Cancer Biology Notes Chapter 1-7 for First Exam
Cancer Biology Notes Chapter 1-7 for First Exam BIOL 3365
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This 39 page Bundle was uploaded by Madeline Abuelafiya on Saturday January 30, 2016. The Bundle belongs to BIOL 3365 at Southern Methodist University taught by Dr. Harrod in Fall 2014. Since its upload, it has received 100 views. For similar materials see Cancer Biology in Biology at Southern Methodist University.
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Date Created: 01/30/16
Ch1, The Biology and Genetics of Cells and Organisms Through research in genetics and a and molecular biology cancer biology became a discipline that could not just list and categorize types of malignant diseases but could go deeper and describe how and why they occurred These revolutions in genetics and molecular biology stem from Watson and Crick’s DNA double helix model and this type of research into why things happen the way they did stems from Darwin and Mendel 1) Mendel establishes the basic rules of genetics (1) Mendelian genetics still apply today, basically unchanged from when Mendel studied pea plants, to both metazoa and meta phyta multicellular animals and plants (2) Mendel discovered that genetic information seems to be inherited in a particulate form to offspring, (a) 20km.7 (b) Genome is organized into genes, tiny packets of genetic information (i) Humans current estimate of our own unique number of genes is 37,000 vs 14,500 in drosophila (3) Mendel discovered that the genetic component of an animal or plant (genotype) is made up of thousands of genes, and that the outward expression (phenotype) is the observable consequence of the genotype (a) Researchers applied Mendel’s technique to higher order eukaryotes: E. coli ,yeast, worms, mice. Genotype determines phenotype remained true (4) Amendments to Mendel, one gene to one physical attribute was not entire true (some traits work together to make a trait), many organisms reproduce asexually, how do they pass information/ change information (5) Mendel realized that genetic information is passed down largely at random from parent to offspring, and that higher level organisms have two copies for each gene thus all higher organisms are diploid. (a) The two copies of the genes could contain different information thus the different versions are called alleles (i) To carry two identical alleles for a gene is to be homozygous (ii) To carry two different alleles for a gene is to be heterozygous (iii) Sometimes the alleles an organism carries can conflict, from this Mendel learned of dominance 1. A dominant allele is a gene that when present will always be shown/ put forth 2. A recessive allele is a gene that when present will only be shown when paired with another recessive allele 3. Codominant alleles are genes that instead of both being expressed independently or one being dominant will form a hybrid phenotype (red + white = pink) 4. Incomplete penetrance – a dominant gene is present but cannot express due to the action of another gene in the genome 5. Incomplete dominance where both alleles will be expressed, (A + B = AB blood) (6) How does cancer uses dominance and excessiveness? (a) Harmful or defective alleles are not advantageous to an organism thus, not seen in general public, however the presence of a defective allele can be masked by a dominant allele thus heterozygotes are not apparent in the public, but if two heterozygotes mate their child could become homozygous recessive and be afflicted by the harmful trait 2) Mendelian genetics helps to explain Darwinian evolution (1) 1920s and 1930s genetic information corruptible (2) Mutations random changes in the genetic information of an organism can be + or neutral (can lead to new alleles, deletions etc) (3) Most common allele is the wild type (usually compatible with normal structure and function of the animal) (a) Mutations allow species to try out new traits and pick the best to survive, all the different alleles generated via mutation or other wise in a species is the gene pool (i) Older the species the larger the pool, ex. Chimpanzees have a larger gene pool than humans who are <150,000, thus younger (b) Natural selection insures that phenotypes generated from these mutations in the genotype that provide advantages will be passed down (4) Discovery that large portions of our DNA does not code for genes, we call it junk DNA though it is still useful (a) 1.5% of mammalian DNA encodes for proteins (b) ~2% encodes regulators for gene expression (c) b/c true genes and junk DNA are mutated equally more often than not a mutation will prove neutral to the organism (neutral mutations remain silent in the phenotype) (d) genetic polymorphisms – interindividual, functionally silent differences in DNA sequence that make each human genome unique (e) 3) Mendelian genetics governs how both genes and chromosomes behave (1) Mendel’s genes and chromosomes both exist in pairs, found that the chromosomal array karyotype is duplicated in all complex organisms when a cell wants to duplicate (a) Length of chromosome is proportional to the number of genes it carries (b) Genes are localized to specific sites on the chromosome also called locus (i) Mapping of genes to specific loci was done extensively in late twentieth centuries b) Evolutionary forces dictate that certain genes are highly conserved i) Some specific genes are vital for organismic viability, and were optimized hundreds of millions years ago, thus mutations in these genes tends to be deleterious and often fatal (1) Thus some genes highly conserved down eukaryotic life, (a) Genes that code for the majority of proteins were first seen probably in single cell eukaryotes (b) Seen as our genes seem to have genetic counter parts in other lower organisms (i) Eye placement in flies and humans is controlled by genetic counterpart genes. Mammalian embryogenesis, physiology and biochemistry is very similar in all mammals c) End (1) germ cells reproductive cells that are haploids (carry a single copy of chromosomes/ genes) eggs and sperm generated in the testes and ovaries (a) each germ cell receives randomly one half the DNA of the parent cell, and during fertilization the cell becomes diploid as egg and sperm combine (b) genes alter phenotype by acting locally (2) sex chromosomes violate the rule of pairs, in females there are 22 autosomes and a pair of X chromosomes that act autosomal. But men have 22 autosomes and a single X and single Y chromosome. X and Y are sex chromosomes (3) females having the extra copy of the gene allows more robust biology, if one of the pair is defective the other will work instead. This is why color blindness, and baldness are much more men things as only one X (a) X codes for 900 genes vs 78 genes on the Y (4) Problem with dosing, if both X were active in females probably cell death, thus during embryogenesis one of the two X chromosomes is turned off randomly. Off gene becomes the barr body 4) Chromosomes are altered in most types of cancer cells (1) Which individual changes in the chromosome is way to tiny to see with a light microsope, the size of a chromosome can be seen, this is useful in finding cancer cells that have irregular size/ number of chromosomes (a) Cancer cells are mutant cells gone wild (2) Euploid karyotype is the normal configuration of chromosomes 22 autosomes pairs and 2 sex chromosomes (3) Aneuploidy is seen in many cancer cells sometimes a miss number of chromosomes is purely symptomatic of a cancer cell but if the copied chromosomes contains say a growth signal gene proliferation can run wild and cell is cancerous 5) Mutations causing cancer occur in both the germ line and the soma (1) Germ line (egg and sperm) this is hereditary line for cancer to pass to the offspring 5% of all cancer is this, (1112* more likely for the offspring to develop cancer if mutation is germ line and non junk) (2) Soma cells are anything not your germ/ gametes. All other cancer types are somatic mutations – not able to be passed from parent to child (a) Somatic mutations affect cell they occur in and the daughter cells that are born from it, and can eventually effect entire tissues. The daughter cells are called clones as they all have the same ancestry (3) Normally the cell is supper good at tracking down mutated DNA and either fixing it or killing itself via elaborate repair systems. It will replace mutations with the wild type if found, kill itself, or guess at what was probably there (a) When the repair system goes wrong is when unregulated mutations leading to unregulated proliferation and reproduction can occur. Cancer/ malignant cells 6) Cancer cells are often aneuploidy i) Changes in cell genotype often accompany and sometimes cause uncontrontrolled proliferation of malignant cells (1) Normal chromosomes can start accumulating in 3 or more copies thus become aneuploidy (2) Other types of chromosomal mutations not complete copies of chromosomes (a) Translocation a broken off segment of one chromosome binds to a different chromosome (b) reciprocal translocations two different chromosomes lose a part and swap segments (3) these mutations are not exclusive, thus translocation of a extensively copied segment of chromosome is possible resulting in long mixed up chromosomes of a variety of origins. (a) Flipping – chromosome segment detaches then reattaches upside down (b) mFISH Fluorecent in sita hybridization (c) segment becomes detached and copies multiple times becoming DM or double minutes (d) DM and other coping problems lead to gene amplification this can potentially lead to copying of a growthpromoting gene and thus proliferation of malignant cell (e) Interstitial deletion –chromosome loses a segment and it is destroyed, very bad if growthinhibiting factor is deleted 7) Genotype embodied in DNA sequences creates phenotype through proteins a) How does genotype affect phenotype? (1) How does a code of nucleotides decide the morphology or shape/ form of a cell? (2) 1944 DNA is blueprint, 1953 double helix 1964 sequences of DNA’s nucleotides determines the order/ sequence of amino acids in proteins (3) Proteins create phenotype: (a) assemble into a cytoarchitecture – also known as the cytoskeleton, (b) secretion of proteins into the ECM extracellular matrix enables cells to bind, communicate, and form complex tissues, (c) proteins can function as enzymes that catalyze the thousands of biochemical reactions in a cell also called intermediary metabolism (d) proteins through selective constructing and deconstructing of actin filaments and the cytoskeleton can enable a cell to have motility (e) communication between cells is a huge job of proteins (i) receive signals> process signal> pass to another protein and so on to reach target. Process called transduction (4) almost all aspects of a cell and its actions are determined by proteins b) therefore, genotype is the sequence of bases in DNA and phenotype is derived from the actions of proteins c) How does DNA code for proteins? (1) Eukaryotes and Prokaryotes alike use RNA molecules as the method to transfer genetic information into the cytoplasm (2) DNA to RNA is called transcription, where genes make a one to one complimentary copy in RNA of a DNA segment (a) Genes being transcribed actively are being expressed while non transcribed is repressed (b) RNA is then translated by protein synthesizers called ribosomes into amino acid chains (i) The chains will eventually fold according to the primary structure’s self interactions (3) Posttranslational (anything after translation) modification is also possible (a) Phosphates, complex sugar chains, and methyl, acetyl, and lipid groups are most common (i) Proteins being secreted to the cell surface are almost always glycosylated or bound to sugars (ii) Ras proteins, key in cell signaling and also cancer development usually have lipids bound (b) Ubiquitin tagging, allows the protein protease to come and cut apart other protiens into recyclable pieces d) How is RNA transcriptedst (1) The RNA 1 made in the nucleus is called the premRNA or (pre messenger RNA) (2) PremRNA contains both introns (non coding genes) and exons (coding genes) before transport to cytosol must remove introns (3) Splicing is the process of fusing together the remaining exon segments (4) hnRNA heterogeneous nuclear RNA initially synthesized RNA molecule and its derivatives found at various stages of splicing, together with nuclear RNA transcripts being processed from other genes, also called premRNA (a) final product may be super small (5) mRNA template for protein translated by ribosomes in cytosol (a) potentially only 1% original length (6) alternative splicing like normal splicing but alternative, (not always in original order on purpose) (a) this causes new combinations and thus the DNA codes for more proteins without having the sequence in that specific order (b) sometimes this can lead to production of a protein that may instruct the cell to proliferate unchecked (become a tumor) 8) gene expression patters also control phenotype (1) all cells in the body are decedents of the fertilized egg/ zygote (2) but then why does a skin cell not act like a brain cell if they have the same DNA? (a) Selective reading of the genome (3) Cells in the embryo begin to specialize in certain functions or differentiation (a) These differentiated cells are committed to become only one type of tissue rather than another. (4) Genes in mammals are grouped into two: (a) Housekeeping genes – encode for proteins universally needed in mammal cells (majority) (b) Tissue specific genes production of proteins and thus phenotype that are associated specifically with this cell type (5) Maybe 3005000 housekeepers while 1000 tissue specific 9) Histone modification and transcription factors control gene expression (1) Cells use transcription factors to determine what is transcribed and what is not (i) These proteins bind to specific DNA sequences and determine if the gene will be transcribed (ii) TFs bind to sequence motif 1015 nucleotide long promoters/ start points (b) TFs may repress or facilitate transcription (2) The ability of a single TF to elicit multiple changes in a cell or organism is called pleiotropy (a) There are about 1445 distinct genes in the human genome that make TFs, this does not count alternative splicing. (b) If a TF goes back it leads to potentially a cascade of effects inside a cell ii) Many genes require multiple TFs to transcribe RNA, and each TF must bind to a specific enhancer site to collaborate active gene expression (a) gene Expression program the coordinated expression of multiple genes within a cell (2) something strange, TFs use DNA pol 2 in not just one direction. Often it will start transcription then after 6080 nucleotides turnaround and transcribe backwards transcriptional pausing (i) Myc is a oncogene, it encodes DNA pol 2 to stop after pausing and not go backwards or forwards. If this goes wrong a lot of extra genes get encoded (ii) (3) TF also deal with chromatin , a mix of DNA and structural proteins (4) Beads on a string and the 8 part histone molecule (2x H2A, H2B, H3, H4)+ one H1 binding agent, this structure is super common in chromosomes (5) Modification of histones (i) Sometimes a core histone molecule will be swapped like H3 for H3.3 or H2A with H2AZ (ii) Post translational modification of the standard 4 histones. These will affect the Nterminus ends of the beads on a string 1. Typical modifications: methylation, acetylation, phosphorylation, ubiquitylation (iii) Methylation generally represses, acetylation generally activates, and phospohorylation is for condensing during mitosis 1. Methylation of H3 does activate though (6) Modified states of chromatin can be passed from mother to daughter cells through idk ways 10) Heritable gene expression is controlled through additional mechanisms (1) Epigenetic inheritance alternative means of maintaining such modifications in a stable and transmitting way from one cell generation to another (probably by biochemical mechanisms) ii) How we maintain methylation, (1) DNA methyltransferase enzymes see hemimethylated segments of fresh copied DNA, scroll on down it and will methylate any unmethylated CpG dinucleotides that are complementary to already methylated CpGs 11) Unconventional RNA molecules also affect the expression of genes (1) How RNA can regulate itself (a) MicroRNAs 2125 nucleotides long, (b) Once formed the miRNA will become part of the RISC RNA induced silencing complex nucleoprotein (c) From here it will either bind to complementary mRNA to destroy it or may (d) Bind to mRNA to stop translation to protein (2) Thought to regulate at least 1/3 of all genes in human genome, at least 650 known, (3) A single RISC can act in multiple ways and multiple times, thus acting pleiotropy (4) Because mRNA does not affect phenotype but is instead regulated by proteins, RISC play an important role in the outcome phenotype as they are a sort of final barrier stopping potential gene expression (5) It has been shown that micoRNA Let 7 is responsible for suppressing Ras in both humans and some worms, Ras is a critical protein that is the key to develop many typesof human cancers (a) Lose a bunch of miRNA and you probably will get cancer ii) incRNA long non coding RNA, in nucleus and cytoplasm. Poorly understood but does regulate gene expression iii) dude most of our DNA is made to stop us from over making shit b) 1 post transcriptional processing of pre mRNA transcripts (splicing and alternative splicing) c) 2 stabilization or degradation of the mRNA product d) 3 regulate mRNA translation e) 4 post translational modifications, stabilization or degradation of the protein product 12) Metazoan are formed from components conserved over vast evolutionary time periods (1) Important gene functions for proliferation of an organism were figured out millions of years ago and if it aint broke don’t fix it shit. Thus there are genes in yeast/mice/ worms/ other things that may be functionally identical and also look alike to some of our genes/ proteins ii) Eye genes in flies and humans are the very similar when deciding where to put eyes 13) Gene cloning techniques revolutionized the study of normal and malignant cells i) 1 we used virus genomes and studied them cuz it was easy to purify and filter them then came gene cloning ii) Genomic libraries (1) Basically an entire organisms genes and all the possible alleles it could have? (2) Cloning of DNA fragments and thus cloned genes (3) Kool stuff (a) We took mRNA and then paired it with reverse transcriptase this then gave us a copy of the complimentary DNA used (b) Vb bn]to make those particular exons Ch 5 growth factors, receptors, and caner How normal cells receive signals from the environment that surrounds them. And how these signals deregulate over time making cancer cells 1) Normal metazoan cells control each other’s lives i) For a multicellular body to function correctly no one cell should have the ability to decide to grow/ divide based on its own needs, it must work for the benefit of the tissue/ organism (1) Cells talk to each other through growth factors GF small proteins released by some cells in intercellular space, impinge on other cells to deliver specific biological messages. (2) The cell’s neighbors will either release a growth simulating factor or a growth inhibitory factor (3) Even if you place cells in a petri dish full of all the nutrients they need for proliferation they will not grow, they require another medium of GF telling them to multiply ii) Looking at minor wounds and how they are healed (a) PDGF platelet derived GF, is the GF released by platelets. (b) PDGF is a potent stimulator for fibroblasts affecting even the basal lamina beneath the epithilia (c) PDGF is and other GF that stimulate growth are known as mitogens (i) Mitogen indicates ability to induce cells to proliferate (ii) PDGF first attracts fibroblasts then tells them to proliferate (d) Without PDGF fibroblasts will continue to maintain homeostasis but will not divide 2) The src protein functions as a tyrosine kinase (1) Once we know that v src was an oncogene the race for how it worked was on. How did it get rid of anchorage depence?, what about the large increase in glucose absorption?, and how did it cause cell proliferation (a) 1977-78, (i) In a petri dish it was observed that v SRC would phosphorylate antibodies if ATP was present 1. SRC is a kinase an enzyme that takes a high energy phosphate group from ATP and transfers it to a suitable protein substrate (ii) V src itself is also a phosphoprotein it carries phosphate groups covalently attached to its amino acid side chains 1. So it either did autophosphorylation or served as the substrate of another kinase. (iii)Kinases were known to be able to modify multiple proteins (SRC) affects 50 different proteins 1. Each protein modified could lead down a long chain of events and cause all the conditions necessary for tumor growth. (iv) Other kinases, attach phosphates to side of chains of serine and threonine AA substraits (v) SRC phosphorylated certain tyrosine residues of its protein substrates 1. Most of the cell’s phosphoproteins were phosphor serine and threonine. Phosphotyrosine is strange a. This phosphotyrosine is unique to SRC 2. Increased phosphotyrosines to over 1% of all phosphoproteins in cell 3. Found that phosphotyrosines are almost only used in mitogenic signaling pathways in mammals (2) How does the phosphorylation of tyrosine kinase cause cancer then? 3) The EGF receptor functions as a tyrosine kinase (a) Next jump came from cell bio. (i) Researchers looking for how cells talk with signaling proteins that were strangely similar to oncogene proteins (b) Starting with EGF the first GF discovered. (i) EGF binds to the surface of cells it signals so there must be a receptor. (ii) EGF is known to have mitogenic effects when applied to some epithelial cell types (iii)Isolating the EGF-R was hard cuz they are normally in low numbers 1. Use tumor cell that makes lots of EGF-R to study (c) Physiology of EGF-R, 1. large ectodomain- extracellular side, involved with binding to ligand and recognizing ligand a. n terminus 2. Transmembrane domain sticks into PM a. Has a group of continuous stretch hydrophobic AA in middle of AA sequence.k 3. Third layer, c terminus side extend into cytosol 4. Form gives away use, ectodomain binds EGF ligand, and PM side activates sending the signal inwards a. It phosphorylates tyrosine in the cell interior to cause cell proliferation (d) Other similar receptors have their own GF ligand that will bind to their receptor, can cause cell shape change, cell survival and cell motility 4) An altered growth factor receptor can function as an oncoprotein (a) 1984 scientists realize that EGF is very similar in sequence to oncoprotein made by the erbB oncogene, thus the oncogene was making oncoproteins (i) AEV retro virus induces leukemia of RBC (erythroleukemia) (2) While normal EGF-R and the protein encoded by erbB are similar, key difference = lack of a/ modification of the N terminous / extracellular side of the receptor protein (a) It is unable to bind EGF yet remains continually expressed anyway. Suggesting the N side was inhibitory (b) Later this kind of ectodomain deletion was found in 1/3 of all human glioblastomas and in smaller numbers other tumors (3) Cousins of ErbB/ EGF-R found to be very active in human breast cancer in the forms ErbB2, HER2 and Neu (i) There genes lead to poor prognosis ii) Discovery of ErbB showed why cancer cells were less dependent of GF, they had receptors that continually act like they are bound to the GF ligand thus always tell the cell to grow. (a) Other ways an oncoprotein can be continually simulated (i) The cancer cell makes a ligand that can activate its own receptors 5) A growth factor gene can become an oncogene: the case of sis (i) Scientist discover AA sequence of PDGF platelet derived GF, the B- chain of the PDGF very similar to oncoprotein made by v-sis from the simian sarcoma virus (b) PDGF unlike EGF, stimulates the proliferation of mesenchymal cells connective tissues, fibroblast, adipocytes, smooth muscles, glial cells (c) Thus tumors that originate in the epithiala have EGF-R while those that originate in the mesenchymal have PDGF-R 1. PDGF-R is also a tyrosine kinase receptor ii) How the PDGF-R receptor works (a) The simian sarcoma virus doesn’t code for the PDGF-R broken receptor (b) Instead it produces copious amounts of PDGF to be released extracellularly, it autocrine signals itself to proliferate 1. Thus the receptor is normal just shit tons of stimulant, maybe a antigen could help stop this cancer? (ii) So not paracrine local signaling to surrounding tissue (iii)Not endocrine long range signaling through blood vessels (iv) Yes autocrine signaling, the cell stimulates self a. Cell produces own mitogen (c) Human cancers that operate the same was as simion sarcoma virus 1. The GFs: transforming GF alpha (TGF alpha), stem cell factor (SCF), insulin like growth factor 1 IGF-1 a. All of these act as ligands in an autocrine loop of cell growth (2) Worst autocrine tumor kaposi’s sarcoma (a) Makes PDGF, TGF beta, IGF-1, Ang2, CCl8/14, CXCL11 and endothelin. Also makes the receptors for these ligands (b) This sarcoma caused by HHV8 human herpesvirus-8 (i) There are some cells that use autocrine looping normally, but in the cancer cells that express it, it is out of hand 6) Transphospohrylation underlies the operations of receptor tyrosine kinase (i) So we have seen 2 things that cause lots of either activation or made broken receptor tyrosine kinases RTKs, but is there maybe something also in the way they tranduct a signal too? (b) how do GF-R use their tyrosine kinase domains to emit signals in response to ligand binding? (i) clues 1. Noticed some proteins being phosphorylated as soon as IGF or other GF bind. 2. They also noticed that while it does phosphorylate other proteins, it tends to mainly autophosphorylate 3. GF ligands were dimeric (made od two identical protein subunits (homodimers) or very similar but not the same heterodimer units 4. The tyrosine kinase proteins are capable of PM movement. (c) Soln. (i) No ligand, receptors exist monomeric single sub-units living alone. (ii) Homodimer binds and now receptor hunts for its other half (iii)Binds to other half, crossing the homodimers making an active ligand and producing a signal 1. The ligands induce receptor dimerization (iv) Next each half kinase phosphorylates the other so not autophosphorylation but transpohosphorylation 1. The phosphorylation can potentially unblock the active domain of the RTK (d) Heterodimers (i) HER2, ErbB2, Neu the over expressed RTK in breast cancer will bind with HER1/ EGF- R or HER3. 1. HER1 and 3 are thought to not even need a ligand bound to dimerize a. Also HER3 is broken on the C end, but with HER2 it works good enough (2) Over expression of a RTK can lead to ligand independent dimerization, b/c of the high number of RTK halves it isn’t unlikely for them to hit themselves and dimerize randomly (3) Jk, its little more complicated then just hitting each other. EGF-R actually naturally will dimerize slightly before the EGF binds allowing for more rapid a response. (a) When the RTK bind like this before ligand they become high affinity receptors for the ligand, sucking it in. thus that may be why EGF-R is hyper responsive to EGF and TGF- alpha in cancer cells (4) Jk again, there are actually some RTK that will dimerize and fire ligand independently (a) Human Met receptors, present in high amounts in liver cancers (ligand to HGF hepatocyte GF/ scatter factor SF) (b) Put in mouse with no HGF/SF but the cells still grew and divided thus it has to be ligand indedended processes, spontaneous dimerization of overexpressed Met receptor molecules (5) Another case with ligand independed dimerization occurs (i) Point mutation in Neu proto oncogene in mice (the ortholog human HER2/ErbB2) causes neuroextodermal tumors in infant mouse pups. (ii) Point mutation causes a favoring of dimerization of the receptors due to the replacement of valine with glutamic acid 1. The dimerized receptor acts as a potent oncoprotein (b) EGF and FGF receptor point mutations also occur, these mutations lead to dimerization b/c of a change in the PM spanning region of the protein (c) Gene fusion in another source of oncoproteins, (i) Causes truncated ectodomains, fusion to unrelated proteins that are prone to dimerization (ii) Ex Met for hepatocyte growth factor HGF and TrkA for nerve GF b) RTK mutations not involving dimerization of receptor (i) Rotation or stereochemical shift. (b) Some RTK are always dimerized and the ligand only exists by a concurrent rotaiotn of both the ectodomains and cytoplasmic domains c) Of all the genes humans have, only 59 encode proteins with structure like EGF and PDGF receptors. (1) Only a minority of these 59 are known to cause cancer, (2) Some RTK mutations make it into the germ line cells too 7) Yet other types of receptors enable mammalian cells to communicate with their environment (1) Janus kinase Jaks, separate polypeptides that associate w/ cytoplasmic domains of the receptors through noncovalent links (2) Erthroptoietin receptor EPO-R, regulate red blood cell production (3) Thrombopoietin – TPO-R receptors control the development of the precursors of blood platelets called megakaryocytes ii) These are all cytokine receptor molecules (1) When they dimerize in response to ligand binding, Jaks phosphorylate and activate C- terminal trails of receptor modules. iii) Transforming GF beta (1) A heterodimer, binds serine/threonine rather than tyrosine (2) Surpress normal epithelial cell proliferation, (3) Made of type I and type II ligand units (a) Ligand binds to type II, closes in on type I (b) Type I phosphorylated (c) Type I phosphorylates cytocolic proteins, then sends to nucleus where they will trigger expression of certain target genes iv) More primitive form of transmembrane signaling, Notch receptors and ligands NotchL, Delta, Jagged (a) Are non-moving receptors that require close proximity for interaction between two cells to occur (b) This signal transduction = juxtacrine signaling (c) Notch receptor binds, attaches to bound ligand of other cell, other cell rips out the receptor and bring it inside the signal emitting cell 1. Cleavage by protease. (d) PM domain portion of receptor now detaches and travels into its host cell and functions as part of a complex transcription factor. v) Patch receptors- multi cytoplasm passing receptor protein. (a) Often are called serpentine receptors (i) Its ligand is Hedgehog class proteins Hh (b) When ligand binds a second serpentine receptor moves away this second g protein receptor is smoothened Smo, (i) Cells have very small cilium on their surface, a cilia with a Gli and smoothened cycle back and forth out of the primary cilia 1. When Hh binds to Ptc smoothened and Gli accumulate in primary cilium 2. Gli converts from transcription depressor to transcription activator and goes to nucleus b) Wnt factor independent signaling system (1) Named for drosophila mutant wings and related gene in mice tnt-1 (2) 19 different human Wnt species. ii) They are tethered tightly to ECM and w/ a lipid tail to cell membranes iii) Not freely diffusible like other GF, iv) Wnt receptors are the Frizzeled (Frz) receptors that are also serpentine v) With out Wnt signaling the glycogen synthase kinase 3 beta GSK3beta will phosphorylate several key proliferation promoting proteins, like beta catenin marking them for death. Wnt will stop this phosphorylation thus the proteins will not be destroyed (1) Canonical Wnt signaling, control of beta catenin by Wnt receptors vi) Non canonical Wnt signaling. Controls various cancer cell phenotypes: motility, invasiveness, and maintenance of self renewal potential. (a) Depends on non canonical Frizzled to activate G proteins that work as on off switches on w/ GTP bound and off with GDP bound (b) Ligand binds, G protein release GDP by alpha unit and binds GTP instead. Alpha and beta + gamma units go into cell and interact w/ effector proteins- a protein that transmit signals further into the cell interior. (c) Alpha’s effector is phosphodiesterase PDE and beta + gamma effector is phospholipase C beta PLC beta (i) Alpha subunit turns self off by hydrolyzing GTP making GDP, it then joins beta and gamma again. (2) Some GPCRs called chemokine –R are important for recruiting cells into the tumor associated stroma 8) Nuclear hormone receptors sense the presence of low molecular weight lipothilic ligands (1) Small hydrophopic ligands that can pass the PM (2) Will directly bind to nuclear DNA binding proteins (3) Common receptors are the estrogen, proestrone and androgen receptors 1. Important in breast ovarian and prostate carcinomas (4) Anatomy of DNA binding protiens (a) Three parts; DNA binding domain (i) Hinge region (ii) Conserved ligand binding domain (b) Some will stay in cytoplasm until bind to ligand and others are always bound to chromatin and just wait (i) Bind as homo or heterodimers to a pair of recognition sequences in the DNA in or near the promotors of genes whose expression they control 1. Hormone response elements HREs the recognition sequences in the DNA (ii) Example Estrogen receptor ER and 17 beta estradiol (estrogen) causes conformational shift, alpha helix near ER ligand binding pocket 1. This will then attract co activators and release co repressors 2. Work by modifying histone a. This modified DNA will either allow or deny RNA pol II from copying genes (iii)Selective estrogen receptor modulator SERM, tamoxifen. Cause ER to lose co activators and gain co repressors blocks signal of E2/ estrogen. (c) Allowing tamoxifen to block E2 signaling reduces breast cancer in post menopausal women 9) Integrin receptors sense association between the cell and the extracellular matrix. (a) Normally cells that have become disconnected to surrounding tissue and/or their anchorage point will apoptosis or programed cell death via anoikis (b) In a petri dish cells don’t anchor to the actual dish, they attach to a sort of ECM made of collagens, laminins, proteoglycans and fibronectins. This attaches to the actual dish (c) Sensing of collagen fibers and thus anchorage/ contact with other cells is done by discoidin domain receptors (d) Sensing of ECM is done by surface receptors called integrins hold cell together and signal different parts (2) After integrins bind their ligands they cluster forming focal adhisions, these will then (a) Integrins reduce the likelihood of anoikis by releaseing anti apoptotic signals (b) Cancer still uses integrins for both survival and proliferation thus it suggests that it is critical for tumor development (3) Weird things about integrins (i) signals that originate in the nucleus affect membrane proteins by controlling the binding affinities of integrins 1. causes already bound ligands to fuck off and new ones to join (ii) cancer can use this as a sort of walking method? Through rapid modulation of extracellular contracts allows the cell to free themselves from one microenvironment within a tissue and move into another (iii)some fibroblasts lack focal adhesion kinase FAK a signaling molecule, associated w/ cytoplasmic domains of integrins, are unable to remodel their focal contacts and lack motility (4) for a cell to begin to divide it must receive two things. One is the presence of mitogenic factors and the second is the existence of adequate anchoring sites (i) a ras oncogene is known to fulfil both of these conditions 1. is the ras oncoprotein able to mimic the signals of ligand activated GF and signals of integrin in the ECM components? 10) The ras protein, an apparent component of the downstream signaling cascade, functions as a G protein (a) Ppl noticed that if someone had a mutant Ras protein that it caused a bunch of the effects seen from erbB and sis oncoproteins, did this mean that ras sat at the head of a cascade effect of cytosolic cellular signaling? (i) Ras proteins are bound by a lipid C terminal tail to the interior of the PM 1. Like G proteins ras was found to be able to hydrolize GTP to GDP, thus ras’s mode of action is quite different from that of Src and erbB tyrosine kinases. But these three oncoproteins all act similarly in the cell (ii) Things ras can do 1. Bind GDP in an inactive state. 2. It ejects the GDP after receiving some stimulatory signal from upstream signaling cascade 3. Takes up a GTP after ejecting GDP 4. Is activated while GTP is bound 5. Turns GTP to GDP after a short while through GTPase activity. (iii)Chain of ras activation 1. Mitogenic signal acts on TKR, activates guanine nucleotide exchange factor GEF for ras. 2. Swap GDP for GTP 3. Ras emits signaling downstream at target(s) 4. Ras acts like GTPase and after a short period turns GTP to GDP a. GAP proteins can short cut this and influence ras to hydrolyze the GTP faster 5. Example of ras going wrong, (this is a point mutation) Harvey sarcoma virus, is able to bind GTP but isn’t able to turn GTP into GDP thus after being turned on once will stay on forever a. So instead of slowly and gradually releasing GF a cell with broken ras will continue to make GFs b. The point mutation leading to Hsv and human oncogene are missense mutations – amino acid substitution, and weren’t nonsense mutaitons cause premature termination of the growing protein chain st i. These mutations affect the 12, 13 or 61 codons of the reading from of the ras gene ii. b/c ras is a inducing gene it now made sense why a simple point mutation caused so much problems, a deletion in the ras gene would actually hurt the cancer cell cuz no more GF, but a altercation would greatly help it iii. also the mutations are specific to 12, 13 and 61 other mutations may do nothing or turn ras off permanently 11) Tumor Viruses Viruses exist to multiple inside a host. And do most of their damage multiplying Cytopathic- the cell killing effect of viruses, this with their ability to invade and infect other nearby hosts makes them very effective at proliferation Some viruses have a different game plan. They want to remain inside the host undetected for as long as possible and force the host cells to proliferate uncontrollably, these are sometimes called tumor viruses and can make cancers Research into these types of cancer didn’t show how cancers formed but how they formed, which genes/ proteins went wrong and why Most viruses are very simple, some genetic material RNA or DNA if RNA some reverse transcriptase, and a protein encasement Yet viruses still are able to infect cells with a vastly more complex genome and redirect cell growth. Apparently their genes must be pretty potent 1) Peyton rous discovers a chicken sarcoma virus a) 1908 copenhagen discover a filterable (means it is a virus cuz so small) agent from chicken leukemia cells. They transferred this agent to other birds and sure enough they developed cancerous cells b) 1909 Rous starts studying his chicken breast sarcoma . (1) 1 he transplanted some cells of the growth to another and it did begin to grow like it did on the 1 animal (2) Homogenized and filtered the cancerous cells then injected filtrate into young hens. Tumors began to grow sometimes as thrly as a few weeks (a) He repeated this to a 3 and 4 generation of hens and they all developed the same type of breast sarcoma ii) Noticed few things: filtrate meant that the agent must be very small (virus), this could lead to many advancements in cancer study as they now had an agent that could induce cancer on a strict time schedule iii) The sarcoma virus he found named Rouse sarcoma virus RSV iv) Soon a few other tumor viruses were discovered some that were also chicken sarcoma inducing (1) Some jumped the gun and went to say that all diseases were due to infectious agents and cancer is now one like cholera, tuberculosis, rabies (2) 1913 Dane Johannes Grib Fibiger shows a carcinoma that developed via a parasitic spiroptera worm v) Cancer are infectious diseases (1) Later due to the discovery that Fibiger’s rats didn’t have cancer but a metastatic stomach epithelia embarrassed the cancer community and ppl thought it is impossible for cancer to be infectious like a disease 2) Rous sarcoma virus is discovered to transform infected cells in culture i) Harry Rubin at California Institute of Tech in Pasadena, observed that chicken embryos infected with the RSV virus survived indefinitely (1) Unlike other viruses that- invade multiply and kill the RSV instead acted like a parasite. (a) It infected and caused cell to continually at a slow pace produce more viruses while maintaining cell growth and division. (b) They also displayed many cancer traits. Grew foci after infection (foci = clusters of cells_ (i) Cells in the foci were morphologically round (undifferentiated) and had a metabolism similar to that of cells taken from tumors. (c) Cell transformation from normal to tumor wasn’t limited to the complex environment of the body but was capable of being caused in a petri dish. (2) This lead to seeing cancer as a disease of malfunctioning cells not of abnormal tissue growth as it is on a cellular level ii) Robin and temin, experiment (1) How do cancer cells continue to proliferate while other cells can’t? (2) Normal cells on a agar stock, will continue to divide until they cover the bottom of the petri dish in a monolayer (a) these are called confluent cultures (b) contact inhibition- cells touching another on all sides will stop dividing (c) density inhibition- cells in a high density area will stop dividing (d) topoinhibition- another way cells can tell they are tightly packed and stop dividing (3) now how to transformants with RSV induced foci act in a petri dish (a) loss of contact and density inhibition. And thus continue to proliferate and pile higher making multi-layer clumps. These clumps all generate from one original cell that mutates due to the virus thus the clumps are made of clones (b) begin to see that tumors are monocolonial 3) the continued presence of RSV is needed to maintain transformation i) does RSV transform its child cells by continuing to affect them or is it a hit and run situation (event causes mutation permanently? (a) Robin and temin show that RSV genome is inside the daughter cells of the original effected cell (2) 1970s Berkley- used a RSV gene that made temp sensitive proteins good at 37C bad at 41C. (a) Temperature sensitive mutants will remain or become mutants when a set temp is held (b) Denaturation – the unfolding of a protein deactivating it (3) Allowed generations of the transformed cell to grow at 37C. then switch to 41C what happens? (a) The cells that were once showing symptoms of being cancerous turned back to normal, the daughter cells act as if they had never been transformed into a RSV infected cell (4) Viral transforming gene is needed both to initiate and maintain the transformed phenotype 4) Viruses containing DNA molecules are also able to induce cancer i) Richard Shope’s virus causes papillomas (warts) on the skin which were benign lesions that rarely mutated to full tumors ii) It used a clearly different mechanism to induce the transformed phenotype as Shope’s virus contained DNA while RSV ‘s genetic material is RNA (1) Also RSV had a lipid coating on its protein coating while Shope’s didn’t iii) DNA viruses related to Shope’s SV40 a simeon virus , and polyomavirus (1) Sv40 uses large virus filled vacuoles to store its shit before it decides to pop the cell host (2) Classified as a tumor virus cuz its lytic cycle (attacking phase) in rats hamsters and other mammals when it was able to infect would turn the cell into something very similar to RSV infected cells b) Papovavirus- class of DNA tumor viruses, the genes in these viruses are circular dsDNA i) Circular-ness helped researchers to search for it in homogenate vs other normal cell DNA c) Herpesvirus one and two, not tumorigenic i) Have super long DNA strands compared to others d) Poxvirus includes smallpox, is tumorigenic 5) Tumor viruses induce multiple changes in cell phynotype including acquisition of tumorigenicity (1) Cancer cells – lack contact inhibition, will proliferate withought growth stimulating factors. And undergo immortalization (2) Anchorage independence- a trait that means the cell does not require attachment to a anchor point to grow (a) Normal cells require a tethering point to solid and are anchorage dependent ii) Testing for cancer the early days (1) Inject mouse cells that r transformed in an invitro transformation into host mice of same strain (a) If the immune system of the syngenic host mouse could not tell the transformed cells were foreign thus they grew into tumors (i) Tumor rejection- the process a tissue will use to try to eliminate the foreign cells iii) How to test tumor virus in a host animal that has ability to reject the tumor cells? (1) Use of immunocompromised host – can’t reject the tumor cells (a) Nude mice, no hair for mites to live in, no immunesystem (2) The tumor cells are usually injected subcutaneously or under the skin b) How did just a few genes of RNA genome cause so much mutation? Pleiotropically the genes must be acting in multiple places 6) Tumor virus genomes persist in virus transformed cells by becoming part of host cell DNA (1) Berkley studies showed that RSV genome is required to become tumorious, but how did the daughter cells get it too? (a) The gene material is passing from mother to daughter cells, if not then cell returns to normal function (2) DNA viruses like polyomavirus and SV 40 are required to remain in a cell for it to be phenotypically mutant ii) Discovered that SV40 inserts self into host DNA to be replicated along with host, not form an extra chromosome to duplicate. Sv40 integrates its viral genome into host cell chromosome (1) This guaranties the presence of viral genome in daughter cell iii) Only the oncogenic portion of the viruses genome is inserted into its host’s DNA, the remaining genetic material sits in small fragments in the cell. (1) This portion outside the chromosome is the information that codes for replication and construction of the virus particles (virons) (2) Oncogenes are genes that contribute to cell transformation, proto-oncogenes are genes that if they mutate will become oncogenic iv) Two strategies of maintaining viral infection (1) Integrate chromosome into telomeric repeats, it remains here hidden slowly stopping active replication and then after a delay will excise themselves and launch active replication (2) Two; DNA viruses maintain their genome as episomes (unintegrated gentic elements) over many cell generations. These genomes latch themselves on to chromatin during mitosis so they enter a new cell equally. 7) Retroviral genomes become integrated into chromosomes of infected cells (1) Temin in late 1960s wondered how RSV got into daughter cells, he concluded the viral RNA molecule contained a enzyme/ protein that could turn the viral provirus (viral genome, for RSV in RNA) into DNA which would then integrate into the host genome (2) Reverse transcription – ppl thought Temin was crazy until 1970 when he and other scientific groups confirmed his hypothesis (3) RSV is a retrovirus- their cycle of replication involves going “backwards” to DNA from RNA, so like SV40 and polyomavirus (DNA viruses), RNA viruses also integrated their DNA in host (4) Butt, distinction in mechanism of integration (a) Retroviruses contain gene specifying an integrase- an enzyme made from viral polymerase gene, it integrates its entire genome into the host’s DNA, (b) DNA tumor viruses only integrate portions of their DNA 8) A version of the src gene carried by RSV is also present in uninfected cells (1) Genomes of viruses are very small, long enough for ten genes, yet they do some many things (2) Discovered that RSV’s reverse transcriptase and integrase is coded by a single set of genes and its replication ability and construction of progeny virions is directed by a different set (a) Mutant RSV some times had the genome to replicate but their integration and DNA making genes were mutant. Also reverse, some were able to integrate into genome but couldn’t replicate cuz mutant genes ii) 3 retroviral genes in viral replication (1) Assembly of virus particles (2) Structural proteins (3) Reverse transcriptase RT enzyme and integrase enzyme iii) This third set of genes is all coded by one gene src gene (1) Experiment- Bishop and Varmus make a src gene probe, that would bind to src and follow it in replication (a) However results showed that the probe bonded to non RSV infected cells (b) Normal cells already have a src gnene! (i) Tests showed that it was 100% the chicken’s gene and not put there by another thing 9) RSV exploits a kidnapped cellular gene to transform cells (a) In one case c src is compatible with normal cell life (b) 2d cast v src is potent oncogene, what gives? (2) What if src wasn’t evolved in the virus ancestrial line and instead it was a host cells c src that was stolen (3) ALV – avian leucosis virus, also a retrovirus that causes tumors, but no src gene (a) ALV only becomes oncogenic when it inserts itself next to the c src gene in a chicken thus a hypothesis ii) Ancestrial ALV is infecting a host cell and accidently sips the src gene adding it to the viral genome (1) Now the virus doesn’t need to find src and insert to be oncogenic, in carries its own oncogene iii) C src is a proto oncogene this was revolutionary at the time, normal wild genes in organisms can be converted to oncogenes through slight modification b) Revolutions in cancer studies at the time i) If vsrc is able to mutate the cell as it does can other types of mutations/ damage to the csrc cause the same results? Is that how other forms of cancer are made? (1) The genetic code for making cancer is already inside us, it just needs to go ahead to kill us ii) Also a single vsrc is able to lead to mutagenesis and tumorigenesis, via shape, vetabolism and growth behavioral changes in a cell, (1) Src acts pleiotropically causing multiple different changes depending on where it acts (2) Only a small number of genes need to be changed for tumorgenesis iii) Are there more proto oncogenes hidden away in normal healthy genomes? 10) The vertebrate genome carries a large group of proto-oncogenes (1) After RSV other retroviruses were found in other animals, did each of these viruses have its own oncogene present in its genome? ii) MC29 myeloctyomatosis virus, induces a bone marrow malignancy in chicken (1) Contained v myc an oncogene that was like v src from the chickens own genomic c myc (2) This was likely also a ALV ancestor, thus retroviruses were concluded to be good at stealing genetic info (a) ALV keeps stealing genes until it steals a proto oncogene (3) Feline leukemia virus and the fes oncogene = makes feline sarcoma virus (4) Rat/mouse retrovirus contains H ras and Kras oncogenes (Harvey and Kristen sarcoma viruses). (5) >30 distinct vertebrate proto oncogenes were discovered during this time iii) Many oncogenes were present in different animals with slight varations, there is a chicken, mouse, and human version of the c myc proto oncogene (1) Soon it was clear that proto oncogenes are something that evolved a long time ago and were essential in nearly all vertibrates 11) Slowly transforming retrovirus
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