question

oncogenes

answer

mutated normal genes that promote cell growth and/or decrease apoptosis and can cause cancer. mutations are gain of functions. cancers of this type are acquired not inherited.

question

tumor suppressor genes

answer

normal genes which protect us from cancer by aiding in the repair of DNA, slowing the cell cycle, or stimulating apoptosis. loss of these can lead to cancer but both copies have to be mutated on the 2 chromosomes. mutations are loss of function and is part of the 2 hit model. some of the changes to these are caused by nonmutational changes like epigenetics. cancer of this type can be inherited if denovo mutation in germ cells.

question

gate keepers and 3 examples

answer

a type of tumor suppressor that inhibit cell proliferation and/or promote cell death. ex: RB, p53, APC

question

caretakers and 3 examples

answer

a type of tumor suppressor that enable DNA repair. ex: XP gene for excision repair, BRCA1 and BRCA2, ATM

question

Cell fusion experiments

answer

experiment in 1960 by Georges Barski and was the 1st indication that there are protective function genes that was lost. 1. fused a cancer cell and a normal cell by treating cells with inactive Sendai virus. 2. The fused hybrid cell divides and has a new nuclei formed containing chromosomes from both original cells and divisions are normal, because the normal cell provided the lost rumor suppressors for the cancer cells. 3. further divisions ended with cancer cell with uncontrolled growth because there's too much genetic material and doesn't divide evenly so lose chromosomes. can help to identify dominance (oncogene) or recessiveness (tumor suppressor) of cancer cell phenotype.

question

2 Fusogenic agents for cell fusions experiments and purpose

answer

1. polyethylene glycol 2. viral glycoproteins (inactive Sendai virus) Purpose: fuse cells so that one plasma membrane surrounds the contents of both cells.

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syncytium

answer

a term for cell fusion experiments. a cell that has many nuclei.

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Polykaryon

answer

a term for cell fusion experiments. a Giant cell with many nuclei inside due to many cells fusing.

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Heterokaryon

answer

a term for cell fusion experiments. a fused cell with nuclei from different cells.

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Selection

answer

a term for cell fusion experiments. using antibiotics or other drugs to select for the cells carrying resistance genes.

question

3 parts of studying tumor suppressors

answer

1. identify the tumor suppressor (usually via families with hereditary patterns of cancer). 2. determine the tumor suppressor's normal function. 3. determine how the loss of function of this gene lead to cancer.

question

Retinoblastoma (Rb)

answer

gene on chromosome 13q associated with cancers of the retina and is characterized by thickening of the optic nerve due to extension of the tumor. displaced normal retina and red and white eye reflection in pictures. thickening of the optic nerve has to be caught early before it reached the brain and can be treated with enclosed gene therapy or have to remove the eye. 40% of the cases are familial. diagnosis happens before 5-6 years of age because cells are still in the blast stage and dividing to catch mutations but will arrest in G0 in maturity.

question

secondary cancers that Rb mutations are associated with and why?

answer

1. osteocarcinoma 2. leukemia 3. melanoma 4. lung cancer 5. bladder cancer this suggests that Rb has bigger functions than simply in the eye.

question

Incomplete penetrance

answer

a carrier who did not develop retinoblastoma because only had mutation in 1 chromosome but can still give it to the kids. ex: 1 in 10% of carriers would be unaffected this way.

question

Loss of Heterozygosity (LOH) and 5 mechanisms

answer

the mechanism in which the second copy of a gene is mutated after the 1st one is mutated. there are 5 ways this happens: nondisjunction (chromosome loss), nondisjunction and duplication, mitotic recombination, gene conversion, deletion, point mutation.

question

Unilateral Rb

answer

most likely associated with the nonhereditary (sporadic) form of retinoblastoma because the mutation is in somatic cells and it not likely to spread to the other eye. part of the 2 hit model where it was easier to get the second hit after getting the first.

question

Bilateral Rb

answer

most likely associated with the hereditary (familial) form of retinoblastoma because the mutation is in the sperm cells and is likely that both eyes are affected. one hit model. will be more likely to get other cancers because they already began with one tumor suppressor down.

question

Mitotic recombination

answer

a mechanism of LOH. this happens more often than deletions that inactivate tumor suppressors. 1. There is a mutated Rb allele and replication in S phase as normal so there are 2 chromatids with the mutation. 2. but in G2 and M of the cell cycle where mitotic recombination happens, the switching of the two chromosomes put the mutated sections in both chromosomes. 3. when segregation of chromatids at the end of mitosis happens, some of the resulting germ cells has retained the Rb heterozygosity but others did not and lack functional gene copies.

question

Gene conversion

answer

a mechanism of LOH. happens when DNA polymerase starts to replicate on a template strand but jumps to a template strand on the homologous chromosome in the middle and copies a portion before jumping back to finish the replication on the original template strand. this causes the new replicated strand to be missing a locus if the strand it jumped to was missing it or even have it mutated.

question

Chance of getting 1st and 2nd hit to tumor suppressors

answer

1:1 million chance of 1st hit. 1:1000 chance of 2nd hit.

question

Cellular Mechanism of Rb and 3 ways it is affected

answer

normally active Rb binds to E2F and inactivates it so that E2F can't go into nucleus and activate gene transcription needed for S phase. a growth factor signaling through the Ras-MAPK pathway will activate Cdk-cyclin, which will phosphorylate Rb so that it can't bind to E2F. Active E2f goes to nucleus and causes gene transcription and translation for enzymes and proteins needed for S phase. Affected by: 1. Heredity 2. Environmental causes of mutations in the gene 3. viral oncogenes: HPV-E7, SV40-large T antigen attack them to have more hosts to infect

question

APC

answer

a tumor suppressor associated with familial adenomatous polyposis coli and sporadic colorectal, pancreatic, and stomach carcinomas as well as prostate carcinomas.. the gene codes for beta catenin degradation and is on chromosome 5p21. tumor suppressor associated with smaller than 5% of familial colon cancers and less than 1% of colon cancers (FAP) in the US.

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p16 INK4A b

answer

a tumor suppressor associated with familial melanoma and sporadic cancers. the gene codes for a CDK inhibitor and is on chromosome 9p21. smoking is more likely to increase methylation of this gene's promoter though it is not as frequent as LOH. it is encoded for by the CDKN2A gene's alternative splicing. the inhibitor of CDK-cyclin prevents Rb from being phosphorylated and the restriction point is held to prevent loss of cell cycle control.

question

p14 ARF c

answer

a tumor suppressor associated with all types of sporadic cancers and codes for a p53 stabilizer on chromosome 9p21. the alternative reading frame of the CDKN2A gene, it inhibits and promotes the breakdown of Mdm2 so that p53 accumulates without destruction to arrest cell cycle and cause apoptosis. without this, the lack of apoptosis could cause fatal tumors.

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PTEN d

answer

a tumor suppressor associated with familial Cowden's disease, breast and gastrointestinal carcinomas and sporadic glioblastoma, prostate, breast, and thyroid carcinomas. the gene codes for PIP3 phosphatase and is on chromosome 10q23.3

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Rb

answer

a tumor suppressor associated with familial retinoblastoma and osteocarcinoma and sporadic retinoblastoma, sarcomas, bladder, breast, esophageal, and lung carcinomas. the gene codes for transcriptional repression control of E2Fs on chromosome 13q14.

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TP53

answer

a tumor suppressor associated with Li-Fraumeni syndrome and many types of sporadic cancers. the gene codes for TF proteins and is found on chromosome 17p13.1

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DNA methylation and 2 types of genes associated

answer

an epigenetic method of inactivating a tumor suppressor by methylating promoter sequences not the actual exons so that the promoter can't recruit transcription complexes and the tumor suppressor can't work. the methyl on DNA recuits deacetylases to remove acetyls to histones and cause the DNA to be wound tighter and cause repression. CpG sequences have C methylated normally in our genome by de novo methylases and maintenance methylases. Ex: APC and RB

question

de novo methylases

answer

enzymes that are responsible for methylation of DNA to inactivate genes.

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Maintenance methylase

answer

methylases that are active during the S phase and will methylate new strands of DNA to keep parent epigenetic changes since polymerase will only copy nucleotides.

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Histone deacetylase inhibitors

answer

a novel drug that is used for treatment of cancer by preventing the removal of acetyls so that DNA is kept from winding up tightly and covering up tumor suppressors.

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RA (retinoic acid)

answer

an example of DNA methylation. this usually binds RARbeta2 and stimulates cell cycle arrest and cell differentiation but can't halt proliferation if it is methylated. treatments for breast cancer cells are refractive to treatment of this because of methylation.

question

TSA (trichostatin A)

answer

an example of DNA methylation this binds to HDAC and inhibits it so that RARbeta2 is no longer suppressed and responds to RA or RA treatment for breast cancers.

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HDAC (histone deacetylase)

answer

enzyme that binds to methylated CpG sites and removes acetates from histone, making chromatin inactive and can cause cancer if it suppresses a tumor suppressor.

question

NF1 (neurofibromatosis type 1) syndrome characteristics

answer

cafe au lait spots on the skin that looks like spilled coffee. lisch nodules in the iris benign neurofibromas that can become malignant peripheral nerve sheath tumors (MPSNST) other related cancers like astrocytoma, pheochromocytoma and myelongenous leukemia.

question

NF1 gene function

answer

neurofibromin interacts with RAs and increases Ras's GTPases activity more than a thousand times. it is a GAP. the ras oncoprotein is activated by a GEF (guanine nucleotide exchange factor) that put a GTP on it. a GAP (GTPase activating protein) is necessary to inactivate Ras because it takes off the phosphate making GTP into GDP. if the gene is absent or unable to interact with Ras, Ras will stay activated. even heterozygotes have increased Ras activity because they are haploinsufficient in this gene function.

question

Familial Adenomatous Polyposis (FAP)

answer

disease associated with APC mutations where patients develop hundreds and thousands of nonmalignant polyps (precancerous hyperplasia lesions) and then cancer of the colon because it is folded and exposed to the digestive system to absorb fluids to has to do many cell turnovers. other cancers caused because of APC's functions outside of the colon are: bile duct, small intestine and stomach. this is diagnosed by family history because it typically is onset before 40 but colonoscopy are only done after 50 so some polyps can be detected and removed. 2/3 of this cancer shows an APC mutation.

question

Knudson's Two hit & multi hit hypothesis and Volgel analysis of colorectal tumors

answer

APC/wnt pathway 20-40 years. 1. normal epithelium 2. APC mutation 3. early adenoma (benign but some extra cells) 4. KRAS mutation 5. intermediate adenoma 6. SMAD4 mutation (signaling protein) 7. late adenoma 8. p53 mutation 9. carcinoma 10. other mutations and epigenetic changes since we lost p53 already. 11. invasion and metastasis since the colon has a rich blood supply to the liver and hepatic portal vein.

question

The Wnt pathway and APC

answer

a signaling pathway associated with colon cancer and APC mutations. normally, without the growth signal Wnt, beta catenin is targeted for degradation by axin-GSK3-APC destruction complex, which phosphorylate beta catenin. Beta catenin is linked to ubiquitin and is sent to the proteosome for degradation and its absence turns off the pathway. normally, with the growth signal Wnt, the Wnt proteins bind to and activate cell surface Wtn receptors, which stimulate proteins to inactivate the axin-GSK3-APC complex and so beta catenin isn't degraded. beta catenin can enter the nucleus and activates target genes that control cell proliferation. cancer cells that are independent of the Wnt protein have no destruction complex activation because of the mutated or absent APC gene. beta catenin accumulates and enters the nucleus, permanently turning on the pathway for cell proliferation. wnt -> frizzled -> dishevelled blocks GSK-3 beta -> beta catenin is active -> cell growth and cell proliferation signals transcribed.

question

beta catenin secondary role

answer

part of the receptor that spans the plasma membrane out to the extracellular space and cytoplasm and has roles in controlling actin filaments in the cell.

question

Ubiquitylation

answer

the process of labeling protein for destruction by proteosome. this is a quick way to known down protein level but RNA and proteins usually have half lives and will naturally degrade though DNA is stable. the process is unidirectional, rapid, fine-tuning, localized and specific.

question

Abnormal beta catenin in colon crypts affect

answer

cells no longer migrate to the top and maintain 3-4 day turnover. cells don't undergo mitosis or migrate. stationary cells form polyps and acquire more mutations to become cancerous.

question

Where in the domain of the APC protein are there the most mutations

answer

binding site for beta catenin and down regulation binding site.

question

Are de novo mutations in germ cells more likely to occur in oogenesis of spermatogenesis and why?

answer

oogenesis because making sperm has more divisions throughout life but oogenesis only divide once in embryo so accumulate less mutations.

question

7 trains gained through genetic mutations

answer

1. faster cell proliferation (APC and beta catenin) 2. evading checkpoint regulation 3. immortality (telomerase) 4. decreased apoptosis 5. angiogenesis (blood vessels for tumor to get out oxygen and nutrients to do glycolysis) 6. resistance to immune system 7. increased invasiveness, ability to survive in bloodstream, and ability to grow a metastasis in foreign tissue though cancers shed cells into the blood and many cells. don't survive in blood to get to other tissues. this can be a good non-invasive way to detect cancer in blood rather than biopsies.

question

Overview of the cell cycle

answer

normal cells divide in 18-24 hours in culture, which is slower than in vivo because may lack signals from the body. prokaryotes divide faster than eukaryotes because they have less DNA and less checkpoints, embryonic and lymphocytes divide in just a few hours. cancer cells will divide more quickly, decreasing G1 from 10 hours to just a few minutes so they have scanty cytoplasm from lack of time to grow.

question

Order of cell cycle stages and durations in culture

answer

1. g1/g0 12-15 hours 2. s 6-8 hours 3. g2 3-5 hours 4. m (prophase, metaphase, anaphase, telophase)

question

What happens when mitosis completes by cytokinesis does not happen?

answer

the cell may have more than one nuclei or too much DNA. cancers cells often have multiple nuclei, chromosome fusions and abnormal numbers of chromosomes.

question

Staurosporine

answer

a drug used in testing cell cycle characteristics. it stops the cell cycle at the restriction point so they can't enter S phase by inhibiting protein kinase. normal cells abiding by the restriction point will be halted in the cell cycle and won't be killed by camptothecin, then will resume normal cell cycle after drug removal. cancer cells that don't respond to the restriction point will not half in this drug's presence and will die under camptothecin. this suggests that combination therapies will work as cancer treatments.

question

Camptothecin

answer

a drug used in testing cell cycle characteristics. it kills cells that are in S phase by binding topoisomerase so it can't decrease supercoiling of DNA. normal cells abiding by the restriction point will be halted by staurosporine and never enter S phase to be killed so can resume normal cycle after drug removal. cancer cells that don't respond to the restriction point will enter S phase even with staurosporine and is killed by this. this suggests that combination therapies will work as cancer treatments.

question

FACS (fluorescence activated cell sorting)

answer

a way to detect aneuploidy and cell cycle characteristics. cells are labeled with fluorophores to see if it is diploid or aneuploid. can determine if the cell is in G1 or G2 by the amount of genetic material present and diagnose cancers by seeing if the majority of cells are accumulating in G2. can also tell if cells are undergoing apoptosis because the genetic material will be condensing into apoptotic bodies for engulfment by other cells. if testing for normal germ cells, should see haploid numbers. can be used to separate tumor stem cells from transit amplifying cells.

question

BrdU (bromodeoxyuridine)

answer

a way to label S phase DNA in vivo and detect cell cycle characteristics. it is usually given to live animals and produced a brown stain in living specimens. it is an analog for thymidine because uridine is not in DNA but does the same binding. When cells do replication for division, it will incorporate this into the new strand and can be used to detect which cells are doing divisions. appear green and usually used with immunolabeling of p27 to see post mitotic cells.

question

Growth factors

answer

materials that stimulate the cell cycle by helping cells get over the restriction point.

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Restriction point and 3 conditions

answer

the point between G1 and S that is not bypassed when there are low levels of growth factors, increased cell density (contact inhibition) and lack of anchorage (prevents anoikis). once pass this point, the division process is automated and cell cannot be stopped from entering S phase. cancer cells bypass this and continue to divide despite DNA damage accumulation.

question

G0

answer

cell quiescent state in which they don't go into S phase and divide. neurons are in this state.

question

5 influences of the cell cycle clock

answer

1. tyrosine kinase receptors that signals the nucleus (Ras) 2. G protein coupled receptors 3. TGF beta receptors - inhibitors of growth 4. integrins - spans the membrane to connect the ECM to the cell membrane of the cell for anchorage. 5. nutrient status

question

4 cell cycle checkpoints and purpose

answer

checkpoints ensure that DNA is repaired if it can be or cell does apoptosis if there is too much damage. without them, there is irregular DNA replication and fragile chromosomes like single stranded breaks and fusions. 1. restriction point 2. cell checkpoint in S phase to see if the DNA polymerase had made errors and the genome is changed to hat further replication. 3. checkpoint between G2 and M to ensure that DNA replication is completed and there are two of everything before mitosis. 4. checkpoint between metaphase and anaphase to ensure that chromatids are properly assembled and attached to the spindle to be separated and prevent nondisjunction.

question

Cyclins and CDKs for each stage of the cell cycle

answer

1. G1 - cyclin D and CDK4/6 2. pass restriction point - cyclin E and CDK2 3. S phase - cyclin A and CDK2 4. S phase and G2 phase - Cyclin a and CDC2/CDK1 5. M phase - Cyclin B and CDC2/CDK1

question

Cyclin

answer

molecules that bind to CDKs and increase their enzymatic activity as well as guide CDKs to their substrates. they are inactivated by ubiquitinylation pathway that involves proteosome degradation. their levels fluctuate with the cell cycle or are externally controlled by things like growth factors and TGF beta. they have no enzymatic activity of their own.

question

CDKs (cyclin dependent kinases)

answer

serine/threonine kinases not tyrosine that helps regulate mitosis proteins. they need cyclins to work.

question

Which cyclin is found in multiple segments of the cell cycle and is regulated by growth factors?

answer

Cyclin D.

question

What is the relationship between cyclin D1 and estrogen receptor?

answer

Cyclin D1 binds to the ER and activates estrogen-related transcription of genes, which is a steroid that signals the nucleus for gene expression and mitosis (mitogens).

question

What induces the D type cyclin expression?

answer

the estrogen receptor with the signaling intermediaries AP-1 transcription factor.

question

cycle example of cyclin and CDK

answer

1. the mitotic CDK is a kinase that is present all the time but cyclin has to be built up. 2. just before the g2-m transition, the cyclin is at high enough concentration to combine with the CDK. 3. active cyclin/CDK complex stimulates nuclear envelope breakdown, chromosome condensation, mitotic spindle formation and targeted protein degradation of prophase. 4. in m phase, the anaphase- promoting complex helps is present if the chromatids lines up properly and cyclin is degraded.

question

Kinases and phosphatases role in cyclin/CDK activation

answer

1. when mitotic CDK and cyclin bind together, they are still inactive. 2. two inhibitory phosphate groups are attached to the CDK molecule by inhibiting kinases. 3. an activating phosphate group is added by an activating kinase but the CDK is still inactive as long as the inhibitory phosphate groups are present. 4. a phosphatase removed the inhibiting phosphate by activating the cdk-cyclin complex. 5. there is a positive feedback loop that stimulated more cyclin/CDKs for that stage of mitosis but will be degraded at the end of the stage.

question

Control of the initiation of chromosome movement by the anaphase promoting complex

answer

1. during metaphase the chromatids are held together by cohesin and the spindle microtubules are attached to each chromatid. 2. the anaphase-promoting complex activates separase. 3. there is movement of the microtubules to pull the chromatids apart for anaphase.

question

Checkpoint control of the anaphase promoting complex

answer

this checkpoint is to prevent nondisjunction. 1. the Mad and Bub proteins attach to chromatids that are unattached to the spindle. 2. the Mad and Bub complex inhibits CDC 20. 3. CDC20 can't bind to the anaphase promoting complex and the complex is inactive and the checkpoint remains on for no progression into anaphase. 4. if there is inhibition of CDC20, it binds to the anaphase promoting complex and activate it for the checkpoint to turn off and there is progression into anaphase.

question

CDK4/6 inhibitors

answer

in G2 before restriction point. p16 INK4A, p15 INK4B, p18 INK4C, and p19 INK4D

question

CDK2 and CDC2 (CDK1) inhibitors

answer

in G1 after Restriction point. p57 KIP2, p27 KIP1, p21 CIP1 (controlled by p53 to stop cycle)

question

Transforming growth factor Beta (TGF beta)

answer

growth factor that increases p15 INK4B (inhibitor of kinase), which will inhibit CDK 4/6 and is the only effective inhibitor before the restriction point. it inhibits growth in early and mid-G1. it is a strong activation of p15, which will inhibit S phase, but is only weak activator of p21 to inhibit other cyclins and CDKs. they are serine and threonine kinases not tyrosine kinases.

question

TGF-beta/Smad signaling pathway and cancers associated

answer

inhibits epithelial cell growth: 1. in the absence of TGFbeta, the type I and II receptors are not clustered or phosphorylated on the cell surface. 2 binding of TGFbeta results in clustering of receptors followed by phosphorylation of type I receptors by type II receptors. 3. the activated type I receptors phosphorylate Smad protein nearby. 4. the phosphorylated Smad bind to other smads and, together, they enter nucleus to activate the transcription of genes that inhibit cell proliferation. 5. 2 of the genes produced are p15 and p21, which halt the cell cycle by inhibiting the CDK-cyclin complexes weakly, whose actions are required for progression through key transition points in the cell cycle. TGF beta mutations are found in colon and stomach cancers. Smad proteins are mutated in pancreatic and colon cancers. inhibition using ligand, receptor or Smad causes issues stopping cell cycle arrest so cell keeps dividing.

question

mitogen as extracellular signal that regulate the cell cycle

answer

mitogen signals though the receptor and activates PI3K and Akt/PKB, which will inhibit p21 and p27. p21 and p27 are phosphorylated and remain cytoplasmic instead of going to the nucleus so they can't stop Cyclin E and other cyclin/CDKs from activating the cell cycle during late G1, S, G2 and M. If in early G1 (before restriction point), p21 and p27 still stimulate cyclin D and cdk 4/6.

question

Phosphorylation of Rb and bypassing the restriction point process

answer

1. a growth factor binds to receptor on the cell surface, leading to signal transduction and the production of active CDK/cyclin. 2. CDK 4/6-cyclin D adds phosphate groups to Rb, using ATP as phosphate group donor, taking it from unphosphorylated to hypophosphorylated and priming it for later phosphorylations 3. after being phosphorylated, Rb can no longer exert its inhibitory influence on the restriction point and cells are free to pass into S phase. if it is not phosphorylated by CDK/cyclin, it will stop the cells at the restriction point. 4. 3. CDK2 and cyclin E hyperphosphorylate Rb after the restriction point. excess cyclin E/CDK2 are associated with cancer because they keep Rb in the hyper state and cell cycle continues out of control. 5. hyperphosphorylate pRb, p107, and p130 releases the E2F transcription factors so they go to nucleus to start cell cycle gene activation. 6. E2F increases the expression of more Cyclin E and CDK 2 for positive feedback of hyperphosphorylated Rb and more of itself.

question

Cycle-dependent phosphorylation of Rb and viral targeting

answer

1. in G1, Rb is hypophosphorylated. 2. at Restriction point, Rb is hyperphosphorylated until it finishes M and starts G1 again. viral proteins bind the hypophosphorylated form of Rb not the hyper form because it targets the restriction point that requires hyperphosphorylation to get through.

question

What viruses target Rb and what proteins target them?

answer

HPV using E7 SV40 using T antigen Adenovirus using E1A

question

3 Rb related proteins (pocket proteins) and their 3 similarities

answer

pRb, p107, and p130 1. all are bound by E1A, E7 and T antigen viral proteins 2. regulated by phosphorylation 3. act on E2F related transcription factors

question

E2F family of transcription factors 4 common domains

answer

1. nuclear localization signal 2. dimerization domain 3. DNA binding domain 4. transactivation domain

question

myc, max, and mad

answer

molecules regulate differentiation and proliferation. for mitogenic signals and active proliferation, myc and max is present on DNA. for increased differentiation and decreased proliferation, mad and max are present on DNA.

question

myc

answer

oncoprotein that inhibits p21 and p27 so cell cycle progresses or goes through the cyclin D and CDK4 pathway it will also progress the cell cycle past the restriction point. 1. ER-myc is usually sequestered in the cytoplasm where it can't access DNA. 2. if estrogen of tamoxifen ligand binds to the ER, they can move to the nucleus. 3. Max is added to bind to Myc so they can bind to DNA to help the cell pass the restriction point.

question

pRb and differentiation

answer

differentiation can be prevented if pRb is hyperphosphorylated and cells can't mature. inhibition of phosphorylation of Rb can stimulate differentiation.

question

Li Fraumeni Syndrome

answer

inherited p53 mutations and deficiencies that lead to a variety of cancers like melanoma, colon cancer, sarcoma, lung cancer, breast cancer, stomach cancer, brain cancer, osteocarcinoma and rhabdomyosarcoma. these people have a 90% risk of developing cancer and are early onset rather than the normal cause which involves age.

question

p53 and mechanism

answer

the guardian of the genome that leads to cell cycle arrest or apoptosis depending on the amount of DNA damage such as ionization radiation or other genotoxic chemicals. 1. the random DNA damage activate ATM 2. ATM facilitates the phosphorylattion of p53 so that it won't be ubiquitinylated by Mdm2 and degraded. 3. p53 goes to the nucleus and acts as a transcription factor and turns on p21 (CDK inhibitor) and PUMA. 4. p21 inhibits CDK-cyclins so the Rb protein is unphosphorylated and the cell cycle arrests. 5. PUMA inhibits Bcl2 so it can't inhibit apoptosis and cell dies.

question

ATM

answer

a tumor suppressor that was first identified in Ataxia Telangiectasia patients. It is activated by DNA damage and will activate p53.

question

Mdm2

answer

a ubiquitin ligase that will tag unphosphorylated p53 so it can by destroyed if there is no activation from DNA damage and ATM. by a negative feedback loops, p53 activates other genes but also this gene to regulate p53 levels in the cell. when p53 is phosphorylated, it is stabilized at this binding domain and this can't bind to it.

question

PUMA (p53 upregulated modulator of apoptosis)

answer

one of the genes activated by p53 transcription factor that will inhibit Bcl2 so that apoptosis happens. it belongs to the Bcl family but only contain the BH3 domain to interact with Bcl-like proteins to activate Bax/Bak oligomerization and apoptosis signaling via the mitochondrial pathway.

question

How was p53 discovered?

answer

by Western blot that identified a protein that was less than 54 kD 1. Radiolabelled 36S methionine for dark bands for 3T3 mouse fibroblast and F9 mouse embryonal carcinoma (EC) cells. 2. the cells were transformed with S40 or not as a control. 3. cells were probed with antibodies with normal hamster serum and serum that was reactive to SV40 transformed cells. 4. identified 94 kD SV40 large T antigen, p53 that is elevated in the transformed cells.

question

3 tumor viruses that perturb pRb, p53, and/or apoptotic function

answer

1. SV 40 with large T antigen targeting p53 and pRb 2. Adenovirus with E1A targeting pRb and E1BSSK targeting p53 and E1819K targeting apoptosis. 3. HPV with E7 targeting pRb and E6 targeting p53.

question

Approximated hose much of the world's cancers are associated with p53 mutations?

answer

50% because it is a tumor suppressor

question

Are p53 mutations mostly acquired or hereditary?

answer

Acquired though Li-Fraumeni Syndrome is hereditary.

question

2 Environmental causes of p53 mutations

answer

1. Tobacco leads to point mutations in p53 2. UV leads to pyrimidine dimers in p53

question

Why do p53 knockout mice survive past birth but Rb knockouts don't?

answer

Because Rb is responsible for stopping cell division enough for differentiation and the failure to do so causes embryonic death but there are p53 independent mechanisms to stop division and cause apoptosis in the cell.

question

What type of mutations are the most common in p53? What are the common mutations in other cancer suppressors?

answer

missense and is often more detrimental than knockout of p53. ex: val 135 mutations. APC, ATM, BRCA1 usually suffer from frameshift mutations.

question

Where are most mutations usually located in p53?

answer

the DNA binding domain so that p53 can't act as the transcription factor to halt proliferation or cause apoptosis.

question

Consequences of mutations of the p53 tetramer and how does this compare to Rb

answer

1. if there are two normal p53 genes, both copies produce normal protein chain that join to form the tetramer for functional p53. 2. homozygous recessive mutation of the p53 so both copies can't produce functional p53 and no protein is produced. 3. heterozygous recessive mutation so only 1 copy is disrupted and normal copy may produce enough normal protein chain that join to form a function protein. 4. dominant recessive mutation so that one copy of the gene has a mutation and produced abnormal chains that may assemble with normal p53 chains from the other copy, resulting in nonfunctional p53. therefore, LOH is not even necessary. Rb is a monomer so both copies have to be mutated or knocked out to cause complete loss of function.

question

6 causes of p53 activation

answer

1. lack of nucleotides 2. UV radiation 3. ionizing radiation 4. oncogene signaling 5. hypoxia - lack of oxygen 6. blockage of transcription p53 may also play a role in metastasis

question

4 effects of p53 activation

answer

1. cell cycle arrest which leads to either senescence or return to proliferation. 2. DNA repair 3. block of angiogenesis 4. apoptosis

question

After DNA damage, does p53 or p21 peak first and what does it mean?

answer

p53 peaks first and may trigger p21.

question

What does p21 inhibit?

answer

Cyclin E and CDK2.

question

What is the half life of p53?

answer

5-20 minutes

question

2 family members of p53 and their significance

answer

p63 and p73 have longer half-lives and slightly different functions.

question

CDKN2A

answer

a gene that encodes for 2 mRNAs using alternative splicing, leading to p16 and ARF tumor suppressors. the gene is mutated in breast, lung, pancreatic, and bladder cancers.

question

Tamoxifen = OHT

answer

an estrogen analogue that binds the estrogen receptor, that when fused to the E2F, will trigger pro-apoptotic gene transcription. with this there is a greater cell percentage having less than normal size due to them forming apoptotic bodies. without this, E2F is sequestered in the cytoplasm and remains inactive but with it E2F is transported to nucleus to work. this is because pRb inactivates E2F, which activates ARF, which inhibits Mdm2, which inhibits p53 so that apoptosis happens.

question

Apoptosis

answer

normal programmed cell death that involves no immune response and no inflammation. cytochrome C of the mitochondria is released to the cytoplasm because of Bcl2 and DNA is cut at exposed areas between the nucleosomes. 1. the cell shrinks and chromatin condenses 2. the membrane blebs 3. nuclear collapse and continued blebbing. 4. formation of apoptotic bodies. 5. neighboring cells or macrophages engulf the apoptotic bodies. other characteristics are fragmented organelles and nuclear fragmentation.

question

What does the DNA latter of Apoptosis and Necrosis look like?

answer

Apoptosis: because of internucleosomal cleavage of DNA, there are distinct bands approximately every 80 bps on the gel. Necrosis: random cleavage of DNA so there is a just a smear when the gel is ran.

question

Normal examples of apoptosis

answer

1. loss of interdigital webbing in embryos. 2. cell turnover at the top of colon crypts. 3. death of immunoreactive cells in development to avoid autoimmune disorders. 4. death of excess neural cells in development since some parts of the brain develop faster and don't form enough neural connections. 5. death in normal cells that have done anoikis.

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Where is cytochrome C normally located?

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in the inner membrane of the mitochondria and is released to the cytoplasm in apoptosis by the action of Bcl2, which makes the mitochondria leaky.

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2 important domains of the Bcl2 family

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1. TM - transmembrane domain to stick into the mitochondria membrane. 2. BH3 domain is a death domain.

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Breeding transgenic mice for Bcl2 and myc and results

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1. IgG-myc and IgG-Bcl2 is cloned and injected into fertilized mouse eggs. 2. eggs are injected into early stage mouse embryos (blastocysts) 3. some embryos yield mice carrying transgene in all their cells, including gametes. 4. breed those with each other. 5. results is mice carrying IgG-myc, mice carrying IgG-Blc2 and mice carrying both. results: bcl2 doesn't really affect survival but myc alone does decrease survival and cause cancer, though not as much as myc and Bcl2 combined. this is because the mice get aggressive tumors and the cells don't die and keep dividing to accumulate damage. Can also be done with mmTV-myc and MMTV-ras to show gene collaboration for cancer.

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Bax and Bak

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pro-apoptotic molecules that are clustered together on the surface of the mitochondria during apoptosis. they activate caspases in response to death receptors and granzyme signaling activating tBid.

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Apoptosome

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complex that is formed when cytochrome c goes to the cytoplasm and binds to Apaf-1 adaptor protein and caspase 9 by the death domain

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The apoptotic caspase cascade

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1. the mitochondria channels are closed but a pro-apoptotic death signal causes the opening of the channel. 2. cytochrome c and Smac/DIABLO is released into the cytoplasm. 3. cytochrome c, Apaf-1 and pro caspase 9 forms the apoptosome. 4. caspase 9 is activated in the complex - initiator caspase activates (this is the last place to stop apoptosis before cell commits to die. 5. procaspase 3, 6, 7 - executioner caspases act on cellular function to stimulate apoptosis.

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Caspases

answer

a group of enzymes that cleave aspartate residues of other proteins by using cysteine residues and regulates apoptosis. they cleave at consensus sequences of 4 amino acids. they can work independent of p53.

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4 substrates of the caspases

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1. ICAD - inhibitor of caspase-activating DNAase that fragments DNA. 2. Lamin - protein in the nucleus that causes nucleus condensation and chromatin condensation because it normally maintain the structure of the nucleus unless cut. 3. vimentin - a cytoskeleton protein that causes blebbing and the formation of apoptotic bodies. 4. actin - a microfilament of the cytoskeleton that causes blebbing and the formation of apoptotic bodies.

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death receptors mechanism and 2 examples.

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part of the external apoptotic pathway and requires ligand binding such as that of a lymphocyte to activate apoptosis. 1. the ligand binds and signaling thorugh these signal through FADD and DISC adaptor molecules clustering. 2. caspase 8 and 10 are activated from procaspases as alternate initiator caspases to caspase 9. 3. the caspase cascade continues and results in apoptosis through death substrates ex: FAS and TNFR1

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Cytotoxic cell apoptosis mechanism

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1. Or a cytotoxic cells delivers granzymes through the perforin pore formation mechanism to the cell. 2. caspase 3 and 8 are activated as executioner caspases. 3. they signal more executioner caspases or Bid to open the mitochondria and release cytochrome C. 4. executioner caspases work on death substrates or the apoptosome is triggered and caspase 9 works on the substrates.

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How does p43 activate apoptosis using the caspases?

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p53 can turn on Fas genes which will activate caspase 8 and 10. or it can turn on Bax genes which will release cytochrome c from the mitochondria and form the apoptosome to activate caspase 9.

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2 Anti-apoptotic strategies used by cancer cells

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1. using growth factors to keep Bad phosphorylated and inactive so that caspase 9 is inactive and apoptosis can't happen. 2. using growth factors to signal to the mitochondria so that cytochrome c can't be released using Bcl2.

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Autophagy

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when cells digest their own organelles in lysosomes and is seen as an organelle with multiple membranes under microscopy. this is because cancer cells do mostly glycolysis and is inefficient so need lots of sustenance and this is a way for them to get energy in their quiescent state.

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7 factors of multi-step tumorigenesis

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1. time - usually years unless it is chromothripsis 2. histopathology 3. genetics - usually involves multiple genes 4. cancer stem cells 5. number of cells in the multicellular organism 6. carcinogens - mutagens and other chemicals that just interfere with signaling. 7. inflammation

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time

answer

one of the factors of mult-step tumorigenesis. states that cancer usually takes several decades to form, and according to Knudson, requires 5-6 limiting steps must occur. and the duration of exposure is more important than age at first exposure to carcinogens.

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Histopathology example for multi-step tumorigenesis

answer

1. normal colonic crypts 2. early adenomatous crypt 3. small tubular adenoma or villous adenoma 4. large tubular adenoma with or without stalk attached to head of polyp wall of colon or invasive carcinoma which can form liver metastasis. 5. some tubular adenoma that can form invasive carcinoma and then liver metastasis through the heptatic portal vein draining nutrients to the liver.

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Polyps

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part of histopathology in multi-step tumorigenesis. an adenoma that is a gland-like tumor that is benign. some of them have a stalk connecting to the colon wall and some are directly attached to the colon wall. it is characteristic of adenomatous polyposis coli (APC tumor suppressor) and familial adenomatous polyposis (FAP). pro-active removal is shown to reduce incidence of colorectal cancer from 3-4% to almost none.

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Field cancerization

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when multiple, apparently independent tumors appear in an organ. they are appear like they are due to familial inheritance like APC from germ line mutations but are due to somatic mutations. the tumors share common mutations and can be explained by multi-step tumor progression.

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Barrett's esophagus

answer

part of genetic factor in the multi-step tumorigenesis. when stratified squamous epithelium of esophagus start to show signs of columnar epithelium of the stomach and can be indicative of cancer.

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Darwinian Evolution and clonal succession

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part of genetic factor in the multi-step tumorigenesis. says that the cancer cells with the most ability to divide and evade death will have the ability to persist more and accumulate more damage that gives them an advantage to other cells and the body. this selects for more invasive and aggressive tumor cells.

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Are single mutations enough to induce cancer?

answer

no. we have several thousand new point mutations of Ras oncogenes activated each day by random but not everyone will get cancer from that. we need multi-hits to the genomes like myc and ras or myc E1A and ras.

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Cancer in monozygotic twins

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part of the genetic factor in the multi-step tumorigenesis. identical twins share placental circulation so when a translocation event in a somatic blood cell occurred in utero and was transferred to the other twin. Additional somatic mutations occurred after birth but the twins are usually diagnosed soon after each other.

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How many hits to the genome does a mouse or chicken need to get cancer? and compare to humans

answer

2 in the mice and chickens but at least 5 in the human.

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5 cell signaling pathways that is disrupted for transformation of human cells to grow tumors in SCID mice

answer

part of genetic factor of multi-step tumorigenesis. 1. Ras using ras mutations 2. pRb using CDK4 and D1, SV40 large T antigen, and HPV E7. 3. p53 using p53 and SV40 large T antigen and HPV E6. 4. telomeres stabilization using hTERT and myc + SV40 large T antigen 5. PP2A phosphatase tumor suppressor that inhibits the Wnt pathway. uses SV40 small antigen and sometimes myc, Akt/PKB +Rac 1, PI3K and B56 shRNA. human cells are highly resistant to transformation

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2 capabilities of cancer pRb transformation leads to

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1. resistance to growth inhibition 2. immortalization

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3 capabilities of cancer p53 leads to

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1. resistance to growth inhibition 2. apoptosis evasion 3. immortalization

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capability of cancer hTERT leads to

answer

1. immortalization

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3 capabilities of cancer Ras leads to

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1. apoptotic evasion 2. mitogenic independence 3. angiogenesis 4. metastasis/invasion

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stem cells

answer

cells that are able to do asymmetric divisions in order to give rise to a new stem cell to replenish themselves and a transit-amplifying cells and post mitotic differentiated cells. we lose this count as we age as a protective measure against cancer because these cells can give rise to new tumors and are immortal but we won't have as many cells to replenish tissues. a mutation of these can carry on dividing forever and accumulate more mutations to give rise to the tumor population of transit amplifying cells and more stem cells.

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Most cancer cells behave like these cells.

answer

transit amplifying cells or progenitor cells.

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Breast cancer stem cells sorting

answer

using FACS analysis and surface antigens CD24 and CD44, cells that express little CD24 but high CD44 are more tumorigenic than cells with high levels of both markers. the tumorigenic population are stem cell like (12%) will form tumors in NOD/SCID mice, and the other population behave like transient cells and won't form tumors in the mice. similarly, brain tumor stem cells express more CD133 antigen.

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Mutator phenotype

answer

the situation in which it is easier to acquire other mutations after the initial mutation. this leads to clonal diversification due to the high mutation rates after the initial common mutation.

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Why is it more important to identify chemotherapies to target cancer stem cells than the non-stem cells in a tumor

answer

because tumor stem cells can create new tumors and are resistant to genotoxic therapies by using similar mechanism of normal stem cells to protect themselves such as free radicals or other cells as protection.

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Animals that don't get cancer or infrequent cases

answer

1. naked mole rats 2. chrondrichthyes like sharks and rays 3. raccoons though there is a breakout of cancers due to a rare tumor virus that causes brain cancer and neurological problems so they act rabid.

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3 stages of carcinogen inducing cancer

answer

1. initiation or exposure to carcinogens (mutagens) like BP, DMBA, or 3-MC 2. promotion that stimulates proliferation of the initiated cells like TPA and PMA. 3. progression of additional genetic stresses.

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inducing skin carcinomas in mice: example of carcinogen causing cancer

answer

1. paint with initiator -> no change 2. multiple painting of promoter -? no change 3. paint with initiator -> multiple painting with promoter -> papilloma (wart) 4. paint with initiator -> multiple paintings with promoter in adjacent area -> no change 5. paint with initiator and then paintings with promoter -> papilloma -> stop promoter painting -> no change because benign with no progression 6. paint with initiator then painting with promoter -> paint papilloma with extensive promoters -> stop promoter painting -> persistent papilloma. 7. paint with initiator and multiple painting of promoter -> paint papilloma with initiator -> carcinoma that is invasive.

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What genes were involved during initiation and promotion of mouse skin cancer model

answer

Ras at the initiator stage and then p53 at the papilloma stage.

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Cytotoxic Agents

answer

tumor promoting agents that has a dose-dependent effect. it has a threshold point rather than a constant increase between dose and biological effect rather than that of genotoxic mutagens.

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7 known human tumor promoters and sites of action

answer

1. estrogen at endometrium 2. estrogen and progesterone at breast 3. ovulation at ovary 4. testosterone at prostate 5. hepatitis B/C viruses at liver 6. chewing tobacco at oral cavity 7. asbestos at mesothelium and lung.

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Menstrual cycles and risk for cancer

answer

the more menstrual cycles the higher the risk of breast cancer because estrogen may be a tumor promoter. those with earlier menstruation and later menopause are more prone to cancer. those with no children because they had no pauses in estrogen cycling have a higher risk of cancer.

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Chronic Inflammation and examples

answer

normal response to fight infection and repair tissues but the release of cytokines and growth factors involved can cause tissue damage and affect neighboring cells. chronic cases may lead to tumor promotion. ex: helicobacter infection in the stomach and mdr in the liver.

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Mdr (multi-drug resistance)

answer

knockout of this gene cause chronic liver inflammation and can see immune cells for inflammation under the microscope. ibuprofen can help alleviate the inflammation by inhibiting prostaglandins.

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What kind of cellular changes are stimulated by NFkappaB as a result of Mdr and chronic liver inflammation?

answer

1. TNFalpha for more inflammation 2. cyclin D1 and myc for mitogenesis 3. anti-apoptotic genes which all cause dysplastic hepatocyte nodules that can lead to heptatocellular carcinoma.

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prostaglandin E2

answer

comes from arachidonic acid of inflammation and promotes tumors. it causes loss of contact inhibition, anchorage independence, loss of E-cadherin and cell proliferation. aspirin may prevent the synthesis of the H2 and this type so has anti-inflammatory and cardiovascular benefits but is blood thinning and stomach irritant

question

NSAIDS (non-steroidal anti-inflammatory drugs)

answer

drugs that inhibits the Cox-2 enzyme that is responsible for converting arachidonic acid to prostaglandines G2 and H2 before E2. it is involved with cyclooxygenase and peroxidase activity.

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