Cell Cycle Regulation and Oncogenesis

Question 1

Cyclin B-CDK1 Regulation and Function

Answer 1

In G2, Cyclin B-CDK1 is constitutively repressed until it is phosphorylated by Cyclin A-CDK1.
Active Cyclin B-CDK1 degrades the lamins in nuclear envelope, and enables condensin to condense chromosomes.
Degraded by APC and mitotic exit occurs.

Question 2

Anaphase promoting complex

Answer 2

Degrades Cyclin B to remove cell from mitosis and indirectly leads to the separation of sister chromatids by cohesin cleavage.

Question 3

Cell Cycle Checkpoints

Answer 3

DNA synthesis checkpoint (in G2).
2 DNA damage checkpoints at the beginning of M and S phases.
Incomplete spindle formation checkpoint (during M).
Restriction point (G1).

Question 4

Restriction Point

Answer 4

A cell cycle checkpoint in G1 that connects extracellular signaling and cell cycle control. Growth factor signaling leads to the upregulation of Cyclin D, causing Rb phosphorylation and E2F release –>DNA synthesis.
This regulation is lost in cancer.

Question 5

DNA Damage Checkpoint

Answer 5

P53 is normally bound to MDM2 (a ubiquitin ligase), which usually targets P53 for degradation. Normally P53 levels are low.
When DNA damage occurs, numerous acetylases and kinases disrupt the interaction between MDM2 and P53, preventing P53 degradation. Concentration of P53 increases, which acts as a transcription factor for P21, which inhibits CDK activity and inhibits phosphorylation of Rb. Cell arrests prior to S phase. P21 also inhibits CyclinB-CDK1, so DNA damage can also arrest cell prior to M.
Also, P53 increases transcription of pro-apoptotic factors PUMA and NOXA.

Question 6

DNA synthesis checkpoint

Answer 6

CDC25 not able to remove inhibitory Wee1 phosphates until it is phosphorylated as a result of complete DNA synthesis.

Question 7

Spindle Checkpoint

Answer 7

The cell can sense unoccupied kinetochores. When it does, it keeps the anaphase promoting complex inactive, so cohesin doesn’t split. Loss of a single MT is enough to stop M, so only low doses of anti-MT drugs like Taxol are needed.

Question 8

Immortalized Cells

Answer 8

Phenotypically normal, unlimited proliferation

Question 9

Transformed Cells

Answer 9

Cells have started to accumulate certain properties of tumorigenic cells:
Cell looks more like a fibroblast than epithelial cell.
Cell depends heavily on glycolysis (Warberg effect).
Cell is no longer dependent upon integrin signaling.
Loss of contact inhibition.
Loss of growth factor dependence.
Changes in ploidy.
Angiogenesis.

Question 10

Senescence

Answer 10

M1. When a cell does not divide, but does not die. Cells look like fried eggs. Senescence is irreversible (growth factors don’t cause growth), as opposed to quiescence. This occurs when telomeres in somatic cells get too short and the cell believes that DNA damage has occurred. P53 will be upregulated and the cell cycle will be arrested.

Question 11

If cells have p53 or Rb ablation…

Answer 11

They will continue to grow until M2 (crisis), where the cells will die. If, however, cells are treated with telomerase, M2 can be overcome.

Question 12

Do cancer cells have greater or fewer telomeric repeats than germ cells and senescent cells?

Answer 12

Fewer. Cancer cells have already hit crisis, but then overcome cell death.

Question 13

ALT

Answer 13

Alternate means of telomere maintenance. Tumor cells will continue to maintain telomeres in the presence of telomerase inhibitor. Something must be happening here…

Question 14

How do RNA tumor viruses cause cancer?

Answer 14

1) Viral genome contain the oncogene (i.e. Rous Sarcoma Virus)
2) Insertional mutagenesis- if viral genome sits next to proto-oncogene, overexpression can occur.

Question 15

Proto-oncogenes

Answer 15

Normal genes that can become oncogenes if overexpressed.

Question 16

Methods of proto-oncogene activation

Answer 16

Deletion or point mutation in the coding sequence (hyperactive protein generation).
Regulatory mutation (overexpression).
Gene amplification (overexpression).
Chromosome rearrangement (overexpression or fusion protein).

Question 17

Double Minutes

Answer 17

Products of gene amplification

Question 18

3 ways that gene amplification leads to oncogenic activity

Answer 18

1) A limiting protein is greatly overexpressed (Cyclin D, CDK4).
2) Growth factors receptors are overexpressed and dimerize spontaneously (Her2, EGF-R).
3) Autocrine Loop, where a product is made in high amounts in a cell and causes endogenous activation of receptors.

Question 19

2 ways that gene rearrangement leads to oncogenic activity

Answer 19

1) Regulatory regions are moved around, causing new expression levels (Burkett’s Lymphoma: t(8,14))
2) Novel fusion proteins (t(9,22) causes CML by generating BCR-ABL, which signals cell proliferation).

Question 20

Gleevec

Answer 20

Blocks BCR-ABL’s ability to phosphorylate a substrate protein that causes cell proliferation.

Question 21

Cooperative Oncogenes

Answer 21

Some oncogenes only trigger transformation if they are expressed at the same time (myc+ras).

Question 22

Experiment suggesting existence of tumor suppressors

Answer 22

Tumor cells fused with normal cells. Resulting cells were normal.

Question 23

Inheritance pattern of inherited tumor syndromes

Answer 23

Generally autosomal dominant, but incompletely penetrant due to an inheritance of just susceptibility.

Question 24

Difference between RNA and DNA tumor viruses

Answer 24

DNA tumor viruses contain novel oncogenes that don’t have human cell counterparts.

Question 25

SV40

Answer 25

Simian virus 40. Expresses large T antigen that inactivates p53 and pRb.

Question 26

Adenoviruses

Answer 26

Express E1A and E1B that bind to pRb and p53.

Question 27

HPV

Answer 27

Human Papilloma Virus. Expresses E7 that inactivates pRb and E6 that targets p53 for degradation.

Question 28

Sporadic vs Hereditary Retinoblastoma, and why.

Answer 28

Sporadic is associated with single eye defects and no secondary tumors. Hereditary RB is bifocal and causes secondary tumors. This occurs because of the two hit hypothesis. In familial Rb, there is an inherited susceptibility on one RB locus. Early on, a second mutation develops. Both of these hits cause many tumors. In sporadic, the two hits occur spontaneously and are localized to one tissue.

Question 29

Difference between oncogenes and tumor suppressors

Answer 29

Oncogenes generally represent “gain of function.” Only requires one mutated allele. Tumor suppressors are “loss of function,” meaning that loss of both alleles is needed for tumorigenesis.

Question 30

ARF

Answer 30

Alternate reading frame protein. ARF inhibits MDM2, preventing p53 degradation and leading to cell death and CDK inhibition.

Question 31

Most common p53 mutation

Answer 31

Missense mutation

Question 32

What happens to a protein after a frameshift mutation?

Answer 32

Generally is truncated.

Question 33

Adenomatous Polyposis Coli mutation

Answer 33

APC protein susceptible to frameshifts. Normally binds to beta-catenin, phosphorylates it, and causes its degradation. If APC is truncated, beta-catenin is not degraded, and it promotes cell proliferation.

Question 34

Most common pRb mutation

Answer 34

Deletion

Question 35

Loss of heterozygosity

Answer 35

When there’s a missense or frameshift mutation on one allele of a tumor suppressor, the remaining wild-type allele is deleted.

Question 36

Haplo-insufficiency

Answer 36

An exception to the two-hit hypothesis, where loss of one allele is enough to cause a tumor. Three ways:

1) Reduced expression below some threshold.
2) Dominant negative effects
3) Epigenetic silencing of the other allele.