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88 Cards in this Set
- Front
- Back
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How was the role of β-catenin discovered?
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It was discovered using the Heidelberg screen.
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What does β-catenin do in drosophila?
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Determines segement polarity.
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What does β-catenin bind to to become a transcription factor?
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β-catenin binds to TCF
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What marks β-catenin for destruction and how?
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GSK3 by phosphorylation
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What does APC stand for?
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Adenomatous Polyposis Coli
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What occurs to β-catenin when APC is absent? What does this indicate?
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The level of β-catenin rises rapidly. This indicates that APC has a role in the destruction of β-catenin.
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What complex is formed by APC?
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APC binds to β-catenin and Axin.
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Describe the Wnt signaling pathway.
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Wnt inhibits binding of APC to Axin and β-catenin, releasing them into the cytoplasm.
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Where does Wnt signaling occur in the colon?
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It occurs in the bottom of crypts.
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Where do precursor colon cells begin to differentiate? Why?
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Colon precursor cells begin to differentiate as they move away from the Wnt signaling sent by stromal cells. The differentiate in the villus.
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What genes are active in the crypts? In the villus?
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The genes turned on by Wnt are active in the crypts while those turned off are active in the villus.
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How does Wnt signaling induce cell proliferation?
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Wnt causes APC to release β-catenin which binds with TCF and acts as a transcription factor for c-myc. C-myc in turn inhibits p21, a protein that stops the cell cycle. Thus Wnt signaling causes the cell cycle to continue.
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What happens if APC is mutated?
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"If APC is mutated, β-catenin is constitutively active and thus p21 is inhibited allowing the cell cycle to continue. If this occurs in the villus, crypt like progenitor cells form in the incorrect location. This clump of dividing, undifferentiated cells is a polyp.
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From which germ layer do mesenchymal cells originate?
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They are formed from the mesoderm.
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What are the building blocks of cell-cell junctions?
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Cell Adhesion Molecules (CAMs)
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What are the CAMs with homophilic interactions?
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N-CAM and E-cadherin
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What are the CAMS with heterophilic interactions?
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P-selectin/glycoprotein and ICAM/integrin
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What is an adherens junction and what does it bind to?
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An adherens junction is a cell-cell junction that binds directly to the actin cytoskeleton.
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Describe the types of extracellular matrix.
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Basal lamina - secreted from epithelial cells, a mixture of fibrous proteins and polysaccharides. Connective tissue - secreted from cells within the tissue, much larger and more varied than the basal lamina.
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What are the two types of proteins contained in ECM? Name an example.
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Structural proteins - collagen
Adhesive proteins - laminin |
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What does GAG stand for?
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Glycosaminoglycan
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What is a proteoglycan?
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A core protein combined with a glycosaminoglycan.
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How do normal cells break though into tissues? What is this process called?
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They break through the way normal cells invade. Epithelial to Mesenchymal Transition.
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When does cell invasion occur naturally?
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Cells migrate during development. Other cells of the immune system actively move into tissues during the inflammatory response.
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Name the steps to cancer metastasis.
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Progression, intravasation, transport, extravasation, micrometastasis, and colonization.
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Where do primary prostate cancers metastasize to?
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Brain, lungs, liver, bone marrow
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Where do primary pancreatic cancers metastasize to?
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Lungs and liver
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Where do primary breast cancers metastasize to?
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Brain, lungs, liver, bone marrow
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Where do primary colon cancers metastasize to?
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Lungs, liver, and bone marrow.
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How does intravasation occur?
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Primary tumors induce angiogenesis, attracting blood vessels. Tumor cells undergo EMT and can enter the blood stream.
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Why is the blood a hostile environment?
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The force of the blood destorys cancer cells. The blood also transports natural killer cells that can recognize and kill metastatic cancer cells.
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How does extravasation occur?
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A tumor cell traveling through the circulatory system gets stuck in a small capillary. This attracts platelets that form a microthrombus which holds the cell in place. The cell is then able to break through the ECM and invade the new tissue.
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What is the first step of colonization?
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Dormant micrometasteses travel throughout the body. Many of these tiny colonies die, but one can eventually become a macrometastasis.
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What allows a micrometastasis to become a macrometastasis?
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If a micrometastasis can cause angiogenesis, it will most likely becom a macrometasis.
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What notable proteins are not expressed in metastatic cells?
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E-cadherin, α-catenin, and γ-catenin
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What do Rho family proteins do?
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Rho family proteins promote actin family remodeling.
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What proteins help tumor cells invade the ECM?
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matrix metalloproteinases (MMPs)
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What is another role of matrix metalloproteinases in EMT?
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They cleave E-cadherin, decreasing cell adhesion.
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Why is metastasis inefficient, but deadly when it occurs?
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Metastasis has a multitude of difficult steps that cause all but the most invasive cells to die. When these maximally invasive cells do for metastases, they are more deadly.
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What are the three major changes in cells during EMT?
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Cell adhesion, cell shape, and secretion of MMPs
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What are the three previously studied cell cycle checkpoints?
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DNA damage
DNA synthesis - active replication forks prevent the cell from entering mitosis Spindle Assembly - chromosomes must be aligned at the metaphase plate and one sister chromatid must be attached to each pole of the cell. |
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What were the two proposals for how the cell cycle was controlled?
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Substrate Product - Physically cannot go into mitosis because of interrepution to the physical contact between the spindle and the chromosome.
Negative Control - Even though a broken chromosome will fit into the transcription machinery, direct signaling causes the machinery to cease their function. |
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What proposal was correct for cell cycle control?
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Negative Control
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Explain how caffeine was used to show that errors can be induced in mitosis, proving negative control.
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When a WT cell is treated with caffeine, there are no effects to the cell cycle. When a cell is treated with hydroxyurea, which causes DNA damage, it causes the cell cycle to stop. When the cell is treated with both hydrobeurea and caffeine, the cell will continue through the cell cycle and one or both cells will be unviable.
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Why is negative control more robust than positive control?
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Negative control allows a binary signal to be detected. When a kinetochore isn't bound to the spindle, the cell cycle can be held by a single signal being sent. In positive control, each kinetochore would send a signal meaning the cell would have to differentiate between two very similar signals. With negative control, the difference between the signals is easier to detect.
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What are the four categories of proteins in the cell cycle checkpoints?
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Signals, sensors, transducers, and effectors
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Describe how RAD9 was discovered in yeast.
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RAD9 was knocked out using a dose of radiation that did not kill the entire organism. A TS Cdc9 was created. This gene codes for DNA ligase which will not occur at the restrictive temperature, causing massive DNA damage. At the RT, WT RAD9 prevents the cell from moving foward in the cell cycle. However, mutant RAD9 prevents the R checkpoint from occuring and the cell moves forward in the cell cycle even with damaged DNA.
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Describe the S. pombe MPF.
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Cdc13/Cdc2 complex that is inactive when it is phosphorylated at Y15 and T161 and active when only one T161 phosphorylated. Wee1, a kinase, inactivates the MPF by phosphorylating Y15 while Cdc25, a phosphatase, activates it by removing this phosphate.
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Describe the equivalent of the MPF in humans.
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Similar to S. pombe MPF. Cdc2/Cyclin B complex is inactive when it is doubly phosphorylated. Cdc25, a phosphatase, removes one of the phosphates, activating the protein which signals for the G2 -> M transition.
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What occurs to the Cdc25 molecule when DNA damage occurs in humans?
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DNA damage causes activation of ATM which signals CHK2. CHK2 then phosphorylates Cdc25. This phosphorylation is detected by 14-3-3 which binds and removes it from the nucleus into the cytoplasm, preventing activation of Cdc2 and thus the G2 -> M transition.
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When is the anaphase inhibitor active?
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It is active when all the kinetochores are not attached to a pole.
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Describe the spindle assembly checkpoint.
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When a kinetochore is not attached, the Mitotic Checkpoint Complex (MCC) activates MAD2. Activated MAD2 inhibits the anaphase promoting complex (APC) from cleaving securin from separase. Once all kinethochores are attached, MAD2 becomes inactive and APC is allowed to break securin from separase. Separase then cleaves the cohesin that holds the sister chromatids together and chromosome segregation begins.
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What is the paradigm of p53 function?
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When the cell is normal, the level of p53 is low. When the cell undergoes stress, the level of p53 increases and the cell either arrests its cell cycle or goes into apoptosis.
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What are the two most mutated genes in human cancers? What mutations occur?
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p53 - 50% of cancers see nucleotide substitutions
INK4a - 40% of cancers see homozygous deletion, nucleotide substitution, small deletion/insertion, or promoter metylation. |
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How was p53 discovered?
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DNA tumor viruses were studied because they need a cell to move into S phase. These viruses produce T antigen which prevents apoptosis and moves the cell into S phase. T antigen was immunoprecipitated and p53 was found to be bound to it.
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What is Li Fraumeni Syndrome?
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A disease caused by mutations in p53. 70% of cases have a germline mutation of p53. It is characterized by various childhood (adrenal) and adult onset (lung) cancers.
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What is Friend Murine Erythroleukemia?
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A retrovirus inserts itself into the p53 gene disrupting it. This causes cancer.
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Where do the majority of mutations in p53 occur?
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They occur in the DNA binding domain.
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How does p53 bind to DNA?
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A tetramer forms and binds to the minor groove.
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What type of mutation is the worst in p53?
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A point mutation causes the most inactivation of p53. A null mutation would cause half of the protein product to form, but it would all be able to tetramerize. If both alleles produce protein and one of the alleles has a point mutation, the protein produced by this allele will poison the good protein created by the other allele because tetramerization won't occur.
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What occurs when a double stranded DNA break occurs?
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ATM/ATR is activated which phosphorlates p53 on S15 as well as CHK2. CHK2 phosphorylates p53 at S20. MDM2is also phosphorylated at S395. Phosphorylated p53 then binds to p21 which inhibits CDK2/Cyclin E
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How was it discovered the p53 binds to p21?
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The luciferase gene was attached to the p21 gene. When p53 is added in vitro, the luciferase is seen. Even when the binding region is removed, some luciferase activity is also seen, indicating that there are multiple binding sites.
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What protein helps to regulate p53 and how does it do so?
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MDM2 binds to p53 and removes it from the nucleus, preventing transcription factor activity.
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How was MDM2 activity shown in vitro?
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A reporter called 50-2 was bound to WT p53. When MDM2 was not present, chloramphenicol was converted into chloramphenicol acetyl transferase (CAT). When MDM2 was added, not CAT was seen.
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How does MDM2 act on p53?
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It causes its destruction by ubiquitylation.
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What is the pathway of ubiquitination of p53?
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E1 takes up ubiquitin from ATP. The ubi is then take up by E2. E3 transfers the Ubi to the target (p53) which is then digested by a proteosome.
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How is ARF transcribed?
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It has an alternative reading frame of the INK4a gene.
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Where does ARF transcription begin? INK4a?
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ARF transcription begins at exon 1β while INK4a begins at exon 1α.
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What is the function of ARF?
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ARF inhibits MDM2, preventing p53 from being moved out of the nucleus and thus activating p53.
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What induces ARF transcription?
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c-myc expression.
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Why has the 5 year survival rate increased?
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Cancer is being detected earlier and treatments have improved.
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What are the three types of therapies? Describe the logic of each.
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Traditional Therapy - kill proliferating cells. However, this kills all proliferating cells.
Targeted Therapy - passive immunization; biology targeted drugs Personalized Therapy - |
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What are the various types of traditional cancer therapy?
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Mustard gas has been used. Chemotherapy.
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What are the various types of chemotherapy? Describe them.
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Alkylating agents - DNA is forced to crosslink, preventing replication.
Antimetabolites - Inhibits nucleotide synthesis. Alkaloids - Inhibits microtubules. |
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Describe MTX. What type of treatment is it?
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Methotrexate is an antimetabolite that disrupts folate synthesis and thus nucleotide formation. MTX binds to DHFR which usually binds to RFC. This prevents the transformation to THF.
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What eventually occurs with MTX treatment?
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MTX will kill any cells that aren't resistant to it. It basically forces natural selection of resistant cells.
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What mutations can cause MTX resistance?
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An RFC mutation can prevent MTX from moving into the cell.
An increase in the activity of PGP, which moves it out of the cell, could move all of the MTX out of the cell before it can prevent nucleotide synthesis. This causes Multi Drug Resistance. The amount of DHFR can be increase by gene amplification to the point at which MTX doesn't have a great enough effect to make a difference. |
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Why is leucovorin used in combination with MTX?
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It provides some folinic acid so that normal cells can still divide. Since cancer cells divide so quickly, they still divide since a relatively small amount if given.
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Why is targeted therapy used?
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It is more efficient and kills only cancer cells as opposed to all rapidly dividing cells.
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What is passive immunization? How does it work?
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Passive immunization uses the immune system to locate and kill cancer cells. An antibody against a cancer cell is created in a mouse. The variable region of the antibody is fused to a human constant region so the antibody itself isn't kill by the immune system. This hybrid antibody is injected into humans.
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What is herceptin? How does it work?
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Herceptin is a passive immunization that works against EGFR, in particular HER2 which is found in breast cancer. HER2-HER2 dimers are ligand independent and induce cell survival in humans because of gene amplification. Since it has no ligand, it is an orphan receptor. By creating a antibody that binds to HER2 which is amplified in cancer cells, they could be marked for destruction by the immune system.
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What is passive immunization effective against? What is it ineffective against?
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It works against proteins created by oncogenes such as HER2.
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What antibody is used against lymphoma?
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Rituxan targets CDC20.
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What are protein specific drugs? Why are they useful?
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They are drugs that target the protein product of certain genes. They can penetrate large tumors and move through the cell membrane because they are low molecular weight. Since they can move into the cell, they can act on intracellular receptors. They are also cheaper to produce than antibodies.
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What are good candidates for protein specific drugs?
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The proteins created by oncogenes, particularly enzymes.
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What is Gleevec and what does it act on?
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It blocks the ATP binding site of the kinase domain on Brc-Abl fusion proteins.
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What is the key property of a drug? What is this called?
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The drug must be effective but not harmful. This is called the Theraputic Index which measures the efficacy versus the toxicity.
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What are the steps in testing a new drug?
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In vitro study
In vivo study Phase 1 - Testing of safety and dosage. Done on patients that haven't responded to other treatments. Phase 2 - Testing efficacy. Does the drug work? Tested on a larger group than Phase 1. Phase 3 - Testing effectiveness. Is the drug better than a previous treatment? Tested on a larger group than Phase 2. |
