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Chapter 6 Cytoplasmic Signaling Circuitry Programs Many of the Trait of the Cancer Slide 1 Normal cells and cancerous cells utilize control circuitry that is almost identical Cancer cells make only minor modi cations of the control machinery operating inside cells Growth factorreceptor signals are processed and integrated in the cytoplasm and then are transmitted to the cell s nucleus where they provide critical input for central machinery that governs cell proliferation A single cell may express 20000 or more distinct proteins which take up approx 30 of its total volume These proteins must be able to talk to one another with great speci city and precision Consequently a signaling protein in a linear cascade must receive and transmit signals from individual upstream and downstream partners ignoring the thousands of other proteins inside the cell Slide 2 A protein component of a signaling circuit must solve two problems 0 Specificity how can it exchange signals only with the small subset of proteins that are its intended partners in the signaling circuit 0 How can this protein gain rapid almost instantaneous access to these signaling partners while operating in the presence of thousands of other proteins within the cell A signaling pathway reaches from the cell surface into the nucleus The growth and division that cells undertake following exposure to mitogens clearly represent complex regulatory programs involving the coordinated actions of hundreds even thousands of distinct cellular proteins Slide 3 Immediate early genestheir expression is rapidly increased within the rst half hour following growth factor or serum stimulation Induction of immediate early genes occurs independently of de novo protein synthesis as a result of altered protein structure con guration and subcellular localization These genes encode transcription factors eg MYC cytoskeletal proteins or growth factors Slide 4 Delayed early genes are expressed dependent upon immediate early transcription factors The expression of these genes is blocked by cycloheximide and requires de novo protein synthesis The rate of protein synthesis increases following serum simulation Cell motility increases and cytoskeletal alterations also occur after serum stimulation Slide 5 These diverse responses indicated that a variety of distinct biochemical signals radiate from ligandactivated growth factor receptors and that these signals impinge on a diverse array of cellular targets eg transcription protein synthesis cytoskeletal reorganization growth factor production The Ras protein stands in the middle of a complex signaling cascade Drosophila eye development the Sevene55 mutant encoded an EGFR homologue and failed to form the seventh cell of the ommatidia Son ofSeveness Sos encodes a Guanine nucleotide exchange factor GEF upstream activator of Ras Slide 6 Other proteins involved in Ras signaling were the adaptor proteins Shc and Grb2 which bind to the phosphorylated growth factor receptor ShcGrb2 form a physical bridge between the Tyr kinase receptor and 05 Tyr kinase receptor gt Shc gtGrb2 gt 05 gt Ras These signaling components represent an ancient mechanism that is conserved among phyla and in many cases they can be functionally interchanged Tyrosine phosphorylation controls the localization and thereby the actions of many cytoplasmic signaling proteins Lessons from Src Tyrosine phosphorylation of growth factor receptors affects the localization of proteins that speci cally bind to PhosphoTyrosine Slide 7 Src contains Src homology domains 1 2 and 3 SH1 SH2 SH3 o SH1 Catalytic domain with Tyrosine kinase activity SH2 acts as a receptor for PhosphoTyr and Cterminal 3 to 6 aa residues ligand SH3 binds to Prolinerich sequences in partner proteins 0 Proteinprotein interaction domain amino acid sequences rich in proline There are dozens of distinct SH2 domain each carried by a different protein and having an affinity for a speci c phospho pepUde Slide 8 Other SH2containing proteins carry catalytic sites that are different from the Tyrosine kinase activity Phospholipase C carries an SH2 domain as does Phosphatidylinositol3kinase PI3K The SHP1 phosphatase also contains an SH2 domain The catalytic domains of the various proteins and their SH2 groups function as independent structural modules The SH2 domains allow these proteins to become localized where they are tethered to their phosphorTyrpeptide substrate Slide 9 Autophosphorylation or Transphosphorylation of the PDGF and EGF receptors creates docking sites for numerous SH2containing adaptor proteins within their cytoplasmic domains image Slide 10 By becoming tethered to a surface receptor molecule SH2 containing proteins are poised to activate their respective cascades Certain SH2containing proteins activate negative feedback loops that shut down receptor signaling Receptorbound SH2 proteins interact with other membrane associated proteins or phospholipids to transmit signals to various downstream cascades Example RasGAP bound to a phosphorylated receptor is tethered in close proximity to membraneanchored Ras o Phosphatidylinositol 3kinase PI3K tethered near the plasma membrane is able to phosphorylate inositol lipids embedded in the membrane Many enzymatic reactions become possible or proceed at a higher rates when enzymes and their substrates are brought into close proximity through tethering to phosphorylated receptors SH3 domains Bind to Prolinerich sequence domains in partner proteins Prorich proteins bound to the Src SH3 domain are substrates for phosphorylation by the Src Tyr kinase Slide 11 Protein interaction domain as modular units of protein structure Modular motifs SH2 SH3 PH facilitate recruitment of various proteins to their binding partners The peptide sequence surrounding the PhosphorylatedAA contributes to the speci city of binding image Slide 12 Another specialized domain PTB recognizes PhosphoTyr however amino acids on the Nterminal side contribute to the speci city of binding The ability of signaling proteins to interact in highly speci c ways with target proteins ensures that signals are passed only to intended targets and not other proteins Src kinase mechanism of activation image Slide 13 SH2 groups explain how growth factor receptors activate Ras and acquire signaling speci city The Grb2 adaptor protein contains one SH2 domain that binds to the Tyrphosphorylated receptor and two SH3 domains that bind to the 05 GEF Sos induces membraneanchored Ras to release GDP and bind GTPcausing activation of RasGTP Limage Slide 14 A cascade of kinases forms one of three important signaling pathways downstream of Ras Growth factors bind to their cognate receptors and activate downstream signaling pathways Cancer cells exhibit deregulated growthproliferation as a result of an autocrine signaling loop or mutant constitutively active receptor The Ras oncoprotein evokes a number of distinct changes in cells transcriptional activation increased protein synthesis growthproliferation cytoskeletal changes Three majors downstream signaling cascades radiate from the Ras protein When Ras binds to GTP an quoteffector loopquot undergoes conformational changes and now interacts with downstream signaling moleculesRas quoteffectorsquot Rafpresent in the cytosol reocaizes to the plasma membrane after binding to RasGTP Slide 15 The Raf kinase is a SerineThreonine kinase that activates downstream components of the MitodenActivated Protein MAP kinase cascade Raf evokes most of the transformation phenotypes induced by the Ras oncoprotein including anchorageindependence and loss of contact inhibition The activated ERK kinases transocate into the nucleus and phosphoryate transcription factors to activate gene expression Slide 16 A second pathway downstream of Ras controls inosito lipids and the AktPKB kinase Certain phosphoipids contain an inosito group a watersoluble carbohydrate molecule nosito can be phosphoryated and cleaved away from a hydrophobic phosphoipid molecule The phosphoinositol IP3 can diffuse away from the membrane and function as a quotsecond messengerquot an intracellular hormone image Slide 17 Speci c kinases phosphorylate a particular hydroxyl of inositol Phosphatidylinositol 3kinase PI3K phosphorylates the 3 hydroxyl of membraneembedded phosphatidylinositol Thus PI3K attaches a phosphate to a phospholipid instead of a protein The product diacyl glycerol DAG can activate Protein Kinase C RasGTP enhances the activity of membranebound Pl3K P3K is activated by a diverse array of signaling agents including PDGF NGF insulinlike growth factors Interleukin3 and ECM attachment Limage Slide 18 Activation of PI3K by Ras Recruitment of proteins to the plasma membrane through interactions with phosphoinositol positions them to activate downstream signaling pathways Many cytosolic proteins that bind to P3K contain Pleckstrin Homology PH domains The SerThr kinase AKT contains a PH domain and is activated by PI3K PHcontaining proteins migrate to PP3 in the Plasma membrane in response to growth factorstimulation Activated AKT has three biological effects on the cell 1 Protection against apoptosis 2 Stimulating cell proliferation 3 Stimulating cell growth cell size image Slide 19 The PTEN phosphatase removes phosphate groups from PP3 and counters the activity of PI3K AKT can become activated in cancer cells through hyperactivation of PI3K or inactivation of PTEN Constitutive activation of AKT results in increased cell growthsize in pancreatic islet cells image Other PHcontaining Guanine Nucleotide Exchange Factors GEFs Rho and Rac or Cdc42 act in a manner similar to Sos Cdc42 reorganizes the actin cytoskeleton in the cell and controls Filopodia Rac involved in the formation of Lameipodiabroad ruf es at the leading edge of the plasma membrane in motile or invasive cells Slide 20 A third Rasregulated pathway acts through Ral a distant cousin of Ras RalA and RalB share 58 sequence identity with Ras image Communication between Ras and Ral is mediated by RalGEFs which stimulate Ral to release GDP and bind GTP and become activated Ras causes RalGEFs to localize to the plasma membrane and conformational changes activate RalGEF activity Ral can activate the Rho proteins Cdc42 and Rac Ral proteins are thought to contribute to motility in cancer cells which enables invasiveness and metastasis Slide 21 The JakSTAT pathway allows signals to be transmitted from the plasma membrane directly to the nucleus lanus kinases phosphorylates Tyr residues within the cytokine receptor Signal transducers and activators of transcription STATS transcription factors which contain an SH2 domain form dimers upon Tyrphosphorylation Limage Slide 22 Cell adhesion receptors emit signals that converge with those released by growth factor receptors lntegrins serve three functions 0 1 Physically linking cells to the ECM o 2 Informing ces whether or not tethering to certain ECM components has been achieved 0 3 Facilitating motility by making and breaking contacts with the ECM Focal adhesions and Focal Adhesion Kinase FAK prevent cell death but also can activate Ras signaling image Slide 23 image be able to draw Chapter 7 Tumor Suppressor Genes Slide 1 Inactivation of Negative Growth Regulators During Neoplastic Transformation The Tumor SUppressors Increased cellular proliferation confers an appreciable risk for the generation of mutations in cancerrelated genes through a combination of errors in DNA replication and repair and abnormal chromosome segregation The expansion of one or more clonal populations under host selective pressure leads to the development of a fully malignant phenotype Factors that stimulate cellular proliferation hormones growth factors mitogens certain viruses are typically clinical risk factors for cancer development Characteristics of Tumor Suppressor Genes 1 Homozygous loss or inactivation in a signi cant proportion of human tumors or tumor types 2 Demonstrated function related to negative regulation of cell growth survival or differentiation 3 Ability to suppress tumorigenicity in cells that do not have normal alleles of candidate gene Slide 2 image Mutations in several tumor suppressor genes segregate with disease in hereditary cancer syndromes One of the hallmarks of tumorsuppressor gene inactivation is mutation of one allele accompanied by deletion of the other aee Such regions are suspected to harbor one or more important tumor suppressor genes The ability to suppress tumordenicitv when reintroduced into cells that have lost normal gene function is the ultimate test for tumor suppressor activity Slide 3 Genes that contain or suppress cell proliferation are called tumor suppressor genes These genes are frequently lost or inactivated during tumor formation The loss of tumor suppressor functions was determined to be as important and in some cases more important as the activation of protooncogenesoncogenes Cell fusion experiments indicate that the cancer phenotype is recessive Meaningful comparisons to two alternative alleles and speci ed phenotypes can occur only when both alleles are forced to coexist with the same cell or organism The process of cell fusion was used to determine whether the transformed cancer phenotype was dominant or recessive compared to the normal cell Cell fusion results in the formation of a syncytiuma single cell or giant cell with multiple nuclei If the cells are of different origins then the resulting hybrid cell is termed a heterokaryon Slide 4 Heterokaryon formation between a cancer cell and a normal cell puts the mutant cancercausing genes in confrontation with genes involved in normal regulation of cell growth The resulting tetraploid cells or subsequent subtetraploid cells were discovered to have lost the ability to form tumors when these hybrid cells were injected into appropriate host animals This meant that the malignant cell phenotype was recessive to the phenotype of normal wildtype growth However when the parental cancer cell had been transformed by a tumor virusthe cancer phenotype was dominant in the hybrid tetraploid cells Therefore if the cancer cell had originally arisen without the involvement of a tumor virus then its malignant phenotype was recessive when this cell was fused with a normal cell The recessive nature of the cancer cell phenotype requires a genetic explanation Slide 5 Normal cells carry genes that constrain or suppress their proliferation During the development of a tumor the evolving cancer cells shed or inactivate one or more of these genes Since the wildtype versions of these hypothetical genes antagonize the cancer cell phenotype these genes came to be called tumor suppressor genes TSGs It is far easier to inactivate a gene by a variety of mutational mechanisms than it is to hyperactivate its functioning through a mutation It seemed highly unlikely that tumor suppressor genes could be fully inactivated during the time required for tumors to form If the probability of inactivating a single gene copy by mutation is on the order of 104 per cell generation the probability of silencing both copies is on the order of 103912 per cell generation exceedingly unlikely The retinoblastoma tumor provides a solution to the genetic puzzle of tumor suppressor genes image Slide 6 Retinoblastomaa rare childhood tumor of the retina arising in the photoreceptor cells is normally observed in about 1 in 20000 children The disease is diagnosed from birth up to 68 years The Tumor Syndrome appears in two forms 1 Sporadic formoccurs in children born into families with no history of retinoblastoma The disease affects a single eye unilateral and is cured by radiation or removal of the cancerous eye 2 Familial formappears in children having a parent who also suffered from and was cured of the disease early in life There are usually multiple foci of tumors affecting both eyes bilateral Treatment of disease does not reduce the individual s chances of developing bone cancers osteosarcomas or other types of cancer later in life The familial form of retinoblastoma is passed from one generation to the next in a fashion that conforms to the behavior of a Mendelian dominant allele image Slide 7 Alfred Knudsen concluded in 1971 that the rate of appearance of familial tumors was consistent with a single random event while the sporadic tumors behaved as if two random even were required for their formation The gene that becomes mutated in retinoblastoma is called Rb Mutations of Rb create inactive recessive alleles of the Rb gene If the tumorpredisposing alleles of Rb are recessive then both copies of the Rb gene must be knocked out before a retinal cell launches the uncontrolled proliferation that results in a retinal tumor During sporadic disease children who possess two wildtype normal copies of the Rb gene must acquire somatic inactivating mutations in the two previously functional copied of the Rb gene Children born into Rb families contain one inactive mutant copy of Rb in all of their cells and therefore these retinal cells need only to acquire a single mutation to launch forth to create a retinoblastoma Thus bilateral familial retinoblastoma exhibits onehit kinetics of occurrence image Slide 8 Incipient cancer cells invent ways to eliminate wildtype copies of tumor suppressor genes How could two copies of a tumor suppressor gene possibly be eliminated one after the other during the formation of sporadic retinoblastomas if the probability of both required mutational events occurring is about 103912 per cell generation A cell that acquires a single mutation of Rb is said to be heterozygous Genetic exchange can occur between the remaining wildtype Rb allele and the mutant inactive allelesuch recombination is termed mitotic recombination This genetic exchange results in the elimination of both wildtype alleles Rb associated with sporadic retinoblastoma Mitotic recombination results in a Loss of Heterozvqositv LOH and occurs with a frequency of 10395 to 10394 per cell generation image Slide 9 Mitotic recombination results in a Loss of Heterozygosity LOH and occurs with a frequency of 10395 to 10394 per cell generation image Nearby neighboring genes of Rb on chromosome 13 also under LOH as a result of mitotic recombination Gene conversionoccurs through template switching during DNA replication by DNA polymerase The remaining wildtype copy Rb is typically inactivated through LOH Slide 10 image Slide 11 image Slide 12 Limage Slide 13 Allelic deletions can indicate the chromosomal positions of tumor suppressor genes associated with LOH in human cancers image Slide 14 Tumor suppressor genes function to directly control the biology of cells by affecting how they proliferate differentiate or die They are referred to as gatekeepers as they either allow or disallow cells to progress through cycles of growth and division DNA maintenance genes control the rate at which cells accumulate mutations These genes are termed caretakers to re ect their role in the maintenance of the cellular genome Promoter methylation represents an important mechanism for inactivating tumor suppressor genes DNA molecules can be altered by the attachment of methyl groups to cytosine bases This modi cation of genomic DNA is as important as mutation in shutting down tumor suppressor genes In mammalian cells this methylation is found only when these bases are located in a position that is 5 to guanosines in the sequence CpG MeCpGL DNA methylation is an epigenetic mechanism nongenetic for controlling gene expression image Slide 15 As the development of a tumor proceeds the overall level of methylation throughout the genome is often found to progressively decrease quotGlobal hypomethylationquot is correlated with chromosomal instability There are localized regions of DNAregions with a high density of Cst called quotCpG islandsquot that become methylated inappropriately in the genomes of cancer cells These CpG islands are usually affiliated with the promoters of genes which means that the de novo methylases are actually silencing certain genes that should remain transcriptionally active eg tumor suppressor genes More than half of the tumor suppressor genes Rb that are involved in familial cancer syndromes because of germline mutation have been found to be silenced in sporadic cancers by promoter methylation image Slide 16 Within a given tumor multiple genes seem to be shut down by promoter methylation Both copies of a tumor suppressor gene might be methylated independently of one another Alternatively one copy might be methylated and the second might then be lost through a loss of heterozygosity LOH image Methylation of a p16lnk in a lowgrade squamous intraepithelial lesion left compared to a highgrade malignant cervical carcinoma right Slide 17 Tumor suobressor genes and proteins function in diverse wavs Tumor suppressor genes and their encoded proteins act through diverse mechanisms to block the development of cancer Some of them function to directly suppress the proliferation of cells in response to a variety of growthinhibitory and differentiationinducing signals eg Rb Others are components of the cellular control circuitry that inhibits proliferation in response to metabolic imbalances and genomic damage eg p53 The NF1 protein acts as a negative regulator of Ras sidnalind Neuro bromatosis NF was rst described by Friedrich von Recklinghausen in 1862 NF is a relatively common familial cancer syndrome with 1 in 3500 individuals affected on average worldwide The primary feature of this disease is the development of benign tumors of the sheath cells around nerves in the peripheral nervous system A subset of neuro bromas progress to malignant neuro brosarcomas Siblings inheriting the same mutant allele of the responsible gene NF1 often exhibit dramatically different disease phenotypes image Slide 18 NFs are composed of a mixture of celltypes Schwann cells neurons perineural cells broblasts and mast cells In benign tumors Schwann cells are the prevailing cell type and are the primary targets of LOH in neurofibromas and exhibit increased paracrine signaling following the complete loss of NF1 function Neurofibromin the protein product of NF1 is a RasGAP Inactivated alleles of the NF1 gene transmitted through the germ line act in a dominant fashion to create disease phenotypes The originally heterozygous con guration of the gene NF139 is converted to a homozygous state NF139 in tumor cells through loss of heterozygosity Neuro bromin is a Ras GTPaseactivating protein GAP which inactivates Rassignaling image Slide 19 Haploinsufficiency of Bone MarrowDerived Mast Cells Contributes to the Abnormal Development of Nervous Tissue in Neuro bromas image Slide 20 NF1 is expressed widely throughout the body with high levels found in the adult peripheral and central nervous systems Upon growth factor stimulation NF1 is degraded which enables Ras signaling to proceed without interference by NF1 Within 6090 min following stimulation NF1 eves return to normal and the NF1 protein that accumulates helps to shut down further Ras signaling a form of negativefeedback control In cells of neuro bromas which are genetically NF139 elevated levels of activated Ras and Ras effector proteins can be found Also a halfnormal dose of NF1 protein can affect ceuar phenotype by increase Rassignaling resulting from a partial loss of NF1 function While the full phenotypic changes of tumor suppressor gene inactivation are felt only when both gene copies are lost halfdosage of their encoded proteins yields subtle but still very real changes in cell behavior haploinsufficiency Apc facilitates egress leaving of cells from colonic crypts The great majority of colon cancers appear to be sporadic gt95 however a small group arise as a consequence of inherited alleles that create substantial lifelong risk for this disease Familial adenomatous polyposis FAP or adenomatous polyposis coli APC an inherited susceptibility to develop adenomatous polyps in the colon These polyps are nonmalignant but are prone to develop into frank invasive carcinomas Slide 21 image In Western populations polyps are often found in low numbers scattered throughout the colon ln FAP patients polyps numbering in the hundreds are found to carpet the luminal surface of the colon The epithelia in the colon ad duodenum are organized to maintain a constant population of stem cells Undifferentiated stem cells yield two distinct daughter cells one remains a stem cell while the other cell and its descendants become committed to differentiate enterocytes These stem cells are protected through their location at the bottom of deep cavities or crypts While some stem cells remain at the bottom of the crypts most migrate upward and out of crypts toward the luminal surface of the epithelium where they brie y function to form the epithelial lining of the gut die by apoptosis and be she into the colonic lumen This process of outmigration takes only 34 days Slide 22 Epithelial differentiation and migration in the colon is regulated by betacateninchLef and APC tumor suppressor functions image Slide 23 The organization of colon epithelia represents a highly effective defense mechanism against the development of colon cancer since almost all cells that have sustained mutations while on duty protecting the colonic wall are doomed to die within days after they have been formed The only mutations that can lead to the development of cancer 1 Block both the outmigration of colonic epithelial cells from the crypts 2 And the cell death that follows soon thereafter Colonic enterocytes that acquire mutations may be retained and survive in a crypt and this cell and its descendants can accumulate there polyp formation APC the product of the adenomatous polyposis coli gene is responsible for negatively controlling the levels of betacatenin in the cytosol When Wntreceptor signaling occurs beta catenin is saved from destruction accumulates and migrates to the nucleus where it binds a group of DNAbinding proteins termed ch or Lef which activate proliferative genes and maintain a stem cell phenotVDe The cells at the bottom of crypts are held in a stem celllike state by the high levels of betacatenin As these cells migrate upwards the level of APC increases which drives down the intracellular level of betacatenin associated with cellular differentiation When betacatenin accumulates in enterocyte precursors because of inactivation of APC function this causes them to retain a stemcell like phenotype which precludes them from migrating out of the crypts These cells can acquire additional mutations to become an adenomatous polyp or malignant carcinoma Slide 24 The Wnt receptor derives from the Drosophila mutations Wingless Wntsignaling activates the Disheveed proteinwhich inactivates the Glycogen Synthase Kinase3Beta GSKBBeta Betacatenin is released from APCGSK 3Beta complexes and promotes cellular proliferation image Slide 25 Cells lacking APC also exhibit a marked increase in chromosomal instability associated with tetraploid karyotypes APC molecules localize to mitotic spindle bers which are responsible for segregating chromosomes during anaphase and telophase of mitosis The acquisition of mutations may facilitate tumorigenesis by accelerating the rate with which premalignant cells acquire advantageous genotypes and phenotypes image Von HippelLindau disease pVHL modulates the hypoxic response Von HippelLindau VHL syndrome is a hereditary predisposition to the development of a variety of tumors clearcell carcinomas of the kidney pheocheomocytomas and hemangioblastomas of the nervous system Mutant VHL alleles result in an incidence of disease of about 1 in 35000 in the general population Slide 26 image Slide 27 The VHL locus undergoes a loss of heterozygosity LOH that results in a VHL 39 genotype in the tumor cells of patients inheriting a mutant germline VHL allele The main task of pVHL the product of the VHL gene is to foster destruction of a subunit of a critical transcription factor termed Hypoxiainducible Factor1 HlFl Under normal oxygen tensions normoxia cellular pVHL provokes the degradation of the HlF1alpha subunit of HlF1 This cycle is interrupted when cells experience hypoxia subnormal oxygen tensions under which HlF1alpha degradation falls to occur and HlF1 levels increase within minutes Ubiquitylation tags cellular proteins for destruction in proteasomes image Slide 28 know every part of this image Slide 29 HlF1 causes expression of target genes whose products are involved in angiogenesis erythropoiesis energy metabolism and glucose transport into cells These products enable a cell to survive under hypoxic conditions subnormal oxygen tensions in the short term and in the longer term to acquire access to an adequate supply of oxygen eg generation of new blood vessels within hypoxic areas of tissues Uncontrolled vascularization in the retina during VHL syndrome left Hemangioblastoma right pVHL binds to HlF1alpha only when either of two critical proline residues of HlF1alpha has been oxidized to hydroxyproline this binding to HlF1alpha fails and HlF1alpha escapes degradation The conversion of the HlF1alpha proline residues into hydroxyprolines is catalyzed by an enzyme that depends on oxygen for its activity image Slide 30 Under conditions of normal oxygen tension normoxia HlF1alpha displays one or two hydroxyprolines is recognized by pVHL and is rapidly destroyed Under hypoxic conditions HlF1alpha lacks these hydroxyprolines cannot be bound by pVHL and accumulates to high levels that support transactivation by HlF1 image Expression of the VEGF target of HIF1 in an in situ localized breast cancen Chapter 8 pr and Control of the Cell Cycle Clock Slide 1 Cellular growth and proliferation quiescence and differentiation are regulated by external signals Almost all types of normal cells will not proliferate unless prompted to do so by mitogenic growth factors Extracellular signal may persuade a cell to enter into a postmitotic differentiated state from which it will never reemerge and resume proliferation A cell s growth regulatory signals are processed by a centralized governor that controls ceuar growthproliferation quiescence or differentiation the CellCycle Clock Positive and negative regulators of cellular growth and division cycles Slide 2 image Slide 3 Oncogenes deregulate cellular growthcontrol signal and converge upon the cellcycle Mutation of negative regulators of the cellcycle eg tumor suppressor genes cause uncontrolled ceuar growthproliferation in cancerceHs Tuberous sclerosis TSC1 is a tumor suppressor that regulates cell growth and proliferation image A cell that has recently been formed by the processes of cell division mitosis and cytokinesis must decide whether it will once again initiate a new round of active growth and division or retreat into a non growing state termed GO This decision is strongly in uenced by mitogenic growth factors in the cell s surroundings CMYC oncogene turns on p53 Tlymphocytes most of these cells aren t dividing need to stimulate them to divide Withdrawal from the cellcycle may be encouraged by the presence of growth inhibitory signals eg TGFbeta Withdrawal from the cellcycle into a quiescent GO state is often reversible Other cells may enter an irreversible postmitotic terminally differentiated state eg neurons in the brain In order for a cell to divide it must first double its macromolecular constituents proteins DNA content 2N gt 4N Cell growth the accumulation of cellular constituents resulting in an increase in cell size Cell division yields two daughter cells from a single mother cell via mitosis and cytokinesis Cell proliferationthe end result of cellular growth and division The cell s genome is duplication through DNA replication during Synthesis Siphase 2N gt 4N often requires 68 hr Sphase is shorter for embryonic cells and activated lymphocytes The rst Gap phase 61 following mitosis and cytokinesis may last from 1215 hr the decision period prior to DNA replication Slide 5 Metaphase alignment of the chromosome Mitosis the process of chromosomal alignment and segregation resulting in the equal distribution of diploid 2N DNA content to two daughter cells last approx 1 hr The duplicated DNA 4N is carried on sister chromatids of the chromosome image Slide 6 The passage of diploid DNA to each daughter cell depends upon 0 1 The faithful replication of a cell s genome during Sphase o 2 The proper allocation of the resulting duplicated DNAs to daughter cells during M phase Checkpoints and checkpoint controls Cancer cells have aneuploidy The cell deploys a series of surveillance mechanisms that monitor each step in cell cycle progression and permit the cell to proceed to the next step in the cycle only if a prerequisite step has been completed successfully If speci c steps in the execution of a process go awry these monitors rapidly call a halt to further advance through the cell cycle until these problems have been successfully addressed Other monitors ensure that once a particular step of the cell cycle has been completed it is not repeated until the cell passes through the next cycle GlZS checkpoint ensures that a cell cannot enter Sphase if the genome is in need of repair i checkpoint halts DNA replication if genome is damaged GZZM checkpoint prevents entry into Mphase if DNA replication is not completed amp checkpoint causes arrest in Metaphase and prevents Anaphase looks for proper chromosomal alignment if chromosomes are not properly aligned or mitotic spindle bers are not correctly attached cancer cells are hypermutile Cancer cells continue to acquire mutations that will confer the greatest proliferative advantage during tumorigenesis An increased mutability of their genomes accelerates the rate at which these cells can acquire advantageous combinations of alleles and thus hastens the overall pace of tumor progression Such mutability and resulting genomic instability is incompatible with normal cell cycle progression since checkpoint controls block the advance to the next cycle if DNA is damaged or if chromosomes are in disarray Many cancer cells have inactivated one or more of their checkpoint controls and can more rapidly accumulate the mutant genes and altered karyotypes that propel their neoplastic growth Slide 9 Cells make decisions about growth and quiescence during a speci c period in the 61 phase Cells communicate with their surroundings and make critical decisions during the early phases of 61 whether to remain in 61 retreat from the active cycle into GO or advance into late 61 and the remaining stages of the cellcycle image Restriction point R point the transition point at which decisions regarding cellcycle progression or growtharrest must be made Normal and cancerous cells proceed through late 61 S 62 and M phases at the same relative rates Slide 10 Cyclins and cyclindependent kinases constitute the core components of the cell cycle clock Progression through various stages of the cellcycle is regulated by phosphorylation Examples Phosphorylation of centrosome associated proteins at the 615 boundary allows their duplication in preparation for Mphase Phosphorylation of other proteins prior to S phase enables DNA replication sites along the chromosome to be activated Phosphorylation of histone proteins place chromatin in appropriate con gurations for S and M phases Phosphorylation of proteins forming the nuclear membrane lamin nucleoporins causes their dissociation in early Mphase Cvclinscvclindependent kinases cvclincdks image Slide 11 How do we make sure cell cycle goes forward and not backwards Cyclindependent kinases cdks are SerineThreonine kinases The activity of cdks is regulated by the levels and availability of their cyclin partners Cyclins are regulated by their rapid degradation by the proteasome dependent upon ubiquitinylation by ubiquitin ligases This ensures that cells that have exited one stage of the cellcycle cannot slip backwards this is why you must degrade image Slide 12 Mitogenic signals stimulate production of cyclin D1 and activate cyclin Dcdk4cdk6 image Slide 13 CyclinCDK complexes are also regulated by CDK inhibitors INK4 proteins p16lnk4a p15nk4B p184C p19nk4D speci cally inhibit cdk4 and cdk 6 P21WafCip1 and p27Kip1 inhibit all other cyclincdk complexes image Slide 14 TGFbeta receptor signaling induces p15nk4B and weakly induces p21WafCip1 to negatively regulate cellcycle progression image Slide 15 Pl3KAKT kinases phosphorylate p21WafCip1 and p27Kip1 to cause their cytoplasmic localization and prevents their import into the nucleus image Slide 16 image Slide 17 p27Kip1 levels maintain a postmitotic arrested state in cerebellar granule cells image Slide 18 Cellcycle dependent phosphorylation of pr image Slide 19 E2F transcription factors enable pr to implement growthversus quiescence decisions image Slide 20 image Slide 21 image p53 and Apoptosis Master Guardian and Executioner Slide 1 TEST 1 QUESTIONS 1 a What is cancer epidemiology The study of cancer incidence and mortality Risk factors associated with incidence and mortality b Which type of cancers has the highest incidence worldwide Reproductive Cancers c Which type of cancer has the highest incidence in the US male population Prostate cancer 2 a Which types of malignancy represents an exception to the correlation fo cancer development with advanced aging Neuroectodermal Malignancies ALL Acute Lymphocytic Leukemia b Why do certain cancers such as pancreatic esophageal and lung carcinomas typically have poor 5year survival rates They are invasive cancers that metastasize c Which type of cancer has the highest incidence in the US female population Breast cancer 3 a Brie y explain what is meant by the phrase quotcancer is a disease of deregulated gene expression and somatic evolutionquot Cancers adapt and gain proliferative advantage b What is an allele A chromosomal gene c What is incomplete dominance with reference to gene expression Blending of phenotypical expression Pink petals red white show incomplete dominance 5 a What type of chromosomal structural change results in the production of homogeneously staining regions HSRs Gene amli cation b Brie y explain why cancer is considered to be a clonal disease Originates from one cell that proliferates with deregulated gene c What are the two major components of a gene Promoter region Coding region 6 a What is histopathology Study of normal cells and tumor cells to determine tissue of origin b How can cancer cells within secondary metastases be traced back to their primary tissue of origin Tissue Characterization c What are benign tumors Benign tumors are nonmetastasizing and noninvasive tumors a What type of tumors are sarcomas Tumors of connective tissues b Describe the major differences between hyperplastic and dysplastic tissues Hyperplastic tissues show increased numbers of normal cells whereas dysplastic tissues have abnormal cells c What is metaplasia Displacement of cells to places they don t belong Abnormalities in the location of cells a How does foci formation represent deregulated growth in oncogenicallytransformed cells Demonstrates that cells have lost contact inhibition b Brie y describe the experiment that demonstrated Rous Sarcoma Virus RSV is needed for oncogenic transformation as opposed to a 39hitandrun mechanism A temperature sensitive mutant of RSV was developed ln nonpermissive temperatures infected cells appeared normal c What are papilloma viruses eg HPV What types of premalignant lesions do they cause Circular dsDNA viruses Cause wartspolyps a What types of malignant diseases are caused by Epstein Barr Virus EBV Burkitt s Lymphoma BL b Why is anchorageindependence generally considered to be an indicator of the tumorigenic potential of transformed cells Demonstrates that cells are no longer dependent on ECM attachment for cell proliferation indicative of transformation Cells acquired motility c Why is it necessary to use immunocompromised mice such as the athymic nude or NODscid strains in xenograft studies of human cancers Immune system won t reject human tumors 11 a What is spectral karyotyping SKY and how is it used to detect karyotypic alterations in cancer cells Chromosomal pointing b What is the major difference between adenocarcinomas and squamous cell carcinomas Squamous cell carcinomas are carcinoma of protective epithelial cells Adenocarcinomas are secretory epithelial cells c Brie y describe how the oncogenic Rous Sarcoma Virus RSV was created from another slow transforming virus Avian Leukosis Virus ALV In the chicken tissue the ALV incorporated the src gene in its genome after integration 12 a Why was it initially believed that human cancers may have been caused by endogenous retroviruses Because ERVs are present in the genome it was speculated that their expression was latent and passed down the germ line until some factor activated the virus leading to cell transformation b Brie y describe how the transfection of DNA from chemically transformed mouse broblasts led to the identi cation of nonviral oncogenes DNA was removed from mice broblasts and cultured Cultured cells showed signs of transformation leading to the discovery that oncogenes can be nonviral c What parameters are indicated by a KaplanMeier Plot Time since diagnosis months vs survival rates 13 a What is a protooncogene A protooncogene is a gene in the host cell that through gene capture can be transcribed in conjunction with a provirus leading to cell transformation b What type of genetic mutation results in the conversion of the normal HaRas protein to an oncogenic protein Point mutation in DNA leads to substitution of valine for the original glycine amino acid c How does NMyc gene expression correlate with the clinical outcome of pediatric neuroblastomas In copies of 10 or more clinical outcome decreases signi cantly with Nmyc expression 15 a How do mutations that delete the ectodomain ligand binding domain of a growth factor receptor affect its activation Truncation of the ectodomain causes constitutive activation always on b What is autocrine and paracrine signaling Autocrine signaling describes a signaling loop in which the cell signals to receptors on its own cell surface Paracrine signaling describes signaling between two or more cells c List the three functional domains of a tyrosine kinase receptor such as the EGFReceptor Ectodomainligandbinding domain Transmembrane domain CterminusTyrosine Kinase domain 16 a Describe how the 39activation loop modulates the catalytic activity of tyrosine kinase receptors With phosphorylation the con guration of Tk exposes catalytic cleft b How do cytokine receptors such as the TNFalpha receptor transmit their signals to other downstream target proteins Recruit tks for transphosphorylation of receptors which leads to phosphorylation of receptor tail and phosphorylation of target proteins c What is the major difference between Transforming Growth FactorBeta TGFbeta Receptors and the EGFReceptor The TGFbeta receptor is a serine threonine receptor and the EGF receptor is a tyrosine receptor 17 a How does the Notch Receptor mediate intracellular SgnaHng Notch receptor recognizes ligands on the surfaces of other cells and causes proteolytic cleavages that release c terminus of receptor to signal to nucleus b What are Serpentine receptors Protein receptors with alpha beta and gamma subunits 7 domain transmembrane receptors c Describe how the Ras protein becomes activated in response to growth factor receptor signaling Ras is activated by GEFs which catalyze GTP binding to Ras to activate it 18 a What are the two major problems that must be solved by any intracellular signaling circuit Speci city and precision b What are Immediate early genes lEGs are present almost immediately after cellular exposure to mitogenic GFs suggesting that they require no do novo synthesis and act as TFs c Why does the activation of Delayed early genes require de novo protein synthesis Delayed early genes require the presence of TFs that activate transcription and therefore must be transcribed and translated de novo upon exposure to GFs TEST QUESTIONS 6 a Describe how cell fusion experiments demonstrated that the transformed cancer phenotype is recessive By fusing the two genotypes and treating with antibodies speci c to 2 different genotypes they are able to select for heterozygous fused cells these fused cells didn t show transformation so the phenotype must be recessive b Explain why the cancer phenotype was dominant in fusion experiments if the cell had been transformed by a tumor virus c List at least two characteristics of tumor suppressor genes Often absent or silenced in tumor cells Ability to halt cell growth and proliferation 7 a Why do patients with familial retinoblastomas exhibit an increased incidence of nonretinal tumors such as osteosarcomas b Explain why familial retinoblastoma associated with bilateral disease exhibits onehit mutational kinetics whereas the sporadic form of disease exhibits twohit mutational kinetics Sporadic formation is depending on two mutational events 110 whereas the bilateral expression only depended on one additional mutational event since the rst was inherited c Describe the process of gene conversion and how it can lead to Loss of Heterozygosity LOH 8 a Describe how epigenetic mechanisms can inactivate tumor suppressor genes in cancer cells Methylation of promoter sequences in TSGS can inactivate them b Describe the clinical features of neuro bromatosis and affected by this hereditary predisposition syndrome Caf du late spots deformation bone marrow c How do mutations of the Neuro bromin1 NF1 protein deregulate cellular signaling associated with increased cell growth and proliferation NF1 is a RasGAP it inactivates Ras Mutations in NF1 causes Ras to be constitutively active causing cell proliferation 9 a What is familial adenomatous polyposis coli FAP lnherited disease characterized by the formation of polyps in the colon can lead to cancer b How does the Adenomatous polyposis coli APC protein regulate Betacatenin protein levels in differentiated cells APC binds beta catenin and brings it in contact with GSK beta to be degraded c Describe the mechanism by which the Von HippelLindau protein pVHL regulates Hypoxia inducible factor1 alpha HIF 1apha expression under conditions of normoxia Under normoxia pVHL binds HlF1alpha and with the help of additional proteins tags it for uniquitylation 10 a How is cellproliferaiton de ned b What does the passage of diploid DNA to each daughter cell depend upon Fidelity in replication during 5 phase Correct allocation of genetic material during m phase c List the four checkpoints for growtharrest during the cell cycle End of 61 DNAdamage checkpoint S DNAdamage checkpoint I M lining of chromosomes in metaphase 11 a What is the Restriction R point of the cellcycle b Which phase of the cellcycle is also known as the decision phase where mitogenic signals exert their in uence 61 c What ensures that cells that have exited one stage on the cellcycle cannot slip backwards Feed forward loops of cyclin proteins that are degraded and synthesized in correspondence with the cycle and their speci c cdk 12 a Which cyclindependent kinases are speci cally inhibited by the INK4 class p1539 quot4b p1639 quot4a p1839 quot4c p1939 quot4d of cdkinhibitors CDK46 b Explain how Pl3KAKT signaling negatively regulates p21Wafl and p27Kipl functions Phosphorylation of p21 and p27 by p3KAKT removes p21 from the nucleus and keeps p27 in the cytoplasm so they can t inhibit cdk function c Explain how cyclin Dcdk46 and cyclin Ecdk2 regulate Retinoblastoma Rb functions to promote Sphase cellcycle entry Dcdk46 initially phosphorylates pr Ecdk2 completes phosphorylation of pr inactivating it 13 a How does the Retinoblastoma Rb protein regulate E2Fdpeendent transcriptional activity pr along with HDAC in its hypophosphorylated form binds 2F and prevents it from being transcriptionally active b How do MycMax transcription complexes affect cellular growth and proliferation Increase growth and proliferation c How do MadMax transcription complexes affect cellular growth and proliferation Decrease growth and proliferation 14 a What cellular functions are regulated by the p53 tumor suppressor Apoptosis DNA repair cell growth b Which type of mutation occurs in the p53 gene sequence in the majority of cancers with p53 inactivation Missense mutation c Explain how mutations of p53 can lead to a dominant negative phenotype that cooperates with oncogenic Ras in foci formation experiments p53 consists of 4 of the same subunit it exhibits a BM phenotype because one mutated unit can in uence the whole protein but since it is still present not null it must cooperate with Ras since foci are shown to form 15 a How does the Murine double minute2 Mdm2 protein regulate p53 functions in normal cells Mdm2 binds and inhibits p53 function via ubiquitylation b How does Pl3KAKT signaling regulate Mdm2 and p53 funcitons in actively proliferating cells c Describe the mechanism by which the p14ARF protein positively regulates p53 expression p14ARF essentially kidnaps Mdm2 so that p53 is able to escape degradation 16 a List at least two microscopic or morphological characteristics of apoptotic cells Release of cytc and other apoptotic proteins from the mitochondria b Name the genetic cancer predisposition syndrome associated with germline mutations in the p53 gene c Explain how the Bcl2 protein inhibits the induction of cellular apoptosis Bcl2 blocks the exit of cytc from the mitochondria 17 a What are the major differences between apoptosis and necrosis Apoptosis is more speci c and directed to one cell rather than a group of cells Necrosis is caused more by physical damage than at request of cellular signaling b What are caspases and how do they promote cellular apoptosis Caspases cause proteolytic cleavage and lead to a cascade of cleavages during apoptosis promoting cell death c How do cytotoxic Tlymphocytes CTLs induce apoptosis or targeted cellkilling in cancerous cells They use receptormediated cell death mechanisms 18 a What is cellular immortalization When cells surpass the regulations of cell aging and division b What three factors in uence the number of replicative doublings that cells in culture can go through before they reach senescence Species Speci c tissue Age c What two mechanisms govern the replicative capacity of cells growing in vitro and in vivo The determination of the physiological state of the cell damage hyperoxia etc Monitoring of the number of cell divisions 19 a What is cellular senescence Cell aging b What is cell crisis Karyotypic crisis Fusion of many chromosomal ends eventually leads to cell death in normal cells c Why does telomere erosion occur as cells replicate their DNA and proliferate 20 a Draw a diagram of how the BreakageFusionBridge cycle leads to karyotypic chaos in cells undergoing crisis b What are the two subunits of the Telomerase enzyme Reverse transcriptase Guide sequence RNA c How do cancer cells which lack Telomerase activity maintain their telomeric DNA associated with immortalization ALT using DNA repair enzymes and copyswitch mechanism to sequence telomeric DNA
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