3 Grading Class Attendance & Discussion (20%) Team Presentation (40%) Final Exam (40%)
4 Presentations 5-member team (self-assembled) Select one paper (Science, Nature, Cell) within the fields of cancer, stem cell or apoptosis, discuss extensively by the team, and prepare for a 20-minute presentation (10-15 slides)Address the following in your presentation:Scientific question the paper was trying to answer;Experimental approaches taken and main techniques used;Major findings and conclusions;Significance of the paper;Your ideas to further the research.
6 Cancer: The Bottom Line In animals, cell numbers are under exquisite controlLoss of this control leads to proliferation of unwanted cellsCancer arises when the above situation reaches a certain degree
7 Cancer vs TumorCancer cells are malignant tumor (vs. benign tumor) cellsAll cancer cells are eventually capable of metastasis
9 Transformed Cells Reduced cell adhesion Round-up morphology Reduced serum requirementsElevated hexose uptakeAnchorage-independent growth (ability to grow in semi-solid media,soft agarLoss of density-dependent growth inhibition (“Contact Inhibition”)Secretion of plasminogen activatorCapable of forming tumor in appropriete setting
10 Causes of Cancer Generalizations Spontaneous genetic mutationsInherited cancer susceptibilitiesCarcinogenic substancesVirusesRadiation
11 Genes, Genes, Genes, Genes, Genes All five of the above listed causes act through genesCancer is due to genetic changes in individual cells which then proliferate and pass on the mutations
12 Oncogenes & Protooncogenes same molecules Called oncogenes for their role in promoting neoplasiaCalled protooncogenes in the normal cellPlay diverse roles in control of cell growthProliferationDifferentiationApoptosis
13 Evidence supporting that cancer results from genetic changes Discussion
24 Development of cancer may require mutations in more than one gene Polygenic ChangesDevelopment of cancer may require mutations in more than one geneCooperativityMany cell-control functions are redundantMutations may affect different aspects of cellular growth control
25 Session SummaryCancer is a fundamental aberration in cellular behavior, touching on many aspects of molecular cell biology. Most cell types of the body can give rise to malignant tumor (cancer) cells.Cancer cells can multiply in the absence of growth-promoting factors required for proliferation of normal cells and are resistant to signals that normally program cell death (apoptosis).Cancer cells also invade surrounding tissues, often breaking through the basal laminas that define the boundaries of tissues and spreading through the body to establish secondary areas of growth, a process called metastasis. Metastatic tumors often secrete proteases, which degrade the surrounding extracellular matrix.
26 Session Summary continued Certain cultured cells transfected with tumor-cell DNA undergo transformation. Such transformed cells share many properties with tumor cells.The requirement for multiple mutations in cancer induction is consistent with the observed increase in the incidence of human cancers with advancing age. Most such mutations are somatic and are not carried in the germ-line DNA.Colon cancer develops through distinct morphological stages that commonly are associated with mutations in specific tumor-suppressor genes and oncogenes.
27 Increased cell proliferation Decreased cell death Cancer GeneticsIncreased cell proliferationDecreased cell death
34 Tumor Suppressor Genes Intracellular proteins, such as the p16 cyclin-kinase inhibitor, that regulate or inhibit progression through a specific stage of the cell cycleReceptors for secreted hormones (e.g., tumorderived growth factor b) that function to inhibit cell proliferationCheckpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormalProteins that promote apoptosisEnzymes that participate in DNA repair
38 Session SummaryDominant gain-of-function mutations in protooncogenes and recessive loss-of-function mutations in tumor-suppressor genes are oncogenic.Among the proteins encoded by proto-oncogenes are positive-acting growth factors and their receptors, signal-transduction proteins, transcription factors, and cell-cycle control proteinsAn activating mutation of one of the two alleles of a proto-oncogene converts it to an oncogene, which can induce transformation in cultured cells or cancer in animals.Activation of a proto-oncogene into an oncogene can occur by point mutation, gene amplification, and gene translocation.The first recognized oncogene, v-src, was identified in Rous sarcoma virus, a cancer-causing retrovirus. Retroviral oncogenes arose by transduction of cellular proto-oncogenes into the viral genome and subsequent mutation.The first human oncogene to be identified encodes a constitutively active form of Ras, a signal-transduction protein.
39 Session Summary continued Slow-acting retroviruses can cause cancer by integrating near a proto-oncogene in such a way that gene transcription is activated continuously and inappropriately.Tumor-suppressor genes encode proteins that slow or inhibit progression through a specific stage of the cell cycle, checkpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal, receptors for secreted hormones that function to inhibit cell proliferation, proteins that promote apoptosis, and DNA repair enzymes.Inherited mutations causing retinoblastoma led to the identification of RB, the first tumor-suppressor gene to be recognized.Inheritance of a single mutant allele of many tumor-suppressor genes (e.g., RB, APC, and BRCA1) increases to almost 100 percent the probability that a specific kind of tumor will develop.
40 More on Mechanism of Oncogenes The growth of a cell is controlled by molecules that impinge on its outer plasma membraneCytokinesLow molecular-weight hormonesGrowth factorsOther proteinsBut how do these external factors affect activities inside a cell and, perhaps, inside the nucleus??
42 Signal TransductionMost commonly, receptors in the plasma membrane bind to specific external factors and then interact with molecules on the inner surface of the membrane.Some external factors can migrate across the cell membrane and bind to ligands in the cytoplasm.
43 Signal Transduction frequently Cytoplasmic EffectsSignal Transduction frequently1. Activates an enzymatic protein e.g. protein kinaseOr2. Enhances nucleotide binding affinity, e.g. enhanced GTP binding
44 Features of Signal Transduction Reliance on multi-protein complexes of signaling moleculesGeneration of second messenger moleculesEventual transduction of the signal into the nucleus
45 The Destination The nucleus is the destination for signals. Those dependent on interaction of plasma membrane receptors with their ligandsandThose initiated by the molecules that migrate into & interact with their ligands within the cytoplasm
46 SignificanceRemember the properties of transformed cells include the reduced requirement for serum, i.e, reduced requirement for external factors .This is due to oncogene products that may replace the normal ligands or otherwise affect the molecular interactions leading to behavior modification
47 Dominant OncogenesTheir presence or activation cause cells to proliferateActing in the membrane (Growth factors & receptors)Acting in the cytoplasmActing in the nucleus
48 Growth FactorsSome cells normally produce mitogens, e.g.platelet-derived growth factor, epidermal growth factor.Genetic changes may enhance this production or make it continuous.
55 GTP Binding Proteins Two Broad Groups Ras SuperfamilyThey are molecular switches that regulate numerous physiologic processes.Two states:On when bound to GTPOff when bound to GDP
56 Normal Regulation by Ras Proteins Mitogenic SignalingCytoskeleton OrganizationNuclear FunctionsMembrane & Protein Trafficking
57 Cell Transformation by Ras DNA proliferationChanges in cell morphology
58 Ras On or OffBound to GDP, Ras proteins are neutral in regard to mitotisBound to GTP, they send mitogenic signals to the nucleus
59 Normal ControlNormal Ras quickly cleaves GTP to GDP by an intrinsic GTPase activityA protein called GAP assists in this activity, enhances the digestion by 10,000 times
60 Ras Mutants Some mutations cause a loss of the GTPase activity Then the protein is constitutively active because remains bound to GTPAlso inactivation of GAP may leave Ras constitutively bound to GTP
61 Ras Superfamily in Cancers Alleles found in many tumor types50% of colon cancers90% of pancreatic carcinomasIn human cancers, virtually all Ras mutations are ones that impair GTPase
62 Neurofibromatosis Type I Loss of a GAP-like protein, neurofibromin,predisposes Ras proteins to remain intheir active state.An autosomal disorder with a wide range of possible symptoms including enhanced susceptibility to benign or malignant tumors
63 Significance in Research RAS mutation in human bladder carcinoma lines was the first identification of a human oncogeneAbility of RAS proteins to bind guanine nucleotides was one of the first biochemical functions ascribed to an oncogene product
64 Hope for TherapyActivation of human RAS genes are the most common dominant mutations in human cancerFunction and cellular localization of RAS proteins require processing steps.These steps provide targets for therapeutic interventions
65 Constitutively Active Cytoplasmic Protein Kinase v-Src
66 Transcription Factor Activation by Growth Factor
67 Session SummaryCertain virus-encoded proteins can bind to and activate host-cell receptors for growth factors, thereby stimulating cell proliferation in the absence of normal signals.Mutations or chromosomal translocations that permit growth factor receptor protein-tyrosine kinases to dimerize lead to constitutive receptor activation in the absence of their normal ligands. Such activation ultimately induces changes in gene expression that can transform cells. Overexpression of growth factor receptors can have the same effect and lead to abnormal cell proliferation.Most tumors express constitutively active forms of one or more intracellular signal-transduction proteins, causing growth-promoting signaling in the absence of normal growth factors.
68 Session Summary continued A single point mutation in Ras, a key transducing protein in many signaling pathways, reduces its GTPase activity, thereby maintaining it in an activated state.The activity of Src, a cytosolic signal-transducing protein-tyrosine kinase, normally is regulated by reversible phosphorylation and dephosphorylation of a tyrosine residue near the C-terminus. The unregulated activity of Src oncoproteins that lack this tyrosine promotes abnormal proliferation of many cells.Inappropriate expression of nuclear transcription factors, such as Fos, Jun, and Myc can induce transformation
69 How about factors that Inhibit Entry into G1 or Progression Through the Cycle permanentDifferentiation InducerstemporaryAntimitogenic factorsCell-to-Cell ContactsAnchorage Factors
71 Cell Cycle New cells formed as a result of mitosis But first need synthesis of new DNAFive stagesG0G1SG2M
72 G0 S G1 G2 M Quiescent Stage: may be permanent or reversible Due to: terminal differentiationanti-mitotic agentscell contactG0SRRestriction Point. Once passed, cell is committed to pass through S, even in absence of mitogenic signalsG1, Transcription of genes whose products are required for DNA synthesis. Progression through G1 dependent on internal & external factorsG1G2M
73 2 functional Synthesis and Mitosis 2 preparatory G1 and G2 Phases2 functionalSynthesis and Mitosis2 preparatoryG1 and G2
74 What factors push the cell through the cycle? ExternalCytokines, growth factors, mitogens, hormones, nutrientsInternalCyclins, enzymes, telomerase
75 Controls at every stage of the Cell Cycle After synthesizing DNA, cells are prevented from synthesizing it again until after mitosisIf there are errors in DNA synthesis, repair enzymes try to fix them.If they persist during the cycle, there are controls that interrupt the cycle.They may just stop the cycle, or they may cause cell death - apoptosis
76 p53 The normal protein p53 acts as a “checkpoint” in the cell cycle. If DNA is damaged, p53 causes arrest in the G1 phase or may cause apoptosis.
78 As An OncogeneLoss of p53 function results in uncontrolled cell cycle progressionIncreased number of cells, damaged DNA, decreased apoptosis
79 Effects of p53 defects Too many cells Genetic Instability Due to loss of control in G1And to decreased apoptosisGenetic InstabilityLoss of control over damaged DNACells can undergo rapid mutation
80 Loss of p53 Function How?Mutation, 50% of human tumors have abnormal p53 genesOverexpression of the p53 binding protein, mdm2Human Papillomavirus protein E6 binds to p53 and increases its rate of degradation
81 Another Checkpoint Gene RbRetinoblastoma Susceptibility GeneThe name does not indicate its protooncogene function
82 pRb Functions The protein product of the Rb gene is expressed in many tissuesControls cell proliferation in normal developmentIs a tumor-suppressor geneLoss of its function is associated with many types of tumors
84 pRbNormal pRb provides another checkpoint in the cell cycle, holding the cell in G1.It binds to proteins that are needed for transcription steps preparatory to DNA synthesisLoss of pRb function loses this checkpointBut, conversely, loss of pRb enhances apoptosisOther genes needed for carcinogenesis
85 Session SummaryOverexpression of the proto-oncogene encoding cyclin D1 or loss of the tumor-suppressor genes encoding p16 and Rb can cause inappropriate, unregulated passage through the restriction point in late G1, a key element in cell-cycle control. Such abnormalities are common in human tumors.The p53 protein is essential for the checkpoint control that arrests human cells with damaged DNA in G1. Replication of such cells would tend to perpetuate mutations.p53 functions as a transcription factor to induce expression of p21, an inhibitor of G1 Cdk-cyclin complexes. .
86 Session Summary continued Mutations in the p53 gene occur in more than 50 percent of human cancers.Because p53 is a tetramer, a point mutation in one p53 allele can be sufficient to inhibit all p53 activity.MDM2, a protein that normally inhibits the ability of p53 to restrain the cell cycle or kill the cell, is overexpressed in several cancers.Defects in cellular DNA-repair processes found in certain human diseases are associated with an increased susceptibility for certain cancers.