BIOL 311 - Exam 4

Historic People

Arthur Pardee (Cell Division) - used 3T3 cells (mouse embryo) to conclude there is a "restriction point" in G1 before the cell continues to the S phase Ruth Sager (I have no clue what section this goes to) - fused normal and cancer cells, observed a loss of the normal phenotype and conversion to cancer phenotype - loss of tumor suppressor genes Peyton Rous - discovered v-src from Rous Sarcoma Rita Levi Montalcini (Cell Death) - used chick embryos, first to see apoptosis, Nobel prize winner John Kerr - coined the term apoptosis, blocked the liver and observed both necrotic cells and apoptotic cells (which he called "shrinking necrosis" at the time)

Cytoskeleton

Definition: - not static; dynamic - Diverse - 3 major proteins - Actin filaments ( microfilaments, F-actin) - Intermediate Filaments - Microtubules - Functions are numerous - Disease states Tensile strength of the three major cytoskeleton proteins: - Microtubules deform and break at weak force, so least tensile strength - Intermediate filaments require most force to deform and break, so most tensile strength - Actin filaments deform and break at medium force F-actin - make up the microvilli, cortex, and adherent belt (E-cadherin) - filopodia, leading edge, stress fibers - phagocytosis - make up to 10% of total cell proteins (muscle cells) - G-actin (building block) - mw = 42,000 - Humans have 3 kinds of actin - alpha-actin - contractile - beta-actin - moving - gamma-actin - stress fibers - F-actin can be "decorated" w/ myosin S1 head -> reveals that F-actins have two ends: the pointed (-) end & the barbed (+) end - F-actin polymerization in a test tube reveals three states: - nucleation - elongation - "steady state" - G-actin is constantly adding and subtracting at both ends - Treadmilling - differential addition and/or subtraction of G-actin on an F-actin polymer - if critical concentration of G-actin at both ends -> no net addition - net addition of G-actin to (+) end is 5x greater than (-) end - Drugs: - Cytochalasin D - inhibits polymerization at the (+) end - Phalloidin - promotes polymerization - fluorescent stain for actin filaments - Thymosin B4 - sequestering protein -> reveals 40% of G-actin is soluble - Profilin - ADP/ATP exchanger - Cofilin - severing protein - Formin - dimer - nucleating protein - Optical tweezer microscope - analyze step size and force - Listeria - food poisoning bacteria, F-actin polymerization making bacteria motile Intermediate Filaments - "rope" - Generally static - 70 genes coding for IF - Keratin filaments in skin, epithelial cells; neurofilaments in axons; lamins in the karyoskeleton (structure for nucleus) - Epidermolysis Bullosa Simplex (EB) - defective keratin filaments - skin sloughs off with minimal shear force Microtubules - Largest cytoskeleton protein - mitotic spindles - typical microtubule has 13 protofilaments - 2 building blocks of microtubules: - alpha-tubulin: non-exchangeable GTP - beta-tubulin: exchangeable GTP - Demonstrates tread milling as well, has plus and minus ends - Play important role in crypsis and distribution of pigments throughout the cell - pigments are tethered to the microtubules and can travel along them - Experiment: Fish pigment cell with pigments distributed throughout cell -> treat with MT inhibitor and decrease [cAMP] -> pigments do not congregate to center - Microtubule Associated Proteins (MAPs) - govern microtubule function - Neuron -> treat with tau antisense -> shortens length of the neuron - Neuron -> treat with MAP2 antisense -> no dendrons on neuron - Kinesin - moves material towards periphery of the cell - Dynein - moves material towards the center of the cell - Drugs: - Colchicine - depolymerizes MT - Taxol/Taxotere - promote polymerization of MT

Experimental Drugs / Disease Treatment Drugs

Cytochalasin D (Cytoskeleton) - inhibits polymerization of Actin filaments at the + end -> loss of structure in treated cell Phalloidin (Cytoskeleton) - promotes polymerization of Actin filaments, prevents depolymerization Colchicine (Cytoskeleton) - depolymerization of microtubules Taxol & Taxotere (Cytoskeleton) - polymerization of microtubules and disrupts the mitotic spindle, triggers apoptosis in dividing cells Lonafarnib (The Nucleus/Cell Division)

Diseases

Listeria (Cytoskeleton, F-actin) Deafness (Cytoskeleton, F-actin) Epidermolysis Bullosa Simplex (Cytoskeleton, Intermediate Filaments) Kartagener's Syndrome (Cytoskeleton, Microtubules) - aka Ciliary Dyskinesia, defect in Dynein arms in Cilia Progeria (The Nucleus/Cell Division)

Cell Death

Necrosis - pathological cell death - outside influence - ex. oligomycin decrease ATP -> necrosis - no ATP required - Many cells die at the same time - Necrotic cells swell, membrane breaks - messy process, inflammation response - caspase activation not required Apoptosis - programmed cell death - internal signal - important in embryogenesis - chemo/radiation therapy triggers apoptosis in cancer cells - strokes can lead to apoptosis of neighboring cells - require ATP - death of single cells - shrink, membrane blebs, form apoptotic bodies that contain DNA - tidy process, caspase activation DNA Gel Electrophoresis - control: full set of DNA bunched at top of gel - apoptotic cell: ladder - non-random DNA cleavage - necrotic cell: smear - random DNA cleavage Apoptosis: History - Rita Levi Montalcini - used chick embryos to first see apoptosis - John Kerr - coined the term "apoptosis," blocked the liver with Ligate, observed "shrinking necrosis" cells (apoptotic cells) and necrotic cells

Normal Cells v. Cancer Cells

Normal Cells 1. Do not grow in soft agar (Commit Anoikis) 2. Typical karyotype (23 sets of 2 chromies) 3. Normal set of miRNA 4. Secrete few proteases 5. Larger (Stuck in G0) 6. Lower nuclear : cytoplasmic ratio 7. Cytoskeleton more organized 8. Extensive ECM 9. Growth to a single later (contact inhibition) 10. Does not cause tumors when injected 11. Serum dependent growth 12. Secretes few growth factors 13. Telomeres shorten with each division 14. 25-50x doublings 15. Few genetic defects 16. Normal glycolysis 17. Normal cell permeability 18. Do not typically secrete angiogenesis factors such as VEGF (Vascular Endothelial Growth Factor) 19. Normal RTKs 20. Normal Apoptosis Cancer Cells. 1. Grow in soft agar 2. Usually aneuploidy (extra/missing chromies) 3. Unique set of miRNAs 4. Secrete more proteases 5. Smaller (constantly in M phase/dividing) 6. Higher nuclear : cytoplasmic ratio 7. Cytoskeleton less organized 8. Little ECM (No fibronectin secreted (must keep moving)) 9. Multilayer (no contact inhibition) 10. Causes tumors when injected 11. Serum independent 12. Can secrete many growth factors 13. Telomeres stay same length 14. Unlimited cell division in vitro 15. Genetic defects in ras, p53, etc. 16. Warburg effect 17. Cell 10x more permeable 18. Can secrete angiogenesis factors 19. RTKs can be aberrant (constitutive) 20. Innate apoptosis (Can be inhibited)

Cancer

Process by which a cell loses its ability to control its cell cycle. 2nd Killer, Heart Disease = 1st, COVID = 3rd Over 200 types, 5000 mutations in a typical cancer. Driver Mutations - Responsible for normal -> cancer transition (5 driver mutations in most cancers) Non-Driver mutations = Passenger Mutations (other mutations) Proto-oncogene = normal gene that controls cell division Oncogene - Gene controls cell division but constitutively regulated Cancer treatment process 1. Identify Driver Mutations 2. Determine how to best intervene w/ driver mutations 3. Best treatment = targets cancer cells only (CAR-T) 4. Billions spent by NIH to understand cancer 5. Understanding basis of cancer (defective RTK and tumor suppressor gene, etc.) is not enough Causes of Cancer 1. Viruses - originally the primary cause because of Rous Sarcoma, but now minor player. 1911 - v-src into animals from rous sarcoma. 1970s c-src in chickens similar to v-src (seemed like viruses got what they needed from this gene from animals). SV40 - DNA virus that can cause cancer. Large T antigen can bind retinoblastoma and p53 (tumor suppressor genes) on exterior so cannot perform their function in the cell. HPV - Can cause cancer. Gardasil first vaccine against viral-based cancer Epstein Barr Virus - Can cause Hodgkin's Lymphoma and Burkitt's Lymphoma 2. Radiation (UV light) - creates thymine - thymine dimers -> corrected easily, but if not or too many = skin cancer. 3. Chemical Mutagens -Base substitutions, DNA cross linking, chromosome breakage. EMS is an example. Tobacco smoke has mutagens and tumor promoters with >60 carcinogens. 4. Defective Cell Cycle Genes (Cyclins) - If one of cyclins is defective, cell cycle may not pause and errors will not be fixed. Cyclin D can cause breast cancer. Cyclin D1 overamplified in >50% of breast cancers 5. Defective Tumor Suppressor Genes (p53) - Defective in >50% of cancers 6. Defective DNA Correction/Repair Enzymes - Can cause Xeroderma Pigmentosa for example 7. Defects in Apoptosis - Too much bcl-2 (anti-apoptotic). Viruses like SV40 can manipulate Rb + p53 as noted above 8. Defects in Growth Factor Signaling Pathway - Mutant Ras - G Protein = effector of many growth factor receptors can be constitutively turned on. rasD found in most human tumors. Loss of TGF beta signaling pathway = antigrowth factor. Defective growth factor receptors that are constitutive (many related to RTK) 9. Defective Telomeres or Telomerase - Mitotic clock, cancer cells = non-shortening telomeres 10. Chromosomal Translocation - Burkitt's Lymphoma. Bcr-abled fusion protein (protein kinase) 11. DNA Amplification - Overproduction of encoded protein. Minute Chromosomes 12. Overproduction/Mutation of Nuclear Transcription Factors Such as C-Jun and C-Fos (G1 early response genes) - Demonstrated with 3T3 cells as natural event when serum added. 13. Cancer Stem Cells - Identified in all cancers. <1% of tumor cell population. Can self renew and differentiate into heterogenous tumor population. Can differentiate into non tumor cell population. Defined by FACS and CD133 (cell surface marker). Tumor microenvironment plays a role. Some tumors metasize or enter remission because of these. Resistant to chemo/rad. Resistant to drug effective against associated tumor usually. What is an oncogene - Can cause cancer, variant of proto-oncogene. Robert Weinberg -> Transfer of DNA from host tumor cell -> normal cell can confer oncogenesis. Also, DNA from human bladder carcinoma can transform mouse 3T3 cells meaning, a gene can cause cancer and it is not specific specific. More than one active oncogene required, tumor microenvironment important. Ha-ras oncogene transform 3T3 to cancer but not REF unless in soft agar meaning tumor microenvironment important. Multi-hit model of cancer induction - Older you get = increased risk of cancer. Essentially, numerous genetic errors accumulate overtime = cancer. Most cancers arise from single mutated cells -> verified by examining female tumors, easy to distinguish, Tumors in women all have same X chromosome inactivated = single cell origin. Mutated Myc and ras show effects of both mutations synergistic (additive) in mice. Myc necessary for much tumor growth. Cancer can be due to sequence of mutations, e.g. colon cancer. After 50 = colonoscopy every 5 years. Oncogenes - Several proteins that participate in controlling cell growth and proliferation. Mutations at many of these proteins can lead to cancer. Most oncogenes originate from normal proto-oncogene -> ras is proto-oncogene that is GTPase controlling cell growth, RasD is an oncogene. Conversion of proto-oncogene to Oncogene = gain of function mutation due to... 1. Point mutations - change in single base pair = constitutively active protein 2. Chromosomal translocation - Fuse two genes = hybrid gene encodes constitutive chimera protein 3. Chromosomal translocation - Brings growth regulatory gene under control of different promoter causing inappropriate expression of a gene 4. leads to production of oncoproteins Examples of oncogenes 1. Special focus on neu oncoprotein and ErbB oncoprotein. 2. Many of the oncogenes associated with cancers are receptor tyrosine kinases a. For Her2, a single point mutation results in a single amino acid change that can convert it to neu oncogene. Now constitutive. b. Overproduction of Her2 can lead to cancer. This is the case for many breast cancers. Herceptin (mAb) is currently being tested as a drug for breast cancer treatment 3. trk oncogene a. Chromosomal translocation results in replacement of most the extracellular domain of the normal trk protein with a non-muscle tropomyosin b. It is constitutively active and in cytosol, not plasma membrane where the normal one would be. 4. Viral activators or proteins can act as oncoproteins a. Ex: Activation of erythropoietin receptor i. SFFV (Spleen-focus-forming virus) induces erythroleukemia - a tumor of erythroid progenitors ii. Gp55 is an SFFV envelope glycoprotein that induces formation of excessive numbers of erythrocytes Defective tumor suppressor genes a. People with inherited defects in tumor suppressor genes have a propensity for cancer b. Example: Rb - retinoblastoma i. First tumor suppressor gene discovered by Robert Weinberg ii. Characterized by retinal tumors that can be bilateral 1. Occurs in childhood and develops from neural precursor cells 2. One child in 200,000 is afflicted 3. 60% not inherited; 40% inherited c. Example: p53 - Guardian of Genome - Li-Fraumeni syndrome - defective p53 that leads to many cancers resulting in a 25 times greater chance of having cancer compared to the normal population d. Example: BRCA1 - Women who have a defective BRCA1 have a 60% probability of developing breast cancer by age 50 compared to 2 percent for the normal population e. Cancers can be caused due to loss of heterozygosity (LOH) - Normal allele is lost, so now mutant allele is expressed - This can occur due to mis-segregation or mitotic recombination Carcinogens and DNA Repair a. most cancer cells lack one or more of the normal DNA repair mechanism b. DNA damaging events occur at the rate of up to 10^6/cell per day - Normal events such as mitochondrial activity and free radicals can cause damage - Environment can cause damage c. DNA mutation can occur through a number of different mechanisms that we won't review here - DNA proofreading enzymes can correct these mistakes d. Some carcinogen/radiation have been linked to specific cancers - WWII survivors - exposed to radiation had high rates of leukemia - Melanoma and other skin cancers - related to high exposure of UV - Carcinogens are considered direct or indirect type - Direct carcinogens a. EMS (ethyl methyl sulfonate), dimethyl sulfonate (DMS) and nitrogen mustards - Indirect carcinogens 1. Cytochrome P450 in the liver usually adds chemical groups to toxicants and detoxifies them 2. But in the case of Aflatoxin, p450 converts it from an indirect carcinogen (doesn't cause cancer_ to a direct carcinogen 3. Benzo(a) pyrene found in cigarette smoke undergoes the same thing Loss of DNA excision repair systems can lead to cancer 1. most stunning example is the inability to correct thymine dimers produced by UV light a. Xeroderma Pigmentosum b. Cell fusion of XP and normal cells - "corrected XP cells" c. Camp Sundown; Midnight Sun What's new in cancer therapy? a. Categories to be discussed - Radiation - Many types that cause excessive damage to DNA causing cells to activate their p53 that results in apoptosis - Brachytherapy - Radioactive seeds - EBRT - external bean radiation therapy - Surgical ablation (resection) - can be surgical removal or thermal (head or cold) ablation - Chemotherapy using non-biologics (Drugs) - Biologics (mABs and conjugated mABs) - Immunotherapy b. Traditional Drugs/treatments - Most drugs like 5-FU block cancer cells through attacking mitosis - Many drugs being developed are more specific, such as attacking one genetic defect such as rasD, but remember that usually more than one defect is responsible for cancers. - The biggest challenge in treating cancer is to selectively kill the cancer cells - now possible using mAbs and immunotherapy discussed under the new era drugs - Another challenge is dealing with multi-hit theory of cancer. That is, how does one tackle the multiple defects in a cancer cell? - Traditional chemotherapeutic regimes affect all cells in the body - 5-FU - 5 Fluorouracil - DNA synthesis - one of the most common - Taxotere/Taxol - Disrupts microtubule spindle in M phase triggering the M phase checkpoint - Radiation. External Beam Radiotherapy. Brachytherapy - radioactive seeds placed inside an organ/tissue New Era Drugs/Treatments - The biggest promise in cancer therapy is mAbs and immunotherapy (Nobel prize for immuno.) These drugs can target select cells. - Synthetic (bispecific) antibodies are being developed that might even be better. They can bind to two different epitopes rather than two identical epitopes like a natural mAbs. - Example - Herceptin - originally produced by Genentech 1. FDA approved Herceptin in 1998. Full course treatment costs $70k. 2. mAb targeted against the Her2/neu receptor that is abundant in 20 to 30% of human cancers 3. Women with this type of breast cancer can have 1 to 2 million Her2 receptors, but normal women have 20k to 100k. 4. Combined with other drugs in "combination therapy" 5. Kadcyla = conjugated mAb of Herceptin with DM1 which is a microtubule disruptor - Avastin otherwise known as Bevacizumab: Anti-VEGF Monoclonal Antibody 1. Tumors need blood and lots of it so secrete angiogenesis factors (VEGF) so the vasculature will supply blood to growing and oxygen starving tumor. 2. w/o blood, the tumor is limited to 2mm diameter. Cells still divide on the outside, but die inside 3. Typical angiogenesis factors are VEGF, bFGF and TGFalpha. VEGF production is induced by hypoxia. 4. Now used to treat macula degeneration (over production of blood vessels in the retina causing blindness) - Example: Rituximab 1. R-CHOP - R stands for Rixtuximab - a drug used in treatment of Non-Hodgkin Lymphomas (NHL). It is a mAb. CHOP = Cyclophosphamide (Cytoxan), Doxorubicin (Ardriamycin), Vincristine (oncovin), and Prednisolone Imatinib - Binds to active site and inhibits substrate binding of Bcr-abl fusion protein that is a constitutively active kinase that phosphorylates multiple signal transduction proteins. Very effective against chronic myelogenous leukemia (CML) Tamoxifen - Blocks the estrogen receptor. Many breast cancers need estrogen to survive. Breakdown products of tamoxifen block the estrogen receptor. Gardasil - First virus vaccine against sexually-transmitted HPV. Controversial and NYS is now involved. Helix BioPharmaceuticals and L-D0547 - Interesting approach because this conjugated antibody binds to the surface of lung cancer cells causing a shift in pH from normal to alkaline (basic) causing the cell to succumb. Unique use of a conjugated mAb. Immunotherapy - A type of cell therapy for treating cancer. Provenge - A cell therapy approach developed by Dendreon (pioneer in this area seldom recognized). Uses dedritic cells to attack prostate cancer. Controversial given that the treatment costs $100k and extends life by 4 months. Insurance companies will not reimburse the costs so Dendreon filed bankruptcy. They were the pioneers of immunotherapy however. Chimeric Antigen Receptor T Cells (CAR-T cells) - Worthy of the 2018 Nobel Prize- 1. Another cell therapy produced but this attacks the 15% of cases of acute lymphoblastic leukemia that do not respond to traditional treatments. 2. Made history on 12/12/12 in Philadelphia where a young patient's cells were collected, genetically altered to attack and kill the cancerous B cells, and then infused back into the patient 3. While she almost died in the treatment, it showed the promise of immunotherapy. 4. But cancer cells have a "Do not kill me" cell surface receptor but Keytruda is a relatively new drug (among others) that are immunomodulators that can block this signal. This is a very active area of research with drugs being developed to overcome the limitation of CAR-T therapy and immunotherapy. - PD-1 is on the T cells and a number of inhibitors can bind to it so that is does not bind to PD-L1 - PD-L1 is on the tumor cells and there are other drugs that can block this receptor so that is does not bind to PD-1

Cell Division

The Cell Cycle - no mistakes are allowed - Cell death is balanced with cell division - 1/2 of RBCs are 120 days, but every second 250k are generated - First proposed in 1953 G0 Phase - "quiescent period" or "resting stage" - non-dividing, typical to differentiated cells G1 Phase - 9 hours of the 24-hour cycle, most variable in length - Arthur Pardee - 3T3 cells - G1 phase required 3 growth factors - PDGF, EGF, Insulin - Early + late response genes - mRNA levels spike earlier with early response, spike later with late response S Phase - 10 hours of 24 hour cycle - DNA doubles - MCM helices unwinds chromosomes G2 Phase - 4 hours of 24 hour cycle - cell verifies all DNA has been duplicated correctly and all DNA errors corrected - chromosome condensation initiated - checkpoints - mitotic cyclins increase in activity M Phase - 30 min of 24 hour cycle - ends in reassembly of nuclear envelope from ER Cell Synchrony - All cells in the same cycle compartment - Methods to accomplish this: - Amino acid deprivation -> remain in G1 - Remove serum -> stall in G1 - Protein synthesis inhibitors -> stall in G1 - Microtubule inhibitors (nocodazole) -> stall in M - DNA synthesis inhibitors -> stall in S - G1 checkpoint - checks fidelity of the cell (enough resources for DNA synthesis) - Restriction point - borders G1 phase and S phase Cell Cycle Kinetics (timing) 1. G1 - tritium-thymidine or BRDU (fluorescent), restriction point between G1 and S, first appearance of radioactive BRDU+ cells 2. S - randomly cycling - 3. M - 30 minutes - percentage of "mitotic figures" Cyclins - control cell cycle transit - first discovered by Ruderman and Hunt - Sea urchin embryos - SDS gels - cyclin A and cyclin B - cyclin B - CDK1 -> phosphorylation of Lamin B - cyclin B only -> nothing - CDK1 only -> nothing - cyclin D is required for G1 cell cycle transit MPF - Maturation Promoting Factor - X. laevis - Mitosis Phase Factor - MPF = Cyclin B - CDK1 Ruth Sager - Fusion of normal and cancer cell -> hybridoma -> normal phenotype -> cancer phenotype - loss of tumor suppressor genes p53 - Guardian of the genome - unstable transcription factor -> phosphorylated by ATM/R and stabilized - checks fidelity of the DNA - if too much damage, initiate apoptosis

The Nucleus

The Nucleus - Seen in the 1800s, powerful enough microscopes - not all cells have nuclei: - RBCs - Skin - the stratum corneum - Lens fibers of the eye are anucleate because if there were nuclei, light would refract differently through the lens and negatively affect vision - nuclear envelope continuous with the ER - ER gives rise to the new nucleus after mitosis - Nuclear pores: - mRNA/protein exchange - Diffusion through nucleus < 62.5k Dalton proteins - Histones - 20k MW - can diffuse - Non-histones e.g. transcription factors - Nucleoplasmin - Found in the eggs of X. laevis (African clawed toad) - 10% of all proteins - First molecular chaperone discovered - 165k Daltons, 5 units 33kD each - Diffuses into the nucleus via nuclear pores - requires ATP and receptor mediation - The nucleus undergoes dissolution during mitosis - Involvement of lamins A, B and C - Caused by MPF -> phosphorylates Lamin B -> triggers dissolution - Some component in mitotic cells triggers MPF - Experiment: combine mitotic cell w/ G1 phase cell -> dissolution of G1 phase cells Lamins - give the nucleus shape (karyoskeleton) - Connects to the chromatin - A, B, C = ~70k Daltons - hydrophobic region critical in anchoring them to the envelope Progeria - aging disease - Farnesyl cannot be clipped off, so Lamin A cannot insert to nuclear envelope - treatment: Lonafarnib