Final

What are the types of cancer?

Carcinoma: Cancers derived from epithelial cells. This group includes many of the most common cancers, particularly in the aged, and include nearly all those developing in the breast, prostate, lung, pancreas, and colon. Sarcoma: Cancers arising from connective tissue (i.e. bone, cartilage, fat, nerve), each of which develops from cells originating in mesenchymal cells outside the bone marrow. Lymphoma and leukemia: These two classes of cancer arise from hematopoietic (blood-forming) cells that leave the marrow and tend to mature in the lymph nodes and blood, respectively. Leukemia is the most common type of cancer in children accounting for about 30%.[86] Germ cell tumor: Cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively). Blastoma: Cancers derived from immature "precursor" cells or embryonic tissue. Blastomas are more common in children than in older adults

Describe the process of Cancer Progression.

Cancer is much like evolution. There is a selection for the "fittest" cell or the cell that has the biggest advantage. Over time, more and more mutations are aquired that make and individual cell more "fit" than it's neighbors. This cell starts to take over and eventually becomes dominant until another mutations makes another cell more fit. Increased proliferation Loss of contact inhibition Deregulation of the cell cycle Increased mutation rates Loss of normal cell morphology Cell adhesion changes Increased migratory and invasive cell behavior

Explain the correlation between cancer and age.

Cancer will affect approximately 1 in 5 people. How many of you have someone they know who has cancer or has had cancer? Why does the incidence of cancer change with age? What is it about age that favors cancer? This is a complicated question that we don't really know the answer to. Cellular replication - if that were the case, would you expect mice or whales to have dramatically different incidence of cancer? Cumulative environmental exposure (accumulation of DNA damage) - Short lived animals would be protected??

Apoptosis vs. Necrosis

Apoptosis is a controlled and systemtic process. Necrosis is often the response to acute injury and frequently leads to inflammation. Notice the difference. During apoptosis, most cellular contents are maintained in apoptotic bodies (little fragment of cell enclosed in membrane). These apoptotic bodies are consumed and digested by phagocytic cells. In necrosis, the cell is reptured and the cellular contents are spilled. This often leads to inflammation. Apoptosis is very common and can happen quite rapidly.

Define apoptosis and its role in development and tissue homeostasis

Apoptosis is used in development to get rid of cells that are no longer needed or are specifically unwanted. Get rid of unwanted cells (sculpting) Maintenance of organ size Cell is infected with virus Cell no longer needed Metamorphosis Immune cells after infection Too much DNA damage Cell is damaged/stressed out

Cell Death

Apoptosis separates the digits of the hand

How does flipase contribute to apoptosis?

Apoptotic cells have a characteristic appearance and are rapidly clear by phagocytosis. Phosphatidylserine is membrane phospholipid that is found exclusively on the inner leaflet of the plasma membrane. During apoptosis, phosphatidylserine is flipped to the outer leaflet of the plasma membrane and is recognized by macrophages as a signal for "consume me".

Cell Division

Asymmetric division occurs when a single parent cell gives rise to two different daughter cells. This occurs when the contents of the cell (proteins, and particularly transcription factors) are inherited differently. One cell receives all of one or two factors, while the other cell receives none. In the example of a simple epithelium. This might occur when the plane of cell division divides the cell so that different regions of membrane (basal vs apical), with different protein contents, are not equally divided amongst the daughter cells. The direction of division plays a critical role in asymmetric cell division. Initially, polarity information of the single cell embryo is conferred by the point of sperm egg fusion. When the sperm fuses, the RhoGAP pyk1 is dumped into the resulting cell. This means that RhoA activity levels will not be uniform. Instead, there is more active RhoA on the opposite side of the cell and little near the site of sperm-egg fusion. RhoA, in its active state, drives myosin-dependent contraction of a cortical membrane skeleton system at the inner cell surface. The result is asymmetric contraction of this membrane skeleton away from the site of sperm-egg fusion. Prior to fusion, Par3/6 coats the entire membrane and is part of that membrane skeleton system. Par ½ is in the cytosol. Upon contraction, Par3/6 is dragged to one side of the cell and par1/2 is free to translocate to the membrane on the other side. The result is the asymmetric distribution of these proteins in the cell. The contraction of the membrane skeleton system is also thought to generate the cytosolic currents that asymmetrically arrange cytosolic proteins.

Antibody regions

Antibodies are made by the adaptive immune system of higher vertebrates (jawed vertebrates and above). They contain a "variable region" that is randomly made in immune cells and then is tested by the body to see if it recognizes something (the antigen). Antibodies that recognize self are quickly eliminated (exceptions to this rule cause diseases like type I diabetes). Antibodies that recognize pathogens are amplified and mutated to try and make them better. Each antibody is composed of 4 peptides that are held together by disulfide bonds.

What is Anoikis

Anoikis: extrusion of apoptotic cells from epithelial tissues. In cells that are part of complex tissues, such as an epithelium, communication between the apoptotic and surrounding cells allows the surrounding cells to extrude the dying cell from the tissue while maintaining tissue integrity.

Nucleosome/histone structure

4 types of proteins, 2 each

Describe why 80% of all cancers arise from epithelial cells.

80% of all cancers are arise from epithelial cells. Why do you think that is? Epitheilial cells are among the most proliferative of our body and are the cells that are exposed to the environment. It turns out are environment is quite toxic. Cells prefer low oxygen concentration (~3%) not atmospheric (~20%). In fact, many animals can only survive for a short time in pure oxygen.

Cell Differentiation

A single cell can become any type of cell in your body Transplantation assays allow us to determine if a cell is specified or determined

Caspases mediate apoptosis signaling

Caspases get their name form the Cysteine (C) in their active site and the aspartic acid (asp) where they cleave target proteins. Caspases are proteases that are inactive when synthesized (called pro-caspases). Initiator caspases are activated by some kind of apoptotic signal. Initiator caspases are activated when they are brought together in a large complex and form dimers. Each initiator caspase cleaves it partner, froming an active caspase. Activated initiator caspase then activate other caspases (executioner caspases) that have multiple targets throughout the cell (including the cytoskeleton [causing blebbing], Laminins, and inhibitor of DNA nucleases). There are two ways in which initiator caspases are activated; the extrinsic pathway and the intrinsic pathway.

Explain examples of how cell division, cell movement, cell differentiation and cell death are essential for development

Cell division - when, how many times, what orientation, etc. are all controlled. Cell movement - Cells will often move within the embryo to help shape things. Cell differentiation - The shape and function of a cell can change Cell death - When and where. Helps shape organs and tissues.

Confocal Microscopy

Confocal Microscopy Great for thick samples (tissue, embryos, whole critters). Images take longer to acquire (scanning) Actual image must be assembled by a computer; it is not seen in real time. Can see inside with orange rind around it.

Ran GAP's location

Cytosolic side

Epitope tagging

Define Epitope the part of an antigen molecule to which an antibody attaches itself. An antigen can have multiple epitopes.

Cell Movement

EMT - epithelial to mesynchymal transition Mesynchymal cell are cells with only loose contacts with their neighbors. They are free to migrate, invade etc. One example of EMT occurs in gastrulation, when the three original parental tissues of the earliest embryo are established. Here cells from the ectoderm are triggered to undergo EMT. They detach, migrate over the ectoderm surface, invade through the primitive streak, and differentiate into non-epithelial mesodermal cells. This event is triggered by signaling from the endodermal cells. Together, the ectoderm, endoderm, and just established mesoderm will generate almost every adult tissue.

Extrinsic Pathway

Extrinsic apoptosis is induced by extracellular triggers (i.e. by immune cells recognizing a cell that must be removed). Perhaps the best understood pathway is the Fas ligand mediated cell death. Immune cells can trigger apoptosis of target cells by secreting ligand (fas) that binds and activates a receptor system and triggers a signal transduction event that leads to apoptosis. Apoptotic signal receptors are called death receptors (queue ominous music) that have a ligand binding domain, a transmembrane domain, and a death domain. Upon binding of ligand, the death receptor trimerizes and then binds an adaptor protein called FADD (Fas associated death domain). FADD binding recruits initiator caspases that then can dimerize and activate each other. The complex is sometime cells the death-inducing signaling complex (DISC).

Forward and Reverse screens

Forward= Tons of mutations looking at specific phenotype. Trying to discover gene sequence. Reverse. Know gene. Manipulate it and watch for change in phenotype

Signaling though PKA

G alpha can also bind and activate, when in the GTP bound state, adenyl cyclase, turning ATP into cyclic AMP. Cyclic-AMP is a short-lived second messenger that has it main effect by activating cyclic-AMP-dependent protein kinase (PKA). cAMP binds to a regulatory subunit of PKA and releases it. PKA has many targets, one is shown above. Phosphorlyation of CREB activates its DNA binding activity and triggers transcriptional activation of target genes. G alpha subunits are classified by their targets. For example, G-alpha that stimulated adenylyl cyclase are called Gs (because the stimulate it). There are all so Gi that inhibit Adenylyl cyclase. There are many classes of G-proteins, we are only going over one.

Understand how cell mechanisms combine in development

Gene regulatory network theory is a framework for understanding how genes regulate each other. The GRN can be thought of as a program (like a computer program) that controls development. Likewise, expression of key parts of the gene regulatory network in the wrong location can lead to ectopic organ formation. This shows their importance in determining the fate of cells in the embryo.

Describe Transcriptional profiling, Next generation sequencing, and genome editing.

Genome sequencing is now becoming more and more common and will likely become standard of care for cancer treatment. Technology continues to drive the cost of genome sequencing down It has created an enormous data overload problem. Changes in tumor DNA can result in gene expression changes. Tumor genome sequencing is an increasingly common tool to analyze tumors. What kinds of things will you miss with genome sequencing? This is a comparison between the old shotgun sequencing method (a) by which the human genome was largely sequenced and next generation sequencing (deep sequencing) by which genome sequencing is done nowadays. This review is a little out of date and sequencing of human genomes is now routine.

Identify some of the specific genes that cancer mutations have been discovered in.

Gleevec was the first truly targetted therapy for cancer (it changed the world). Targetted therapies generally have far fewer side effects.

Phosphorylation occurs during chromatin condensation *histone modification (he mentioned in class to remember HAT and HDAC)

HATs acetlyate, open. HDACS deacytelate.

JAK-STAT signalling

Here is another signaling pathway, the JAK-STAT pathway. Here there is recruitment of signaling proteins to the receptor. Unlike RTKs, it is these same molecules that go into the nucleus and alter gene transcription. There is no cascade; the system is much more direct. Would signaling through this pathway likely be sigmoidal or flat?

G-protein coupled receptors

Here is how G-protein coupled receptors work. G-protein are heterotrimeric proteins (they are a complex of three proteins) called G-alpha, G-beta, and G-gamma. The alpha subunit can bind GDP/GTP. Like other GTPases we have talked about, G-alpha is inactive in the GDP-bound state and is bound to the beta- and gamma-subunits. G-proteins can be found associated with a receptor or can bind to an activated receptor—these receptors are called G-protein coupled receptors (because the are coupled to a G-protein). Once ligand binding occurs, a conformational change causes the receptor to act like a GEF and trigger the alpha-subunit to release its GDP and bind GTP instead. When G alpha is bound to GTP, in no longer associated with G-beta (aka the beta-subunit) and G-gamma and the complex disassociates. The beta-gamma-complex and the activated alpha complex both go on to propagate the signal. More than half of all drugs currently used are targets of GPCRs

Advantages of Monoclonal

High specificity. Detect only one epitope on the antigen. useful for consistency and standardization of experimental procedures and results. Specificity of monoclonal antibodies makes them extremely efficient for binding of antigen within a mixture of related molecules, such as in the case of affinity purification.

Intrinsic Pathway

If DNA damage is too severe, p53 induces the cell to commit suicide (apoptosis) rather than divide and propagate deleterious mutations. Apoptosis is used in tissues of multicellular organisms to remove cells that have extensive damage (genetic mutations, viral infections) that might make them dangerous to the entire organism. An example is in response to p53 stimulation following DNA damage.

Assembly of Apoptosome

In the cytoplasm, cytochrom C complexes with Apaf1 to form the apoptosome (queue dramatic music). This complex recruits pro-caspases (initiator caspases) through its CAspase Recruitment Domain (CARD) and initiates the cascade. Assembly of the complex lead to activation of the initiator caspases and triggers the apoptosis pathway. Caspases activate more and more caspases, which in turn cleave specific target proteins at specific locations. This can inactivate (red) or activate (green) the target protein, thus driving the cellular events of apopotosis. This cascade is generally irreversible.

Demonstrate how apoptosis is initiated and carried out at the molecular level.

In this process, cells break down the chromatin and organelles, fragment into pieces (via membrane boiling or blebbing), and are consumed by phagocytosis by surrounding cells.

Advantages of Polyclonal

Inexpensive to produce. Technology and skills required for production less complex compared to monoclonal antibodies. Production time scale is short. Polyclonal antibodies are not useful for probing specific domains of antigen because polyclonal antiserum will usually recognize many domains.

Intrinsic Pathway cont.

Intrinsic apoptosis is induced by conditions within the apoptotic cell, such as by the DNA damage checkpoint. In this system the interplay between bcl proteins determined whether apoptosis occurs. The intrinsic pathway is regulated by the Bcl family of proteins. Pro-apoptotic bcl proteins (such as bad, bax, bak, nid, and others) are in the membrane of mitochondria and can form a channel that allows leakage of the mitochondrial contents into the cytoplasm, driving apoptosis. Anti-apoptotic bcl proteins (bcl-2 and bcl-x) prevent pro-apoptotic bcl proteins form forming a channel. The main component to leave the mitochondria through the bcl channel is cytochrome C, a boring mitochondrial electron transport protein that forms a complex with apaf1 in the cytoplasm. The Cytochrome C- Apaf-1 complex, or apoptososome, activates procaspases to caspases to drive the events of apoptosis.

Receptor Tyrosine Kinases

Many growth factor receptors are transmembrane molecules, with an extracellular ligand binding domain and an intracellular kinase domain. Receptor tyrosine kinases are a major class. Activation is the result of dimerization. Binding of ligand brings two receptors together. It has the effect of concentrating the receptors together. The question is, what happens next. Does dimerization activate the kinase? Do the receptor phosphorylate in trans or in cis? Some RTKs are always active, it is dimerization that brings them into close enough proximity to result in trans phosphorylation (each receptor phosphorylates the other within the dimer). Other RTKs undergo a conformational change when two receptors dimerize. This conformational change activates the intracellular kinase. Once phosphorylated, protein containing SH2 domains can bind to the receptor. RTKs, once activated, initiate a long cascade of events that results in phosphorylation of target proteins and changes in gene transcription. A benefit of this type of cascade is signal amplification. In the RTK cascade phospho receptors recruit Grb2, which recruits SOS (son of sevenless). SOS is a GEF for Ras, a small GTPase, which in turn activates raf (or MAP KinaseKinaseKinase), which phosphorylates Mek (MAP KinaseKinase), which phoshphorylates ERK (MAP Kinase), which phosphorylates transcriptional activators. This kinase cascade is very important in determining how signal intensity generates cellular responses.

Explain how morphogens regulate patterning in the embryo

Morphogens are signaling molecules that are generated in a specific spot and diffuse through the embryo. The result is a pattern on differentiated cells that respond to this gradient It was once thought that cells just had a dose dependent response to morphogens. This is generally* not the case. Patterns are refined by the downstream effects of genes. Example 1 of morphogens: Dorsal Ventral Patterning in the neural tube Sonic and BMP/Wnt are made by opposite sides of the neural tube. They then diffuse through to create different regions in the neural tubes. Each of these regions makes different types of neurons. Changes in gene expression for these genes lead to a number of defects, like the extra or missing ribs shown above.

Understand how cancer is the result of multiple mutations.

Most cancers are spontaneous. They arise from mutations in cells, not inherited mutations. Most cells that gain mutations die. Most mutations are toxic, or the checkpoints arrest the cell and initiate apoptosis. However, very specific mutations confer a selective advantage. Cancer almost never happen in a day, but requires many sequential steps and disruptions of the pathways we have talked about. In the example above, the first mutation may increase the proliferation rate of the a cell. The second may block apoptosis. The third makes the cell able to migrate and invade other tissues.

Identify some of the benefits of studying cancer.

Much of cell biology was discovered while studying cancer Cell cycle Cell signalling Apoptosis Tissue architecture Studying cancer helps us understand the rules that govern normal cell behavior

Ran GEF

Nuclear side. A

Executioner Capases

Once activated, executioner caspases start cutting up proteins everywhere. Once example is shown above. In healthy cells, the endonuclease CAD is associated with an inhibitor (inhibitor of CAD or iCAD). Activated caspases cleave iCAD, leaving the endonuclease free to go chop up DNA. The characteristic ladder pattern of DNA that comes from apoptosis cells arises from the nuclease cleaving between nucleosomes, leading to a ladder of ~150bps pieces of DNA.

Signaling through PKC

Other G-coupled protein receptors activate and enzyme called phospholipase C (both the alpha and the beta-gamma are required for activiation). This is an enzyme that cleaves specific lipids (our friend PI(4,5)P2) into the lipid tail and head group, which have secondary signaling roles. The head group, I(1,4,5)P3), floats away and causes calcium channel IP3 receptors to open and release calcium ions into the cytosol from calcium stores in the ER. This calcium binds and helps activate, along with the lipid tail resulting from PLC activity, protein kinase C, which initiates further signaling. This demonstrates that it protein-protein interactions, GTPases, kinases, lipids (diacylglycerol), small molecules (IP3), and ions (calcium) that can all combine to mediate more complex signaling pathways.

Understand how mutations make cancer genomes unstable.

Show above is a karyotype (by sky) of a cancer cell. Notice that some chromosomes are missing (i.e. chromosome 6) and others are inappropriately combined (i.e. chromsome 2). The cells now have an "unstable" genome and acquire mutations at an even faster rate.

What is Cancer?

Six hallmarks of cancer: Cell growth and division without the proper signals to do so Continuous growth and division even when there are signals telling them to stop Avoidance of programmed cell death Limitless number of cell divisions Promoting blood vessel construction Invasion of tissue and formation of metastases

Know the difference between Specified, Determined and Differentiated cells

Specified - Cell has received instructional cues to become a particular cell type, but still can be changed Determined - Cell is now set to become a particular cell type. This is difficult to reverse and will happen independent of outside help. Differentiated - Cell has become a specific cell type

Monoclonal vs polyclonal antibodies

What is a Polyclonal antibody? Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. The other method is to isolate the plasma cells and fuse them with a specific type of cancer cell. This results in a cell with two nuclei that are both immortal (from the cancer cell) and produce antibodies (from the plasma cell) call a hybridoma. They can be cultured almost indefinitely and the secreted antibodies collected.

Differentiate between totipotent, pluripotent and multipotent

Totipotent - can become any type of cell, including extraembryonic tissues like the placenta Pluripotent - can become most cell types, usually used to denote stem cells that can make any cell within the embryo Multipotent - can become any of several cell types, but is fairly restricted

Histone modification table

UMA K causes transcriptional activation. Phosphor causes chromatin condensation Arg trans inactivation

Describe how understanding genetic changes in cancer help with rational drug treatment.

Understanding the genetic changes in cancer leads to rational drug treatment. You can inhibit specific pathways necessary for cancers propagation. The CRISPR/Cas9 proteins were adapted from prokaryotic cells where they act as an "immune system" against viruses. This protein/RNA combination allows you to introduce double stranded breaks to just about anywhere in the genome you want. The technology is extremely inexpensive and very easy to use. Double stranded breaks are usually repaired using non-homologous end joinging (NHEJ) which is very error prone and typically results in insertion or deleltion mutations. If you provide a template for repair, cells will also use homology driven repair (HDR) which allows you to introduce any sequence you want, anywhere you want. This is a transformative technology that will be everywhere in a few years. It is not inconceivable that this technology will be used to repair mutations that occur in cancer cells. This technology also has opened up research in many new species (i.e. rats, silk worms, unicorns...).

Widefield Microscopy

Widefield Microscopy Most commonly encountered "normal" type Good for thin slices. Image can be constructed for thick slices. One is computational, the other optical. These three-dimensional microscopic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting the light that comes from out-of-focus regions above and below that plane. Thus one sees a crisp, thin optical section. From a series of such optical sections take and different depths and stored in a computer, a three dimensional image can be reconstructed. The methods do for the microscopist what the computed tomography (CT) scanner does for the radiologist investigating the human body: both machines give detailed sectional views of the interior of the intact structure. This is called image deconvolution." can only see orange rind.

Notch Signalling

Yet another mechanism. This receptor is activated and becomes proteolytically cleaved. The cytosolic piece can then go into the nucleus and exert effects on gene transcription by direct interactions with nuclear proteins. Notch ligands are typically expressed on the surface of cells. So what type signaling would Notch signaling be?