homework

Question 1

Why is high fidelity replication necessary to prevent cancer?

Answer 1

Replication fidelity is the accuracy of DNA replication during mitosis or meiosis. Errors in genome replication lead to genetic mutations that alter the sequence of bases in the genome. If mutations occur in genomic regions that code for proto-oncogenes or tumor suppressor genes or their regulatory regions, cells can become cancerous.

Question 2

Why are genomic repair mechanics necessary to prevent cancer

Answer 2

Mutations occur spontaneously due to errors in DNA synthesis or through environmental factors that damage DNA. A cell’s repair machinery can repair mutations and replace damaged bases to prevent genomic changes that could lead to cancer.

Question 3

What are the different types of environmental causes of cancer?Give at least one example of each.

Answer 3

• Chemical: smoke, PAHs, etc.
• Physical: radiation or inflammation
• Viral / Biological: RNA viruses or DNA viruses

Question 4

describe the difference between a mutagen and a non-mutagenic carcinogen

Answer 4

Mutagens directly change a cell’s genome by altering the DNA sequence, while non-mutagenic carcinogens promote cancer via enhanced division rate to expedite the formation of mutations and select transformed cells within a cell population.

Question 5

describe the genomic level changes for a cell/tissue from normal to cancerous

Answer 5

Cells undergo multiple genetic mutational events in tumor suppressor genes and/or proto-oncogenes that alter cell phenotype. These changes are typically depicted graphically as a Vogelgram to show sequential changes mediating the progressive transition from normal cells to cancer cells.

Question 6

describe the cell population changes for a cell/tissue from normal to cancerous

Answer 6

Singlecellsinanormalpopulationofcellsundergomutational events (initiation) that provide a selective advantage in comparison to their normal neighbors. Over time, the mutated cells divide more rapidly and have a tendency to survive, so their number increases through clonal expansion, yielding more mutated cells in the population (promotion). Over time more mutational events occur that provide cells with greater selective advantage (progression) that ultimately leads to carcinogenesis.

Question 7

describe the tissue pathological changes for a cell/tissue from normal to cancerous

Answer 7

Normal cells in a tissue that become mutated and exhibit more rapid growth lead to a larger mass of cells/tissue compared with normal tissue (hyperplasia). As further mutations occur, these cells no longer have a normal phenotype in the tissue and microscopically the cells appear to be morphologically abnormal, but these cells are fairly rare and interspersed with normal cells (metaplasia). Eventually these abnormal cells dominate the cell population in the tissue (dysplasia). Once the level of abnormality has reached a critical threshold, the tissue is deemed to be cancerous, or a carcinoma in situ (severe dysplasia) if it has not yet invaded locally

Question 8

What types of molecules, cells, ororgan is m changes would you test for if you were to design a test for: early stage cancer

Answer 8

Thisisbeforeinvasionhasoccurred–anychangesstatedtooccur early in carcinogenesis would be appropriate: genomic mutations detected through PCR or FISH, abnormal proteins detected using ELISA, mass spec, or protein chips, or early- stage pathological screens of excised tissue showing hyperplasia, metaplasia, or dysplasia. Imaging or physical examinations can also reveal abnormal masses in the body.

Question 9

Whattypesofmolecules,cells,ororganismchangeswouldyoutestforifyouweretodesigna test for: early stage cancer late stage cancer

Answer 9

Thisisafterinvasionhasoccurred–anychangesrelatedto invasion/metastasis are appropriate: excised tissue show invasion across basement membrane, tumor cells appear in the patient’s blood, lymphatic tissue, or distant locations in the body, or imaging reveals abnormal masses in body locations distant from the primary tumor.

Question 10

Describe the different mechanisms by which viruses cause cancer.

Answer 10

• Insertion of viral oncogene (v-onc) into host cell genome (acute retrovirus)
• Insertional mutagenesis: Insertion of viral DNA into tumor suppressor gene sequence to knock
  out gene (chronic retrovirus)
• Insertional mutagenesis: insertion of viral promoter upstream of cellular proto-oncogene / oncogene (c-onc) to enhance expression (chronic retrovirus)
• Synthesis of proteins that inactivate tumor suppressor proteins (DNA tumor viruses)

Question 11

Itisthoughtthatcancerscausedbyviruseswillbeeradicatedfromthehumanpopulationsooner
than cancers caused by chemical carcinogens. Explain why this might be.

Answer 11

• In general, viruses yield function through viral proteins that are foreign to the body that can be more selectively inhibited with drugs or recognized by immunotherapy because they are not part of normal uninfected cells. Targeted drugs would have few side effects compared with drugs for cancers that arise through non-viral mutations, which act on proteins that are also used by non-tumor cells to perform healthy physiological functions.
• Vaccines can potentially completely prevent viral infections, so cancers related to viral infection could be eradicated preventatively in the future.

Question 12

Apatientpresentedanidiopathic(ofunknownorigin)malignantmass–howwouldyouproposeto determine if it originated from a retroviral infection?

Answer 12

Many answers possible. Best answer: excise or biopsy the mass and isolate cells from the tissue. Assay the cells for viral gene sequences (e.g. gag, pol, env sequences) using PCR, a gene chip, or FISH. You could also screen for viral mRNA transcripts or viral proteins, although the detection sensitivity could be lower.

Question 13

Atumorwassurgicallyexcisedfromapatientandthecellsfromthetumorweregrowninapetri dish. Propose 2 simple tests that do not require molecular analysis to determine whether or not the cells composing the tumor are cancerous.

Answer 13

1) allow cells to divide excessively and see if they exhibit loss of contact inhibition – growing on top of one another after they have reached confluence,
(2) allow cells to grow in suspension and see if they survive and thus exhibit loss of anchorage dependence,
(3) allow cells to divide for >50 passages and determine if they continue to divide, suggesting that they have limitless replicative potential,
(4) inject tumor cells into an immune-compromised mouse and see if they develop into a tumor.

Question 14

ligands

Answer 14

In the context of cell signaling,l igands are small molecules(e.g.steroid hormones)or peptides/proteins (e.g. epidermal growth factor) that bind to a protein receptor inside a cell or on its plasma membrane to change the function of the protein and initiate a signaling process detected by the cell. Ligands are defined by their solubility and origin and can be either soluble (autocrine – same cell, paracrine – adjacent cell, endocrine – distant cell) or bound to matrix or other cells (juxtacrine).

Question 15

receptor tyrosine kinases

Answer 15

RTKs are proteins that span the plasma membrane (transmembrane proteins) and transduce extracellular signals (ligands) to intracellular signals (kinases). They have 3 main functional domains: a ligand binding domain, a dimerization domain, and an intracellular kinase domain. Upon ligand binding to the ligand binding domain, receptors dimerize and the intracellular kinase domain becomes activated, inducing autophosphorylation and recruitment of adaptor proteins.

Question 16

adaptor/bridging proteins

Answer 16

Adaptor proteins serve to link two or more proteins together to induce co-localization. They are primarily important for mediating the interaction between activated receptors and signaling cascade molecules. For example, Grb2 is an adaptor protein that binds to phosphorylated RTKs to allow binding to SOS and then Ras.

Question 17

Ras

Answer 17

Ras is a G-protein (GTPase) that functions as the major regulator of the MAPK pathway, controlling the transduction of signaling from receptors/adaptors to kinases. It is inactive when bound to GDP and active when bound to GTP. GDP bound to Ras is exchanged with GTP to activate Ras by guanine nucleotide exchange factors (GEFs) such as SOS. Ras is inhibited by activation of its GTP hydrolyzing enzymatic domain by GTPase activating proteins (GAPs).

Question 18

Cytosolic kinases

Answer 18

CytosolickinasessuchasRaforMAPKphosphorylateotherproteinstoturn on or off their function. These are part of intracellular signaling cascades that lead to amplification of signals received from outside the cell.

Question 19

transcirption factors

Answer 19

ranscription factors can either promote or inhibit the expression of specific genes and they serve as the ultimate downstream target of cell signaling cascades, leading to alteration in cell phenotype, for example, differentiation, cell division, or quiescence.

Question 20

Cyclin dependent kinases

Answer 20

Cdksregulatethecellcyclebyfunctioningaskinasesthatturnon and off proteins that induce phase changes. Specifically they phosphorylate proteins that mediate DNA synthesis, protein synthesis, and cell division.

Question 21

cyclins

Answer 21

CyclinsregulateCdks,turningontheiractivityduringspecificpartsofthecellcycle. Their phase-dependent concentration is responsible for inducing Cdk-specific activity at appropriate phases in the cell cycle.

Question 22

E2F

Answer 22

E2F is a transcription factor that promotes the transcription of DNA synthesis proteins, as induced by Cdk4/cyclin D and Cdk2/cyclin E during the G1/S transition.

Question 23

RB

Answer 23

RbisaninhibitorofE2Fthatpreventstranscription.ItisphosphorylatedbyactiveCdk4 and Cdk2 to inhibit its activity, allowing E2F to become active.

Question 24

Howdocellcycleregulatorproteinsbecomealteredincarcinogenesis?

Answer 24

[Just one mechanism of the following 3 is sufficient] Cancer is characterized by excessive cell proliferation, such that cells divide too frequently. This may be the result of regulation of the cell cycle itself, such that negative effectors have been deactivated (TSGs, e.g. CIP/KIP) or through the mutation of positive effectors (proto-oncogenes, e.g. Cdk4) such that they have excessive activity. This can also be mediated by excessive production of pro-proliferative signals and reduced production of anti-proliferative signals in other cellular pathways (e.g. Ras/MAPK pathway).

Question 25

what does it mean if The copy number of the gene coding for Cdk4 is 6

Answer 25

Increasingcopynumberincreasestotal expression of the protein. Cdk4 is a pro-proliferative protein, and is thus a proto-oncogene. Overexpression is phenotypically carcinogenic.

Question 26

what does it mean if a retroviral promoter has been isnerted upsteam of a gene coding for an INK4 protein

Answer 26

Retroviral promoters lead to strong constitutive expression. INK4 is an anti-proliferative signal, so over-expression will reduce proliferation, and thus the phenotype of the cell is non- carcinogenic.

Question 27

what happens if Both chromosomes containing the gene coding for Rb have been deleted.

Answer 27

Rb is an anti- proliferative protein, and is thus a tumor suppressor gene. Deletion of both functional alleles enhance proliferation, and thus the phenotype is carcinogenic.

Question 28

what happens if Both copies of cyclinD have deletions of the cyclin box

Answer 28

CyclinsbindtoCdkstoactivate their kinase domains to drive the cell cycle. If a cyclin does not have a functional cyclin box, which binds to Cdk, then the corresponding Cdk will not function, which will inhibit the cell cycle and thus inhibit proliferation. The phenotype is non-carcinogenic.

Question 29

intracellular events that can trigger apoptosis

Answer 29

DNA damage, reactive oxygen species, abnormal cell cycle, cellular damage due to chemotherapy or radiation, disruption of calcium homeostasis, and hypoxia.

Question 30

extracellular events that can trigger apoptosis

Answer 30

pro-apoptotic ligand binding (FasL,TNF) to death receptors on the cell.

Question 31

Caspase role in apoptosis

Answer 31

Caspasesareproteasesthatdirectlyinitiatetheeventsleadingtoapoptosis (caspase initiators) by activating other caspase zymogens or directly degrade proteins to induce cell death (caspase effectors). They are pro-apoptotic proteins, and thus their associated genes are tumor suppressor genes.

Question 32

p53 role in apoptosise

Answer 32

p53isatranscriptionfactorthatisthemajorprotein‘switch’thatmediatescellularstress response by promoting the expression of proteins related to cell cycle arrest, DNA damage repair, and apoptosis. Phosphorylation by stress kinases such as Chk1/2 activates p53, allowing activation of the stress response. p53 is a pro-apoptotic protein, and thus its associated gene is a tumor suppressor gene.

Question 33

role of mdm2 in apoptosis

Answer 33

mdm2 transcription is promoted by p53, and the mdm2 protein inhibits p53 activity by preventing it from binding to DNA (auto-inhibition), and thus it is a promoter of cell cycle progression and an inhibitor of apoptosis. It is a pro-cancer protein and thus a proto-oncogene.

Question 34

Bcl-2 role in apoptosis

Answer 34

Bcl-2isaproteinontheoutermitochondrialmembranethatinhibitsBax/Bak-mediated membrane permeability that allows cytochrome c to leave the mitochondria, which leads the formation of the apoptosome and apoptosis. Bcl-2 is thus an anti-apoptotic protein and a proto- oncogene.

Question 35

Thehumanpapillomavirus(HPV)isaDNAvirusthatisbelievedtoberesponsiblefor70%of cervical cancer cases. Viral infection induces expression of the E6 and E7 viral oncogenes that inhibit p53 and the Rb protein, respectively. Based on this description, describe (a) how infection will impact cell biology and (b) why infection can be more oncogenic than a typical point mutation induced by ionizing radiation.

Answer 35

a. Infection will lead to inhibition of both Rb and p53 . Inhibition of Rb will allow E2F to continuously transcribe genes mediating the G1/S cell cycle transition, yielding unregulated cell proliferation. Inhibition of p53 will prevent the cell from responding to stressors such as DNA damage or cell cycle irregularities, leading to genomic instability.
b. Infection leads to the alteration of multiple major cell signaling pathways with pro-carcinogenic outcomes through a single event (infection). Typical point mutations are fairly randomly induced throughout the genome and are singular. The probability of a single point mutation impacting a proto-oncogene or tumor suppressor gene in the genome is low, and would not impact two major pathways simultaneously.

Question 36

Which single inhibitor would be appropriate for a patient whose cancer cells are found to have Ras oncogene expression

Answer 36

Raf,because it is directly down stream of Ras .As an oncogene, Ras would have a high level of activity, inducing pro-proliferation signals. Shutting down its major target (Raf) would be the best approach if there is no inhibitor of Ras available.

Question 37

Which single inhibitor would be appropriate for a patient whose cancer cells are found to have mutations of a receptor tyrosine kinase such that its kinase domain is constantly active

Answer 37

Grb2/SOS, as it is directly downstream of the phosphorylated (active) RTK. Inhibition of the binding of these adaptor proteins to RTK or to Ras would prevent downstream Ras activation, shutting off the major signals induced by the constantly active RTK protein.

Question 38

Which single inhibitor would be appropriate for a patient whose cancer cells are found to have loss of CIP/KIP activity

Answer 38

Cdk4/cyclin D, as CIP/KIP is an inhibitor of these proteins that mediate the transition between cell cycle phases. Loss of CIP/KIP would allow the Cdks to be constantly active, which is a pro-carcinogenic phenotype, so their inhibition is the best option.

Question 39

Which single inhibitor would be appropriate for a patient whose cancer cells are found to have p53 mutations that prevent DNA binding

Answer 39

fp53cannotbindtoDNA,thenitcannotinducea stress response (cell cycle arrest, DNA damage repair, and apoptosis). The first event mediated by p53 is cell cycle arrest. Thus the best option is to inhibit proteins mediating the cell cycle: the only option available is Cdk4/cyclin D. Bcl2 would also be an option, as its inhibition would promote apoptosome formation and induce apoptosis.

Question 40

describe how DNA damage is different from dna mutation

Answer 40

dana damage is a physical abnormality in DNA such as a chemical modification of a base or a break in the backbone that can potentially be corrected by the cell. A mutation is a heritable change in base sequence of the DNA (e.g. a point mutation or change in gene number) that cannot be corrected.

Question 41

describe why loss of genomic stability is an important part of carcinogensis

Answer 41

Genomic stability is mediated by DNA damage sensors and DNA repair proteins that safeguard the genomic DNA of a cell by preventing mutations that could be carcinogenic. Without DNA damage sensors and repair proteins, damage (e.g. due to replication mistakes, radiation, or chemical carcinogens) would more frequently lead to a mutation and thus a potential change in the cell that could lead to cancer over the course of multiple cell cycles as the mutations add up.

Question 42

describe how genomic instabillity can contribute to drug resistance in cancer

Answer 42

Genomic instability yields both a heterogeneous genotype between cells in an advanced tumor and a high mutation rate. The presence of multiple genotypes in a tumor allows the possibility that some clonal populations are more resistant to certain drugs than other clones, allowing them to survive a drug treatment that shrinks a tumor, and then proliferate to re-form the tumor, which is then resistant to the same drug. The high mutation rate allows the mutation of gene products that mediate the cellular pathways targeted by the drug, such that tumors can develop mutated cells that do not respond to drugs, which can then expand to become the main cells in the tumor.

Question 43

AfterexperiencinganeventthatdamagesDNA,acellnormallydetectsthedamageandinitiates signaling events that lead to repair of the damage. Name 2 proteins involved in these downstream events that, if mutated, would prevent the cell from completely recovering. Describe, mechanistically, how each mutation would prevent recovery.

Answer 43

We are looking for proteins “downstream” of (directly or indirectly impacted by) DNA damage sensors. We studied 3 sets directly downstream: ATM/ATR which activate Chk2/Chk1, which in turn activate p53 (which is negatively regulated by mdm2). p53 then directly transcribes proteins related to cell cycle arrest, DNA damage repair, and apoptosis, which would be appropriate answers as well. If ATM/ATR, Chk2/Chk1, or p53 (tumor suppressor genes) was mutated, DNA damage would no longer activate p53, which would prevent the cell from responding to DNA damage, which lead to unregulated cell proliferation after damage that is not repaired, which could lead to carcinogenesis. If mdm2 was mutated to no longer bind to p53, this would lead to excessive cell cycle arrest, which is not carcinogenic. If mdm2 was mutated such that it no longer responded to inhibitors or stimuli, becoming constitutively active, this would be an oncogenic change, preventing p53 from becoming active after DNA damage, which is pro-carcinogenic.

Question 44

Describe the underlying mechanistic steps mediating the formation of metastases involving cells in primary tumor

Answer 44

Epithelial cells detach from their neighbors via loss of E-cadherins, invade into the basement membrane via expression of matrix metalloproteinases, migrate into the stroma, and intravasate into either lymphatic vessels, blood vessels, or body cavities. These irregular cells evade immune detection and rapidly alter their phenotype.

Question 45

Describe the underlying mechanistic steps mediating the formation of metastases involving cells that have intravasated into blood vesssel or lymphatic vessels

Answer 45

In circulation, tumor cells survive by evading anoikis-mediated death (loss of anchorage dependence) and often form multicellular aggregates that arrest in the capillary beds of distant tissues, where they extravasate into the tissue interstitium. These irregular cells evade immune detection and rapidly alter their phenotype.

Question 46

Describe the underlying mechanistic steps mediating the formation of metastases involving cells that have extravasated into distant tissue

Answer 46

Cells proliferate and induce angiogenesis to establish a metastasis. These irregular cells evade immune detection and rapidly alter their phenotype.

Question 47

How are extracellular matrix molecules and adhesion molecules involved in metastasis?

Answer 47

• In the initial stages in metastasis, normal epithelial cells develop the ability to detach from matrix molecules in basement membrane by altered expression of integrins.
• In the initial stages in metastasis, normal epithelial cells develop the ability to detach from adjacent cells in their surrounding epithelium by losing expression of E-cadherins and gaining expression of N-cadherins.
• Detachment from adjacent cells and basement membrane allows enhanced division and hyperplasia.
• Dissolution of the basement membrane and other matrix molecules by matrix metalloproteinase expression allows invasion across the basement membrane and into surrounding tissue.

Question 47

Provide3reasonswhytumorcellsthathaveextravasatedintothebloodstreamhavealow efficiency in establishing a metastasis.

Answer 47

• tumor cells in blood circulation have lost contact with matrix that they normally require for survival, which can induce anoikis/apoptosis.
• Immune cells (leukocytes) can recognize and destroy circulating tumor cells.
• Shear forces can destroy circulating tumor cells.
• The tumor cells do not rapidly alter their phenotype so that they are not capable of extravasating in distant tissues.
• Tumor cells cannot respond to their new environment in a distant tissue to induce proliferation and/or angiogenesis.
• Cells die in their new site of metastasis due to incompatibility with new local signaling
  BIOE 498/598 TC1, Fall 2023 Homework Solutions
  molecules/chemokines.
• Immune cells can recognize and destroy tumor cells in the metastatic site