crisis by: A. Overexpressing hTERT B. Producing high levels of p53 C. Staying in G1 phase by increasing cyclin D activity D. Eliminating the dependance on hTR RNA involvement for telomerase to function

A. overexpressing hTERT

Why has evolution selected for many diverse TSG pathways in a cell, but less diversity in proto-oncogene pathways? (car analogy)

ANALOGY: Good brakes are more important in a car than good acceleration... to be able to stop the car from crashing = It is more important to be able to put a stop to proliferation in cells than to augment proliferation

ARF-mediated activation of p53 is different than phosphorylation of p53. What is ARF and how does it function?

ARF (Alternate Reading Frame) protein monitors the amount of E2F in a cell. - too much E2F then ARF gets transcribed to sequester Mdm2 and allows p53 to do its role in targeting genes responsible for cell cycle arrest or apoptosis! Take home message: Too much E2F activity means that RB control is lost, therefore cells are primed to respond quickly by arresting or undergoing apoptosis

APC presentation, normal and abnormal functioning

Adenomatous Polyposis Coli (APC) also called familial adenomatous polyposis (FAP) is an inhertied susceptibility to development of adenomatous polyps in the colon. Such polyps, while themselves are nonmalignant, may develop into frank carcinomas. However, in relatively rare individuals suffering from FAP, they have polyps numbering in the HUNDREDS to carpet the luminal surface of the colon. Normal function: APC is essential to regulate beta-catenin signaling (as seen in the Canonical Wnt signalling pathway). -In WT crypt of the intestinal lumen, beta-catenin is normally degraded by APC (and Tcf/.Lef expression remain OFF) which allow for cell cycle arrest of differentiated cells Abnormal function: In APC -/- crypt of the intestinal lumen, beta-catenin is not degraded by APC and Tcf/Lef remain ON, which causes a progenitor-like phenotype and accumulation at site of future polyp formation. There is also failure of mutant cell to continue outward migration.

Inherited mutant TSG alleles can be thought of as recessive at the cellular level and yet dominant at the whole organismal level. What do we mean by this?

At the cellular level, the neoplastic phenotype of human cancer cells is usually recessive because two inactive alleles of a TSG are required (example is sporadic RB). However, if we examine familial retinoblastoma, then only one additional "hit" in a good copy of the RB gene is required for cancer to arise.

When a cell has passed the R point and is committed to completing the cell cycle A. it moves through the rest of the cycle without pausing B. it may pause if errors occur during a phase of the cycle C. RB enables the cell to "back up" to a previous phase before moving ahead D. p53 activity is no longer needed

B. It may pause if errors occur during a phase of the cycle

Which of the following is NOT a common characteristic of human senescent cells? A. Heterochromatic foci present in the nucleus B. Fluctuating levels of cyclins C. Large/expanded cytoplasm D. Loss of normal differentiated morphology

B. Fluctuating levels of cyclins

During which phase of the cell cycle would you expect most of the RB protein in a cell to be hypo-phosphorylated? A. G0 B. G1 C. S D. M

B. G1

Up to 90% of aggressive solid tumors exhibit over-expression of hTERT following a period of great genetic instability because A. hTERT expression encourages BFB mutations B. If BFB is allowed to continue too long, the cancer cells will be so messed up that they cannot survive C. hTERT is an oncogene that drives proliferation D. Normal cells cannot proliferate without hTERT activity

B. If BFB is allowed to continue too long, the cancer cells will be so messed up that they cannot survive

What is the significance of the t-loop at the ends of chromosomes? A. it functions as a hook that attaches chromosomes to the spindle fibers B. it protects the chromosomes form exonuclease activity C. It is made of non-telomeric DNA that codes for genes D. All of the above

B. It protects the chromosome ends form exonuclease activity

During regulation of cellular response to oxygen levels, HIF-1a protein levels may increase in response to: A. Oxygen conditions stabilizing at normal physiologic levels B. Cells experiencing hypoxic conditions C. Over-saturating oxygen levels D. HIF-1a binding to pVHL

B. cells experiencing hypoxic conditions

What best explains the high protein levels of p53 in almost all tumors? A. the cell cycle is permanently stuck in S phase so p53 is leading to DNA damage B. The mutant p53 fails to drive mdm2 expression C. oncoproteins such as E1A, E6 and E7 bind to p53 and prevent its degradation D. none of these are valid explanations

B. the mutant p53 fails to drive mdm2 expression

An increase in cellular cyclin D protein levels may results from: A. The cell entering S phase B. various mitogenic signals from outside the cell during G1 phase C. Dominant-negative RB mutants improperly blocking cyclin-E1A oncoprotein interactions D. the cell staying in G0 phase longer than normal

B. various mitogenic signals from outside the cell during G1 phase

The rate of new mutations that pile up over the course of many generations of a continuously dividing transformed cell will likely be A. steady from one cell generation to the next B. Very slow in early generations, but very rapid in later generations C. Gradually slowing down as the cancer cells stabilize their genomes D. non-existent if the first generation of transforming cells already has critical mutations in them

B. very slow in early generations, but very rapid in later generations

What are the roles of Bcl and Bax/Bim proteins in regulating apoptosis?

Bcl-2 family functions in pro-survival of the cell by keeping mitochondrial channels closed and preventing cytochrome C from leaking out into the cytoplasm Bax/Bim function by opening the cytochrome C release channels (pro-apoptosis)

In familial disease, why do we see mutant TSG alleles (eg. RB, NF1, VHL) transmitted through the germ line, but oncogenic mutations in proto-oncogenes (eg. SRC, MYC, RAS) are not typically passed on?

Because TSGs work in diverse, overlapping pathways there is redundancy if one gene is compromised. With oncogenes, loss of control over single pathway likely leads to non-viable embryonic development

If you see high levels of cyclin D and rising levels of cyclin E and CDK2, what event is the cell likely approaching? A. entry into mitosis B. exiting mitosis C. Crossing the R point D. Completing the S phase

C. Crossing the R point

How do waves of cylcin A, D, B and E levels prevent the cell cycle from "backing up"? A. Cyclins set a rigid timer that each phase of the cell must adhere to on time B. Cyclins are constantly inhbiitng CDKs from stimulating gene expression found in the prior phase of the cylcle C. Cyclins work together with CDKs to emphasize expression of those genes that are needed for the cell to move into the next phase D. cyclins can only work with 1 specific CDK type and that alone determines the cell cycle phase

C. Cyclins work together with CDKs to emphasize expression of thsoe genes that are needed for the cell to move into the next phase

Which statement is most true about RB protein? A. Hyperphosphorylated RB binds tightly to E2F proteins B. Cellular RB protein levels are eliminated as the cell approaches mitosis C. Hypophosphorylated RB is the state in which it inhibits E2F1,2,3 proteins D. RB protein must release E2F proteins in order for them to repress gene expression

C. Hypophosphorylated RB is the state in which it inhibits E2F1,2,3 proteins

Histone deacetylase (HDAC) enzymes A. promote initiation of transcription B. complex with hyperphosphorylated pRB C. Repress E2F family activity D. add acetyl groups to E2F promoters

C. Repress E2F family activity

Which of the following is NOT commonly present at high levels in senescent cells? A. p16INK4A B. p21Cip1 C. SV40 Large T antigen D. All of the above are typically present in high levels

C. SV40 large T antigen

Why does it take years for a cancerous tumor to form if theoretically 40 cell divisions (such as over 40 days) would generate 10^12 cells? A. Cancer cells spend a long time between rounds of mitosis B. several tumor cells must work together to gain immortality C. The vast majority of daughter cells in each round undergo apoptosis D. Re-expression of hTERT only happens once a sizable tumor mass starts forming

C. The vast majority of daughter cells in each round undergo apoptosis

If DNA damage is detected during S phase, what protein signals may occur? A. RB becomes dephosphorylated and quickly inhibits E2Fs B. cyclin D and E levels rise sharply to halt the cell cycle C. p53 is activated through phosphorylation D. Mdm2 interactions with p53 increase

C. p53 is activated through phosphorylation

What is the best description of the role of caspase proteins? A. they pause the cell cycle to allow DNA damage repair B. They destroy Mdm2 so that p53 can be activated C. they carry out the destruction of the cell signaled by the apoptosome D. they are a series of kinases that phosphorylate each other in a chain

C. they carry out the destruction of the cell signaled by the apoptosome

Why does RB tumor first show up as eye tumors in patients with family history?

Cells in the eyes are most exposed to mutagens (harmful UV radiation from the sun)and are much more likely to obtain a second "hit" in the good copy of the RB allele.

Activating of Myc may result in all of the following EXCEPT A. hyperphosphorylation of pRB B. Increased expression of CDK4 C. Degradation of p27Kip1 D. Decreased activation of cyclin E/CDK2 complexes

D.

Cellular inactivation of TSGs can occur as the result of: A. somatic mutation B. promoter demethylation C. loss of heterozygosity D. both A and C

D. Both A and C

Of the following statements which most accurately describes RB activity? A. RB inhibition of E2Fs leads to expression of pro-proliferation genes B. Tumor cells find ways of completely ignoring pathways involving RB during transformation C. RB interactions with E1A, E6 and E7 viral oncoproteins lead to cell cycle arrest D. Once RB is hyperphosphorylated, it ceases to actively control S, G2 and M phases

D. Once RB is hyperphosphorylated, it ceases to actively control S, G2 and M phases

What series of signals would irreversibly start the process of apoptosis?

D. P53 UPREGULATES Bax which releases cytochrome C from mitochondria

Which of the following is most likely true of aggressively dividing cancer cells? A. They do not express telomerase B. They do not express the hTR RNA template for telomere repeats C. They are in a state of senescence due to cell crisis D. Their telomere lengths become stabilized

D. Their telomere lengths become stabilized

A transforming cell that lacks telomerase activity may: A. Be in crisis B. Have shortening telomeric repeat sequences C. exhibit chromosome breakage and improper chromosome fusions D. all of the above

D. all of the above

p53 behaves differently than most TSG in what ways? A. p53 mutations found in tumors tend to be point mutations versus deletions B. p53 double knockout (-/-) is not embryonic lethal compared to most TSG C. p53 protein levels tend to increase in tumors versus decrease D. all of the above

D. all of the above

How can Mdm2 inhibition of p53 be released? A. phosphorylation of p53 in its transactivation domain B. ARF expression leading to removal of Mdm2 away from p53 C. polyubiquitylation of p53 leading to its activity relocating to the cytoplasm D. Both A and B are correct

D. both A and B are correct

What would be the best descriiption of how cyclins control CDKs? A. Cyclin label CDK for degradation B. cyclins directly bind CDK genes and activate their expression C. Cyclins get stabilized by CDK binding and de-phosphorylate RB D. cyclins bind CDKs as a way to direct CDK activity to proper protein targets

D. cyclins bind CDKs as a way to direct CDK activity to proper protein targets

Why were early transfection experiments with p53 and v-ras confusing? A. it was unclear why wild type p53 seemed to prevent v-ras induced tranformation B. it seemed strange the wile type p53 has stronger oncogenic properties than v-ras C. versions of the p53 that were truncated seemed to block v-ras more effectively D. point-mutant p53 seemed to enhance v-ras oncogenicity

D. point-mutant p53 seemed to enhance v-ras oncogenicity

Why do so many tumors over-express high levels of p53?

Defective p53 has no way of being degraded by Mdm2 because mutant p53 tetramers fail to drive its expression, therefore p53 builds up in the cell and is not recycled!

What is familial RB versus sporadic RB disease? Why does one come with little-to-no risk of tumors developing elsewhere in the body whereas the other carries significantly increased risk of other tumor types?

Familial RB is when there is an inherited mutation in one of the alleles of every single cell in the human body of the offspring. Once another "hit" is made to the other allele of RB, this is when the tumor phenotype becomes apparent. Familial RB usually presents bilaterally in the offspring. Sporadic RB is when there are two random mutations in both alleles of the RB gene in a single cell. These cases normally present unilaterally in the offspring. Individuals with sporadic RB, once treated, do not have a higher risk of developing tumors elsewhere in the body.

How does loss of proper TSG functioning prove more fatal than gain-of-function proto-oncogenes? ("Key" analogy)

For example, PTEN is a TSG that undoes the effects of PI3K and Akt. If PTEN is not able to do its proper role in dephosphorylated PIP3 to PIP2, then PIP3 will go onto activated Akt, which will phosphorylate a number of downstream targets to activate transcription and growth. Analogy: A key is meant to open a certain lock. If a key is bent out of shape (mutated), then it is more likely that the key will not be able to open its lock (loss of function), rather than gain the ability to open a different lock (gain of function).

Review that phases of the eukaryotic cell cycles and list what the main cellular activities are in each phase

G0: quiescent arrest/ maintenance of functions (indefinitely) G1: growth and phenotypic functions (12-15 hours) S: DNA replication (6-8 hours) G2: Preparation for mitosis (3-5 hours) M: Mitosis (1 hour)

Is apoptosis always a bad thing?

It is not always a bad thing. It is regularly used during embryonic development as a normal part of tissue development. - Elimination of webbing between fingers - 95% of erythroblasts destroy themselves before maturing into red blood cells - Reduction of 90% in mammary gland tissues after weaning infant

What impact does Large T antigen have on p53 function when it binds?

Large T oncoprotein targets host-cell proteins (such as p53) for binding and sequestering a single molecule of p53 on each of its homo-hexamer subunits. p53 can't do its proper functioning.

Understand the role of promoter DNA methylation in controlling gene expression. Why might over-active DNMT family protein activity promote cancer?

Methylation of promoter DNA shuts down expression of the gene (discourages transcriptional activity) -DNA from a tumor shows that almost all sites have a methylated CpG, whereas DNA from normal individuals shows all sites with unmethylated CpG -DNMTs (DNA methyltransferases) add methyl groups to CpGs to shut down TSG expression, allowing for uncontrolled growth of mutated tissues and decreased growth of normal cells/tissues.

How is Myc controlled in the cell?

Myc expression is controlled by SMAD. -TGF-beta blocks cell cycle progression by activating SMAD proteins to bind Miz-1 and increase CDK inhibitors (p15 and p21) -SMAD proteins also form repressor complexes that block Myc expression

How does NF1 present?

NF1 is a common familial cancer syndrome (many diverse symptoms) -Characterized as a development of benign tumors in sheath cells surrounding neurons. It is presented as "cafe-au-lait" spots on skin of Lisch nodules in the eye -NF1 can eventually progress to neurofibrosarcomas

NF1 normal and abnormal functioning

Neurofibromatosis type 1 - defective GAP (GTPase-activating protein) Neurofibromatin protein NORMALLY functions as a GAP to turn off Ras - specifically, R1276 ("arginine finger") inserts into Ras GTP cleft and encourages GTPase activity. -R1276 mutations reduces NF1's GAP functioning by 1000 fold and the loss of NF1 function in a cell can mimic functionally HYPERactivated Ras proteins that are created by mutant Ras oncogenes. -Individuals who are NF1 (+/-) exhibit HAPLOINSUFFICIENCY (one "good" copy is not enough) and are predisposed to disease.

In what striking ways is p53 mutations seen in tumors different than those of other TSGs?

P53 mutations are point mutations (missense), rather than premature truncations. -p53 knockout (-/-) is NOT embryonic lethal -Detrimental point mutations are common in the sequence-specific DNA binding domain -P53 binds as a homo-tetramer

Describe proliferation and differentiation in regards to Myc.

Proliferation: If the cell is told to actively divide, then the cell will recruit Myc and Max. When Myc and Max are in a heterodimer together and bind to E-boxes, they emit mitogenic signals that upregulate active proliferation by recruiting HATs that will release RNA Pol II from pause sites allowing for transcription to continue Differentiation: If there is decreased proliferation and as cells differentiate, Mxd (Max) levels increase progressively and Myc is displaced by Mxd, resulting in the disappearance of Myc-Max complexes, which would otherwise block differentiation

How does RB function as a tumor suppressor? Compare this mode of action against how PTEN of APC works.

RB protein (pRB) prevents cell cycle progression by binding to the E2F transcription factor (which is responsible for turning on the genes responsible for driving the cell cycle through its different phases). PTEN works as a tumor suppressor as a negative regulator of the PI3K/Akt signalling pathway. It works by removing phosphate groups from PIP3 to prevent this product of the PI3K pathway from binding and activating Akt. APC is a TSG in the Wnt/beta-catenin pathway. APC works by binding to beta-catenin and degrading it when it no longer necessary for transcription in the cell nucleus.

Compare and contrast how the "supervisor" Ras and the "worker" Myc are controlled by the cell

Ras is a cytoplasmic proto-oncogene that, unlike Myc, has a main on/off control mechanism. It is normally in a GDP-bound "off" state, and the structure of Ras has many 3D surface features (functional domains) where different proteins can bind for fine control over Ras action c-Myc already has oncogenic tendencies... Expression and post-translational modifications are its main controls. The structure of Myc oncogene is remarkably simple (bHLH) and is always essentially "ON"

How is the apoptosis program carried out in the cytoplasm? Why are caspases already floating around in a pro-caspase form?

The apoptosome - the wheel of death - is assembled in the cytosol when cytochrome c molecules are released form mitochondria and associate with Apaf-1. This causes the assembly of a seven-spoked wheel, in which Apaf-1 forms the spokes and cytochrome C molecules form the tips of the spokes. Procaspase 9 is attracted to the hub of the wheel and is converted into active caspase 9, which in turn cleaves and activates procaspases 3,6, and 7 into executioner caspases.

When early cancer researchers fused a cancer cell with a normal cell of the same type it originated from, was the resulting hybrid cell phenotypically more like cancer or normal? What does this tell you about what must have happened in the cancer cell in terms of oncogene activation and TSG deactivation?

The hybrid cell will be phenotypically normal (nontumorigenic) indicating that cancer alleles are "recessive" Oncogene activation must have been halted and TSG deactivation must have been reversed in the cancer cell... because "as long as the cancer cell lacks these genes, it continues to proliferate in a malignant fashion. However, the moment that wild-type, intact versions of these genes operate once again within the cancer cell, having been introduced by the technique of cell fusion, the proliferation of the cancer cell, or at least its ability to form tumors, grinds abruptly to a halt."

VHL modulating cell response to hypoxia

Von Hippel-Lindau disease is a syndrome that predisposes to tumors in breast, kidney, adrenal gland, and blood vessels. -pVHL degrades a protein, hypoxia-inducible factor 1 and 2 (HIF1 and HIF2) and regulates the cell's response to low-oxygen levels Normal: in normal O2 conditions, pVHL flags HIF for degradation Abnormal: in hypoxic conditions, HIF turns on genes such as Vascular Endothelial Growth Factor (VEGF)

What is Loss of Heterozygosity?

When a normal heterozygote for a TUMOR SUPPRESSOR GENE gets a mutation in the remaining good gene, they become symptomatic and have 'LOST their heterozygosity.' Mitotic recombination can lead to LOH of a gene such as Rb. Genetic material is exchanged between two homologous chromosomes through the process of genetic crossing over occurring during G2 phase or, less often, during the M phase of a the cell cycle. The subsequent segregation of chromatids may yield a pair of daughter cells that both retain heterozygosity at the Rb locus OR yield two daughter cells that have undergone LOH at the Rb locus.

How is p53 regulated in the cell? What role does Mdm2 have? What role does p53 have in regulating Mdm2 levels?

p53 has a relatively short life-span of <20 minutes. P53 is targeted by ubiquitin-ligase (mdm2) and ubiquitin flags added and degraded by cytoplasmic proteasomes OR p53 will bind to other target genes (housekeeping genes, one of them being mdm2), which will then feedback to create more mdm2 proteins to aid in the degradation of p53.