BI 398
Spring 2022
Study Guide for Test #2
Test #2 will be on Friday, March 25. It will be a similar format to the first test, again worth
100 points. The outline below lists the topics we have covered since Test #1 (Lectures #1220 & Paper Discussion #2). As before, the test questions will be based on what I presented
in class and will not include parts of the reading that I did not talk about in my lectures.
A. Discovery of tumor suppressor genes
1. Cell fusion experiments to test dominant vs. recessive nature of cancer phenotype
(including result)
2. Mechanisms for loss-of-heterozygosity (LOH): mitotic recombination; gene
conversion; chromosomal nondisjunction—explanation for pseudo-dominant
behavior of inherited cancer phenotypes
3. Rb gene: first oncogene discovered; identification of chromosomal deletion in the
region & eventual tracking down of gene
4. Role of promoter methylation in inactivating tumor suppressor genes
5. Examples of tumor suppressor genes: NF1; Apc; VHL (be able to explain why these
are tumor suppressors if shown diagrams or given descriptions of what these
proteins do)
6. APC & familial adenomatous polyposis; crypt-to-villus differentiation of colon
epithelial cells & significance of Apc
7. Mouse knockouts as means of testing tumor suppressor function
B. Cell cycle control & cancer
1. Cell cycle stages: G1, S, G2, M & G0
2. Inputs that affect the cell cycle “clock” and outcomes of those inputs (entry into G0
vs. entry into the active cell cycle; progression from one cell cycle phase to the next)
3. Central role of cyclin-dependent kinases (CDKs) in regulating cell cycle progression
(role of phosphorylation as biochemical switch); regulation of CDK activity via
ubiquitin-proteasome-mediated degradation of cyclins (role of enzymes in
ubiquitinating target proteins)
4. Central role of cyclin D accumulation in growth factor-stimulated cell division;
understanding of what is meant by a “cell-autonomous chain of events” kick-started
by cyclin D/CDK
5. Role of CDK inhibitors (CKIs) in blocking cell cycle progression in response to
differentiation signals or DNA damage—e.g. p21
6. Significance of the “restriction point” in late G1 and the role of Rb & E2F proteins in
regulating passage through it (activating expression of G1/S and S phase cyclins)
7. Ability of viral oncoproteins to bind to Rb and related “pocket proteins” to
accelerate cell division
8. Role of myc in signaling proliferation vs. differentiation; myc as target of mitogens
(to promote proliferative pathways)
BI 398
Spring 2022
C. p53 discovery, regulation & function
1. General phenomenon of inactive p53 accumulation in tumor cells (b/c of Mdm2
negative feedback loop); identification of p53 in cells where it was inactivated by
SV40 tumor virus infection
2. Dominant negative effect of p53 inactivating point mutations due to formation of
mixed tetramers; synergism with oncogenic Ras in stimulating tumor formation
(understand that experiment)
3. Ability of p53 to cause cell cycle arrest, DNA repair and/or apoptosis in response to
various stresses; role of multiple types of post-translational covalent modifications
(phosphorylation, acetylation, ubiquitylation, methylation, etc.) in tailoring p53
function to the specific circumstances
4. Regulation of p53 by Mdm2 (degradation) & ARF (inhibition of Mdm2 in response to
excess proliferative signals, via E2F accumulation)
D. Apoptosis, necrosis & autophagy
1. Apoptosis as “default” outcome in absence of any signaling
2. Functions of apoptosis
3. Series of events in apoptosis & observable features; differences between apoptosis
& necrosis
4. Role of caspases in initiating apoptosis; intrinsic & extrinsic activation pathways
(role of cytochrome c release from mitochondria, apoptosome in former & death
receptors, death-induced signaling complex in latter)
5. Role of Bcl-2 proteins in promoting or inhibiting apoptosis via regulation of
mitochondrial channel opening
6. Use of flow cytometry/fluorescence activated cell sorting (FACS) to assay for
apoptotic cells
7. Occurrence of necrosis in tumors due to hypoxia; ability of inflammation due to
necrosis to stimulate cell division/tumor progression
8. Autophagy as a means to stave off starvation
E. Telomeres, senescence & immortality
1. Definition of senescence; observation of senescence in cell culture
2. CKI expression & “fried egg” morphology as markers of senescence
3. Ability of SV40 large T antigen-mediated inhibition of pRb & p53 to overcome
senescence and continue proliferation; eventual entry into apoptotic crisis
4. Structure of telomeres; reason for telomere shortening with each round of DNA
replication
5. Breakage-fusion-bridge cycle of apoptotic crisis initiated by telomere defects & role
in genetic instability
6. Ability of telomerase to extend telomeres; composition of telomerase (protein &
RNA) & mechanism of action
7. Ability of cancer cells to reactivate telomerase (silenced in most non-stem cells)
8. ALT: another means of extending telomeres w/o telomerase
BI 398
Spring 2022
F. Stem cells & likely cells of origin for cancer
1. Division of stem cells to produce another stem cell plus a differentiated cell or a
transit-amplifying cell
2. Spatial segregation of stem cells (e.g. in intestinal crypts); additional mechanisms to
protect stem cells from genetic damage
3. Likelihood of transit-amplifying cells being the cells of origin for tumors because of
less protection than stem cells & more division than both stem cells & differentiated
cells
G. DNA damage & repair
1. Various sources of mutations: replication errors, spontaneous chemical changes,
side-effects of cellular enzymes (e.g. APOBEC3A), effects of mutagens
2. Proofreading activity of DNA polymerase; tumor promoting effect of mutations that
inactivate this activity
3. Microsatellite instability (MIN) in sequence repeats; role of mismatch repair
pathway in preventing MIN
4. Pathways of DNA repair:
a. Dealkylating enzymes that remove alkyl groups added by chemical carcinogens
b. Base-excision repair (BER) & nucleotide-excision repair (NER) to remove altered
DNA bases/nucleotides (depurinated nucleotides, deaminated bases, oxidized
bases, pyrimidine dimers caused by UV irradiation)
c. Induction of mutations by error-prone repair by “bypass” polymerase
d. Repair of DNA double-strand breaks (DSBs) by homology-directed repair (in late
S/G2 phase) or non-homologous end joining (NHEJ) in G1 when there are no
sister chromatids
5. Hereditary cancer syndromes caused by mutations in DNA repair proteins
a. Xeroderma pigmentosum & NER (existence of multiple complementation groups)
b. Hereditary non-polyposis colon cancer (HNPCC) & microsatellite instability due
to defects in mismatch repair
c. Hereditary breast & ovarian cancer due to mutations in BRCA1/2 (involved in
homology-directed repair as scaffolding proteins)
6. Defects in spindle assembly checkpoint proteins resulting in aneuploidy seen in
many cancer cells
H. Buisson et al. paper—review Reading Guide questions for overview of experimental
purpose, approach & conclusions; be able to explain the figures (just the ones in the
actual paper, not the supplementary ones)