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Media guide t-bo_c2

  1. 1. The Biology of Cancer the biology of S E CO N D ED I T I O N the biology of of f Second Edition CANCER CANCER Media Guide Robert A. Weinberg Thoroughly updated and incorporating the most important advances in the fast-growing field of cancer biology, The Biology of Cancer, Second Edition, maintains all of its hallmark features admired by students, instructors, researchers, and clinicians around the world. The Biology of Cancer is a textbook for students studying the molecular and cellular bases of cancer at the undergraduate, graduate, and medical school levels. The principles of cancer biology are presented in an organized, cogent, and in-depth manner. The clarity of writing, supported by an extensive full-color art program and numerous pedagogical features, makes the book accessible and engaging. The information unfolds through the presentation of key experiments that give readers a sense of discovery and provide insights into the conceptual foundation underlying modern cancer biology. DVD-ROM AND POSTER INCLUDED • The enclosed DVD-ROM includes the book’s art program, a selection of movies with narration, audio files of mini-lectures by the author, Supplementary Sidebars, and a Media Guide. • The revised “Pathways in Human Cancer” poster summarizes some of the key signaling pathways implicated in tumorigenesis and tumor progression in humans. Robert A. Weinberg o r b r Robert A. Weinberg is a founding member of the Whitehead Institute for Biomedical Research. He is the Daniel K. Ludwig Professor for Cancer Research and the American Cancer Society Research Professor at the Massachusetts Institute of Technology (MIT). Dr. Weinberg is an internationally recognized authority on the genetic basis of human cancer and was awarded the U.S. National Medal of Science in 1997. SECOND EDI T I O N EDI T I O N Besides its value as a textbook, The Biology of Cancer is a useful reference for individuals working in biomedical laboratories and for clinical professionals. ISBN 9780815342199 9 780815 342199 T his document contains an overview of the contents of the DVD-ROM packaged with The Biology of Cancer, 2nd Edition by Robert A. Weinberg. It also contains transcripts of the movies and audio files on the DVD. The DVD contains the following media for students and instructors: • Figures from the book (PowerPoint® and JPEG formats) • Supplementary Sidebars (PDF) • Movies (QuickTime® and WMV formats) • Mini-lectures from the author (MP3 format) The media on this DVD, as well as future media updates, are also available to students and instructors at S E CO N D EDI T I O N
  2. 2. 2 Media Guide for The Biology of Cancer, Second Edition Figures from the Book—The Art of The Biology of Cancer F igures, tables, and micrographs from the book are located in the “Art of TBoC2” folder. They have been pre-loaded into PowerPoint presentations, one for each chapter of the book. A separate folder contains individual versions of each figure, table, and micrograph in JPEG format. The folders are called “PowerPoint” and “JPEGs.” In addition to serving as a tool for creating visual presentations for lectures, the PowerPoint files are also useful for printing the figures. If you wish to print all the figures from a particular chapter, open the PowerPoint presentation for the chapter, select the “file/print” menu option, and print “All” the figures. If you wish to print only a select number or just one figure, choose the appropriate slide number in the PowerPoint printer options window, accessed through the menu described above. This process is roughly the same on both Mac OS and Windows operating systems. You may also print the individual JPEGs from the JPEG archive using Adobe® Photoshop® or similar image editing programs. Both Mac OS and Windows have built-in image viewers that also allow printing. Please consult the user manual for these programs for further instructions. The figures can also be imported into Microsoft Word® and other text editing programs through the “insert/picture” menu option or by cutting-and-pasting the figure from one program to another. In Microsoft Word you can re-size the figure to match your document.
  3. 3. Media Guide for The Biology of Cancer, Second Edition 3 Supplementary Sidebars T he Biology of Cancer contains a special feature called “Sidebars,” which consist of commentary that detours slightly from the main thrust of the textual discussion. Often these Sidebars contain anecdotes or elaborate on ideas presented in the main text. In addition to the Sidebars that appear in the text, the author has written the following additional Sidebars, which can be accessed from the “Supplementary Sidebars” folder on the DVD or from the Garland Science website. These Supplementary Sidebars are cross-referenced throughout the text. They are available in PDF format for optimal viewing and printing. Supplementary Sidebar 1.1 Each female cell can access information only from a single X chromosome Supplementary Sidebar 1.2 Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus Supplementary Sidebar 1.3 The network of miRNA-controlled genes Supplementary Sidebar 1.4 Knocking down gene expression with shRNAs and siRNAs Supplementary Sidebar 1.5 Gene cloning strategies Supplementary Sidebar 2.1 Commonly used histopathological techniques Supplementary Sidebar 2.2 The complicated conventions for classifying and naming tumors Supplementary Sidebar 3.1 Is SV40 responsible for the mesothelioma plague? Supplementary Sidebar 3.2 Maintenance of KSHV and HPV genomes in episomal and chromosomal form Supplementary Sidebar 3.3 Re-engineering the retrovirus genome for gene therapy Supplementary Sidebar 3.4 Classic Kaposi’s sarcoma appears to be a familial disease Supplementary Sidebar 3.5 Viruses like RSV have very short lives Supplementary Sidebar 4.1 Endogenous viruses can explain tumor development in the absence of infectious viral spread Supplementary Sidebar 4.2 Boveri and Hansemann independently hypothesized genetic abnormality as the cause of cancer cells’ malignant behavior Supplementary Sidebar 4.3 Southern and Northern blotting Supplementary Sidebar 4.4 Genes undergo amplification for a variety of reasons Supplementary Sidebar 5.1 Making anti-Src antibodies presented a major challenge Supplementary Sidebar 5.2 The protozoan roots of metazoan signaling Supplementary Sidebar 5.3 Lateral interactions of cell surface receptors Supplementary Sidebar 6.1 Systematic surveys of phosphotyrosine and SH2 interactions Supplementary Sidebar 6.2 The complexities of understanding RTK signaling Supplementary Sidebar 6.3 The rationale for multi-kinase signaling cascades Supplementary Sidebar 6.4 Non-canonical Wnt signaling Supplementary Sidebar 6.5 The Hippo pathway and control of stem cell proliferation Supplementary Sidebar 7.1 Heterozygosity in the human gene pool Supplementary Sidebar 7.2 Which is more likely—LOH or secondary, independent mutations? Supplementary Sidebar 7.3 The polymerase chain reaction makes it possible to genetically map tumor suppressor genes rapidly Supplementary Sidebar 7.4 The MSP reaction makes it possible to gauge methylation status of promoters Supplementary Sidebar 7.5 Ubiquitylation tags cellular proteins for destruction in proteasomes Supplementary Sidebar 7.6 Krebs cycle enzymes and cancer development Supplementary Sidebar 7.7 Homologous recombination allows restructuring of the mouse germ line Supplementary Sidebar 8.1 The origins of embryonic stem cells Supplementary Sidebar 8.2 Plasticity of the cell cycle clock Supplementary Sidebar 8.3 Chromatin immunoprecipitation (ChIP) Supplementary Sidebar 8.4 Some tumors increase Id concentrations by de-ubiquitylating them Supplementary Sidebar 8.5 Specific targeting of cell cycle regulators by E3 ubiquitin ligases Supplementary Sidebar 8.6 The major puzzle surrounding the RB gene: retinoblastomas Supplementary Sidebar 9.1 UV-B radiation, HPV, and cutaneous squamous cell carcinomas
  4. 4. 4 Media Guide for The Biology of Cancer, Second Edition Supplementary Sidebar 9.2 The TUNEL assay Supplementary Sidebar 9.3 Dominant-negative functions of mutant p53 alleles: functional interactions between p53 and its p63 and p73 cousins Supplementary Sidebar 9.4 Some mutant p53 alleles cause highly specific tumors Supplementary Sidebar 9.5 Autophagy is critical to post-fertilization development Supplementary Sidebar 10.1 The use of the TRAP assay and adaptations thereof permits the rapid and quantitative assessment of the levels of the catalytic activity of telomerase enzyme in eukaryotic cells Supplementary Sidebar 11.1 Monoclonal antibodies and fluorescence-activated cell sorting (FACS) Supplementary Sidebar 11.2 How does multi-step tumor progression actually take place? Supplementary Sidebar 11.3 Symbiosis between distinct subpopulations within a tumor Supplementary Sidebar 11.4 Comparative genomic hybridization Supplementary Sidebar 11.5 Are rodent carcinogen tests reliable indicators of danger to humans? Supplementary Sidebar 11.6 Does saccharin cause cancer? Supplementary Sidebar 11.7 How does diet affect colon cancer incidence? Supplementary Sidebar 12.1 Hematopoiesis as a model for the organization of many kinds of tissues Supplementary Sidebar 12.2 Stem cell pools may explain the protective effects of pregnancy Supplementary Sidebar 12.3 The conserved-strand mechanism and protection of the stem cell genome Supplementary Sidebar 12.4 Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome Supplementary Sidebar 12.5 How does red meat cause colon cancer? Supplementary Sidebar 12.6 A convergence of bacterial, yeast, and human genetics led to the discovery of hereditary nonpolyposis colon cancer genes Supplementary Sidebar 12.7 Homology-directed repair Supplementary Sidebar 13.1 Localization of growth factors is important for proper heterotypic interactions Supplementary Sidebar 13.2 Ongoing heterotypic signaling in carcinomas Supplementary Sidebar 13.3 Certain highly advanced tumors provide exceptions to the generally observed dependence of carcinoma cells on stroma Supplementary Sidebar 13.4 Myofibroblasts predict clinical progression of cancer Supplementary Sidebar 13.5 A technique for separating stromal from epithelial cells Supplementary Sidebar 13.6 Microvessel leakiness dooms many forms of anti-cancer therapy: optimizing anti-angiogenic treatments Supplementary Sidebar 13.7 The temporary nature of vessel regression created by anti-angiogenesis therapy Supplementary Sidebar 13.8 Kaposi’s sarcoma cells hold the record for the number of documented heterotypic signals they receive Supplementary Sidebar 13.9 Effects of an anti-VEGF-R monoclonal antibody on the growth of a human tumor xenograft Supplementary Sidebar 13.10 Fibroblasts are heterogeneous and can change dynamically in response to signals Supplementary Sidebar 14.1 Visualization of the dynamics of pathfinding fibroblasts followed by squamous cell carcinoma cells Supplementary Sidebar 14.2 Metastasizing cancer cells often take on hitchhikers while traveling through the blood Supplementary Sidebar 14.3 Instruments for detecting circulating tumor cells Supplementary Sidebar 14.4 Hidden micrometastases are revealed through organ transplantation Supplementary Sidebar 14.5 Wolves in sheep’s clothing: when carcinoma cells invade the stroma Supplementary Sidebar 14.6 TGF-β works in conflicting ways during tumor progression Supplementary Sidebar 14.7 Dynamics of EMT induction: the EMT may be controlled in some cancer cells exclusively by their own genomes Supplementary Sidebar 14.8 An example of an EMT relatively late in embryonic development Supplementary Sidebar 14.9 Relatively rapid metastatic dissemination of advanced primary tumor cells Supplementary Sidebar 14.10 Our cells devote an enormous number of genes to regulating protein degradation Supplementary Sidebar 14.11 Peritoneovenous shunts provide dramatic support for the seed and soil hypothesis
  5. 5. Media Guide for The Biology of Cancer, Second Edition Supplementary Sidebar 14.12 Tooth extractions may occasionally become exceedingly painful Supplementary Sidebar 14.13 Tumor stem cells further complicate our understanding of the metastatic process Supplementary Sidebar 14.14 Does Darwinian evolution accommodate metastasis-specific alleles? Supplementary Sidebar 15.1 Rearrangements of chromosomal DNA segments generate a vast array of antigen-binding domains in antibodies and T-cell receptors Supplementary Sidebar 15.2 Virus-infected cells may not always be recognized by the immune system Supplementary Sidebar 15.3 Bizarre tumors reveal how cancer cells can become infectious agents Supplementary Sidebar 15.4 Mice have proven to be far more useful for tumor biologists than chickens Supplementary Sidebar 15.5 An HPV vaccine protects against many cervical carcinomas Supplementary Sidebar 15.6 An unexpected type of anti-p53 reactivity is often found in cancer patients Supplementary Sidebar 15.7 Immune recognition of tumors may be delayed until relatively late in tumor progression Supplementary Sidebar 15.8 Some paraneoplastic syndromes reveal defective tolerance and overly successful immune responses to tumors Supplementary Sidebar 15.9 TSTAs can arise as by-products of chemical and physical carcinogenesis Supplementary Sidebar 15.10 Are melanomas more antigenic than other tumors? Supplementary Sidebar 15.11 Strategies for cloning genes encoding melanoma TATAs Supplementary Sidebar 15.12 Anti-CD47 therapies hold promise in treating lymphomas and other hematopoietic malignancies Supplementary Sidebar 15.13 Cancer cells may thwart extravasation by circulating T cells Supplementary Sidebar 15.14 Herceptin can be modified to potentiate cancer cell killing Supplementary Sidebar 15.15 Bone marrow transplantation and the treatment of hematopoietic malignancies Supplementary Sidebar 15.16 Whole genome sequencing allows a new attack on tumor cells Supplementary Sidebar 16.1 Modern cancer therapies have had only a minor effect on the overall death rate from the disease Supplementary Sidebar 16.2 Prostate cancers usually do not require aggressive intervention—a tale of two countries Supplementary Sidebar 16.3 Clinical practice and our understanding of disease pathogenesis have often been poorly aligned, leading to sub-optimal, often tragic outcomes Supplementary Sidebar 16.4 The ability to assign tumors to specific disease subtypes is critical to the success of drug development Supplementary Sidebar 16.5 p53 germ-line polymorphisms and somatic mutations can complicate the induction of apoptosis by drugs Supplementary Sidebar 16.6 Ras function can be inhibited by interfering with the enzymes responsible for the maturation of the Ras protein Supplementary Sidebar 16.7 Chemical synthesis, compound libraries, and high-throughput screening Supplementary Sidebar 16.8 Evolution can generate huge collections of structurally similar proteins Supplementary Sidebar 16.9 Large-scale screen of the inhibitory effects of a drug on various kinases Supplementary Sidebar 16.10 Epidermal growth factor receptor expression levels predict little about a tumor’s susceptibility to receptor antagonists Supplementary Sidebar 16.11 Akt/PKB function is controlled by multiple upstream signals 5
  6. 6. 6 Media Guide for The Biology of Cancer, Second Edition Movies T he “Movies” folder contains thirty-one movies, available in both QuickTime and WMV formats, that will aid in understanding some of the proteins and processes described in the book. The QuickTime movies will provide the optimal viewing experience. The WMV format is included because QuickTime movies will not work in PowerPoint for Windows, but the WMV formatted movies will work seamlessly. Each movie is accompanied by a voice-over narration. The movie table of contents, followed by the full text of each movie narration, is below: Chapter 1: The Biology and Genetics of Cells and Organisms 1.1 1.2 1.3 1.4 Replication I Replication II Translation Transcription Chapter 2: The Nature of Cancer 2.1 2.2 2.3 Embryonic Origins of Tissues Mammary Cancer Cells Visualization of Cancer I: Lymphoma Chapter 3: Tumor Viruses 3.1 Contact Inhibition Chapter 4: Cellular Oncogenes No Movies Chapter 5: Growth Factors, Receptors, and Cancer 5.1 5.2 5.3 5.4 Cellular Effects of EGF vs. HGF Activation of Kit Receptor Signaling by SCF EGF Receptor Family IGF Receptors and Monoclonal Antibodies Chapter 6: Cytoplasmic Signaling Circuitry Programs Many of the Traits of Cancer 6.1 6.2 6.3 Regulation of Signaling by the Src Protein Signaling by the Ras Protein EGF Receptors and Signaling Chapter 7: Tumor Suppressor Genes 7.1 Intestinal Crypt Chapter 8: pRb and Control of the Cell Cycle Clock 8.1 8.2 Animal Cell Division CDK2 Chapter 9: p53 and Apoptosis: Master Guardian and Executioner 9.1 9.2 p53 Structure Apoptosis Chapter 10: Eternal Life: Cell Immortalization and Tumorigenesis 10.1 Telomere Replication Chapter 11: Multi-Step Tumorigenesis No Movies Chapter 12: Maintenance of Genomic Integrity and the Development of Cancer 12.1 DNA Repair Mechanisms
  7. 7. Media Guide for The Biology of Cancer, Second Edition Chapter 13: Dialogue Replaces Monologue: Heterotypic Interactions and the Biology of Angiogenesis 13.1 Mechanisms Enabling Angiogenesis 13.2 Interactions of Innate Immune Cells with a Mammary Tumor Chapter 14: Moving Out: Invasion and Metastasis 14.1 Adhesion Junctions 14.2 Mechanisms of Brain Metastasis Formation 14.3 Visualization of Cancer II: Metastasis Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy 15.1 The Immune Response 15.2 Antigen Display and T-Cell Attack Chapter 16: The Rational Treatment of Cancer 16.1 Drug Export by the Multi-Drug Resistance Pump 16.2 PI3K 7
  8. 8. 8 Media Guide for The Biology of Cancer, Second Edition Movie 1.1 Replication I U sing computer animation based on molecular research, we are able to picture how DNA is replicated in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called a helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand because it must be copied backwards. It is drawn out repeatedly in loops and copied one section at a time. The end result is two new DNA molecules. Animation produced for DNA Interactive ( © 2003 Howard Hughes Medical Institute, ( All rights reserved. Movie 1.2 Replication II I n a replication fork, two DNA polymerases collaborate to copy the leading-strand template and the lagging-strand template DNA. In this picture, the DNA polymerase that produces the lagging strand has just finished an Okazaki fragment. The clamp that keeps the lower DNA polymerase attached to the lagging strand dissociates, and the DNA polymerase temporarily releases the lagging strand template DNA. As the DNA helicase continues to unwind the parental DNA, the primase becomes activated and synthesizes a short RNA primer on the growing lagging strand. The DNA polymerase binds to the DNA again and becomes locked in by the clamp. The polymerase uses the RNA primer to begin a short copy of the lagging-strand template DNA.The polymerase stalls when it reaches the RNA primer of the preceding Okazaki fragment, and the entire cycle repeats. Animation: Sumanas, Inc. ( Music: Christopher Thorpe
  9. 9. Media Guide for The Biology of Cancer, Second Edition Movie 1.3 Translation W hen the mRNA is complete, it snakes out of the nucleus into the cytosol. Then in a dazzling display of choreography, all the components of a molecular machine lock together around the RNA to form a miniature factory called a ribosome. It translates the genetic information in the RNA into a string of amino acids that will become a protein. tRNA molecules‚ the green triangles‚ bring each amino acid to the ribosome. The amino acids are the small red tips attached to the tRNAs. There are different tRNAs for each of the twenty amino acids, each of them carrying a three-letter nucleotide code that is matched to the mRNA in the machine. Now we come to the heart of the process. Inside the ribosome, the mRNA is pulled through like a tape. The code for each amino acid is read off, three letters at a time, and matched to three corresponding letters on the tRNAs. When the right tRNA plugs in, the amino acid it carries is added to the growing protein chain. You are watching the process in real time. After a few seconds the assembled protein starts to emerge from the ribosome. Ribosomes can make any kind of protein. It just depends on what genetic message you feed in on the mRNA. In this case, the end product is hemoglobin. The cells in our bone marrow churn out a hundred trillion molecules of it per second! And as a result, our muscles, brain, and all the vital organs in our body receive the oxygen they need. Animation produced for DNA Interactive ( © 2003 Howard Hughes Medical Institute ( All rights reserved. Movie 1.4 Transcription T ranscription is the process by which DNA is copied into RNA in the first step of gene expression. It begins with a bundle of factors assembling at the start of a gene, that is, a linear sequence of DNA instructions, shown here stretching away to the left. The assembled factors include an RNA polymerase, the blue molecule. Suddenly, RNA polymerase is let go, racing along the DNA to read the gene. As it unzips the double helix, it copies one of the two strands. The yellow chain snaking out of the top is the RNA, a copy of the genetic message. The nucleotide building blocks that are used to make the RNA enter through an intake hole in the polymerase. In the active site of the enzyme, they are then matched to the DNA, nucleotide by nucleotide, to copy the A’s, C’s, T’s and G’s of the gene. The only difference is that in the RNA copy, thymine is replaced with the closely related base uracil, commonly abbreviated “U.” You are watching this process, called transcription, in real time. Animation produced for DNA Interactive ( © 2003 Howard Hughes Medical Institute ( All rights reserved. 9
  10. 10. 10 Media Guide for The Biology of Cancer, Second Edition Movie 2.1 Embryonic Origins of Tissues C ells of this developing frog embryo rearrange in a dramatic ballet of orchestrated cell movements. In a continuous motion, cells from the outer layer of the embryo sweep towards the vegetal pole and start invaginating, forming a deep cavity in the interior. The paths of the cells and the topology of these rearrangements are best seen in this animation of an embryo that has been sliced open. The different cell layers that are formed in this way have very different fates. Cells that line the newly formed cavity, called the endoderm, develop into the lining of the gut and many internal organs such as liver, pancreas, and lung. Cells in the middle layer, called the mesoderm, give rise to muscle and connective tissue. Cells remaining on the outside, called the ectoderm, go on to form the outer layer of the skin, as well as the nervous system. Jeremy Pickett-Heaps University of Melbourne From “From Egg to Tadpole” Jeremy Pickett-Heaps and Julianne Pickett-Heaps Cytographics ( Movie 2.2 Mammary Cancer Cells N ormal human mammary epithelial cells can be suspended and grown in a gellike medium. Under these conditions, they form structures that resemble the little sacs of cells in the mammary gland, called alveoli, in which milk is made. Cells assemble into a well-organized, polarized epithelium that forms a closed sphere with an internal lumen. In the mammary gland, this space would be connected to ducts, and cells would secrete milk into it. By contrast, these human breast cancer cells grown under the same conditions divide aggressively and in an uncontrolled fashion. They are also more migratory and grow into disorganized clumps, which would form tumors in the body. Original script: Peter Walter, Howard Hughes Medical Institute, University of California at San Francisco Mina J. Bissell, Karen Schmeichel, Hong Liu, and Tony Hansen Lawrence Berkeley Laboratories
  11. 11. Media Guide for The Biology of Cancer, Second Edition 11 Movie 2.3 Visualization of Cancer I: Lymphoma M agnetic resonance imaging tomography enables the visualization of tumors. In this 3-D reconstruction, we see a lymphoma in living tissue that has been artificially colored red. The lymphoma was generated by cells that initially lacked p53 function; in the absense of p53 expression, the lymphoma grew to be quite large. In this experiment, p53 expression was reactivated in the lymphoma at Day 0. 18 days after p53 reactivation, we can observe significant regression of the tumor. This regression illustrates that p53-activating signals were present in the lymphoma cells during its initial growth, but failed, in the absence of p53 expression, to halt tumor growth. That is, without p53 expression, these signals alone could not trigger apoptosis in the tumor cells. However, once p53 was expressed in the tumor cells, it functioned powerfully to induce tumor regression, presumably by causing widespread apoptosis in the lymphoma cells. Andrea Ventura, Tyler Jacks, and David G. Kirsch The David H. Koch Institute for Integrative Cancer Research at MIT Jan Grimm and Ralph Weissleder Massachusetts General Hospital By the 28th day of p53 reactivation, the tumor cannot be seen at all. 3.1 Contact Inhibition W hen normal cells are introduced into a Petri dish at low numbers, they begin to grow and divide. As the cells begin to touch one another, they stop dividing. This response is called contact inhibition. Once the cells fill up the bottom of the dish, all cell division ends, creating a state called confluence. Contact inhibition ensures that the cells create a layer only one cell thick—a monolayer. The behavior of cancer cells is quite different. If a cancer cell is seeded among normal cells, all of the cells will proliferate as before. However, once confluence is reached, the normal cells will stop multiplying while the cancer cells continue to divide, yielding a clump of cells often called a focus. Contact inhibition can be demonstrated in vitro by removing cells from a confluent monolayer. In this experiment, cells are removed by scratching the monolayer with a needle. The surviving cells at the edge of the wound now do two things. First, they begin to proliferate again, since they are no longer fully contact-inhibited. And second, they migrate into the empty area of the wound, attempting to fill it up. Animation: Sumanas, Inc. ( Sheryl Denker and Diane Barber University of California at San Francisco
  12. 12. 12 Media Guide for The Biology of Cancer, Second Edition Movie 5.1 Cellular Effects of EGF vs. HGF E pidermal Growth Factor, or EGF, has been added to these liver epithelial progenitor cells in culture. EGF binds specifically to the Epidermal Growth Factor Receptor on the surface of the cells. This stimulates its tyrosine-kinase activity and a downstream signaling cascade that promotes DNA synthesis and cell proliferation. Note how the cells cling to one another and form a clump of cells as they divide. In this movie, another growth factor, Hepatocyte Growth Factor, or HGF, has been added to liver epithelial progenitor cells in culture. HGF binds to the c-Met receptor on the cell surface, and similar to EGF, it stimulates tyrosine-kinase activity and a downstream signaling cascade that promotes cell proliferation. However, unlike EGF, HGF has an additional effect on cells by promoting cell motility. This causes the dividing cells to scatter. Because of this effect, HGF is also known as Scatter Factor or SF. Andrea Bertotti and Paolo M. Comiglio Institute for Cancer Research and Treatment, University of Torino, School of Medicine The differences in the effects of EGF and HGF illustrate the quite different responses that these two growth factors elicit from cells. Movie 5.2 Activation of Kit Receptor Signaling by SCF I n the absence of ligand, a growth factor receptor exists in a monomeric (single-subunit) form embedded in the plasma membrane. Many growth factor receptors, such as the Kit receptor shown here, have lateral mobility in the plane of the plasma membrane and are relatively free to wander back and forth across the surface of the cell. Like many growth factor receptors, the Kit protein can be divided into 3 major domains. The ectodomain, which sticks out of the surface of the cell and plays a key role in ligand binding; the transmembrane domain, which is extremely hydrophobic and spans the plasma membrane; and the cytoplasmic domain, which can initiate signaling inside the cell. In the example of Kit, the signaling process begins when Kit encounters its ligand, called stem cell factor, or SCF. SCF is composed of two identical protein subunits. When presented with SCF, Kit binds to one of the two identical subunits. When the SCF/Kit complex encounters an unbound Kit monomer in the plasma membrane, the unbound Kit monomer can bind to the other subunit of the SCF ligand, and form a dimeric receptor. Upon dimerization of the ectodomains driven by SCF, the cytoplasmic domains of the two Kit proteins are brought into close proximity. The kinase domain of each Kit receptor then phosphorylates tyrosine residues present in the cytoplasmic domain of the other Kit receptor, a process called transphosphorylation. Transphosphorylation triggers a cascade of signaling activity inside the cell that can affect cell growth and proliferation. Animation: Sumanas, Inc. ( Joseph Schlessinger Yale University School of Medicine
  13. 13. Media Guide for The Biology of Cancer, Second Edition 13 Movie 5.3 EGF Receptor Family E pidermal growth factor receptors, also called EGF receptors, constitute a family of four similarly structured receptor tyrosine kinases that interact with one another to promote general cell growth and proliferation. When EGF receptors bind with their ligands—such as EGF, TGF-α, or NRG—they form homodimers or heterodimers, and activate their cytoplasmic domains. Once activated, the cytoplasmic domains emit signals inside the cell that activate cytoplasmic signaling cascades that function, in turn, to promote cell growth and proliferation, inhibit apoptosis, and increase cell motility. In cancer cells, signal emission by EGF receptors becomes deregulated and enables them to promote tumor growth, resistance to chemotherapy, and tumor metastasis. In the absence of ligand, normal EGF receptors exist in a monomeric form, and move laterally in the plane of the plasma membrane. Even if they collide, they cannot form stable dimers. When an EGF receptor binds with its ligand, it undergoes a dramatic conformational change in its extracellular domain. This enables the altered extracellular domain of this receptor to bind to the extracellular domain of a second receptor molecule. Dimerization brings the intracellular kinase domains of the two receptor molecules close together. The tyrosine kinases become activated, and each tyrosine kinase phosphorylates the cytoplasmic tail of the other receptor molecule. This process is referred to as transphosphorylation. The resulting phosphorylated tails then serve to recruit and activate a series of signal transducing proteins that in turn activate multiple downstream signaling cascades. Animation: Sumanas, Inc. ( Mark Sliwkowski Genentech, Inc.
  14. 14. 14 Media Guide for The Biology of Cancer, Second Edition Movie 5.4 IGF Receptors and Monoclonal Antibodies T he surface of all cells is studded with a wide variety of receptor tyrosine kinases, among them the receptors for insulin and insulin-like growth factors, called IGF-1 and IGF-2. Upon binding their insulin or IGF ligands, these two receptors transduce signals into the cell, including those that encourage cell growth and prevent apoptosis. The key molecules involved in the IGF system are: the soluble ligands IGF 1 and IGF 2, a series of IGF-binding proteins, the cell surface IGF receptors, type 1 and type 2, and the insulin receptor. Both of the ligands, IGF1 and IGF2, exert their biological activities through binding to type-1 IGF receptors. They can also bind to the insulin receptor, but with about 1000 times lower affinity. The type-2 receptor is believed to function as a decoy receptor, to draw IGF2 away from the type-1 receptor. It is considered to act as a tumor suppressor, and its expression is frequently lost during tumor progression. In addition, a series of six binding proteins associate with IGFs in plasma to regulate the amount of free IGFs that are available to bind and activate the type-1 IGF receptor. The type-1 IGF receptor tyrosine kinase can bind either IGF1 or IGF2 ligands. This receptor exists on the cell surface as a pre-formed dimer. It is composed of an extracellular alpha subunit, which contains the IGF binding site, and a beta subunit, which extends through the lipid bilayer of the cell membrane to the inside of the cell, where the tyrosine kinase enzyme is located. The insulin receptor is structurally and functionally very similar to the type-1 IGF receptor. As a result, the type-1 IGF receptor and insulin receptor can form hybrid, heterodimeric receptors containing an IGF receptor subunit and an insulin receptor subunit. Studies show that these hybrid receptors preferentially act like type-1 IGF receptors and bind IGFs with high affinity. When IGFs bind, the receptor tyrosine kinase activity on the beta subunit is activated, causing tyrosine residues to become phosphorylated. As is the case with all other tyrosine kinase receptors, these phosphotyrosine residues then recruit signaling proteins to turn on a variety of downstream signaling pathways, such as the MAP kinase proliferation pathway and the AKT survival pathway. Therefore, binding of IGFs to the type 1 receptor provides signals to tumor cells that may confer resistance to killing by cytotoxic drugs. Blockage of binding to the type-1 receptor represents one therapeutic strategy to stop cell proliferation and affect tumor cell survival. Anti-type-1 IGF receptor monoclonal antibodies can be generated that are highly specific for binding to the alpha subunit of the type-1-IGF receptor, thereby blocking its function. Monoclonal antibodies have been developed that have high selectivity and affinity for the type-1 IGF receptor, and do not recognize or bind to insulin receptors. Due to the selectivity for the type-1 IGF receptors, they can also recognize and block hybrid receptors formed between type-1 IGF receptors and insulin receptors. Treatment of tumor cells with such an antibody creates a direct blockade of IGF binding to type-1 receptors and inhibits downstream signaling. It also causes type-1 receptor molecules to be removed from the surface of tumor cells. Ultimately, it is hoped that clinical use of IGF receptor-blocking antibodies can reduce tumor cell growth and increase apoptosis in tumors. ImClone Systems
  15. 15. Media Guide for The Biology of Cancer, Second Edition Movie 6.1 Regulation of Signaling by the Src Protein T he Src protein kinase acts as a molecular signal integration device. Src consists of two clearly demarcated regulatory domains: an SH3 domain and an SH2 domain. The catalytic domain is structured much like other tyrosine kinases. In addition, a linker and the short C-terminal tail play regulatory roles. To be an active protein kinase, the active site of the Src kinase domain must first become accessible to substrate proteins. In the inactive state, the active site is blocked by an activation loop. The activation loop must be phosphorylated, most likely through the actions of another SRC kinase molecule, not shown here. Phosphorylation causes the activation loop to swing out of the way and rearrange so that other substrate proteins can bind and become phosphorylated. Src is kept inactive by an interaction of its SH2 domain with the C-terminal tail peptide. A phosphorylated tyrosine on its C-terminal tail is buried in a deep binding pocket in the SH2 domain. To activate Src, this phosphate group is removed by specific phosphatases. Upon dephosphorylation of the tyrosine, the C-terminal tail is released from the SH2 domain. In this cartoon view, Src is represented in its inactive state. The linker segment, shown in red, connects the SH2 and SH3 domains on the one hand, and the kinase domain on the other. The activation loop, shown in dark green, drapes across the catalytic sites and, in this configuration, blocks the catalytic domains access to the substrate of the kinase. Several other intramolecular interactions hold the Src kinase in an inactive configuration. The SH2 domain, in blue, binds a phosphotyrosine at residue position 527. The SH3 domain, in light green, binds the linker segment. When a PDGF receptor becomes activated by ligand binding, its C-terminal tail becomes phosphorylated on tyrosine residues. One of these phosphotryrosines can be recognized and bound by the SH2 domain of Src. The SH2 domain, which until now participated in an intramolecular binding, switches and forms an intermolecular bridge by binding the phosphotryrosine on the C-terminal tail of the PDGF receptor. The SH3 domain follows suit. It too breaks its intramolecular binding and binds to a proline-rich segment on the PDGF receptor. These shifts liberate the kinase domain of the Src protein. In a series of concerted reactions, the tyrosine residue at position 527 of Src is dephosphorylated, and the activation loop becomes phosphorylated on a tyrosine residue. This last alteration forces the activation loop to move out of the way so that it no longer obstructs the catalytic cleft of the Src enzyme. This allows Src to phosphorylate a diverse array of protein substrates on their tyrosine residues. Original Storyboard by Peter Walter, Howard Hughes Medical Institute, University of California at San Francisco Animation: Sumanas, Inc. ( 15
  16. 16. 16 Media Guide for The Biology of Cancer, Second Edition Movie 6.2 Signaling by the Ras Protein T he Ras protein is a representative example of the large family of GTPases that can function as molecular switches. The nucleotide-binding site of Ras is formed by several conserved protein loops that cluster at one end of the protein. In its inactive state, Ras is bound tightly to GDP. As a molecular switch, Ras can toggle between two different conformational states depending on whether GDP or GTP is bound. Two regions, called switch 1 and switch 2, change conformation dramatically. The change in conformational state allows other proteins to distinguish active Ras from inactive Ras. Active, GTP-bound Ras binds to, and activates, downstream target proteins in the cell signaling pathways, such as RAF, PI3K, and GDS. A space-filling model shows that the conformational changes between the GDP and GTP bound forms of Ras spread over the whole surface of the protein. The two switch regions move the most. Ras hydrolyzes GTP to switch itself off; that is, to convert from the GTP-bound state to the GDP-bound state. This hydrolysis reaction requires the action of a Ras GTPaseactivating protein, or Ras GAP for short. RasGAP binds tightly to Ras burying the bound GTP. It inserts an arginine side chain, sometimes called an Argenine finger, directly into the active site. The arginine, together with threonine and glutamine side chains of Ras itself, promotes the hydrolysis of GTP. Ras then sits in its inactive GDP-bound state, awaiting a stimulatory signal that will cause it to evict GDP and acquire GTP. Molecular modeling and animation: Timothy Driscoll Original script: Peter Walter, Howard Hughes Medical Institute, University of California at San Francisco Animation: Sumanas, Inc. (
  17. 17. Media Guide for The Biology of Cancer, Second Edition Movie 6.3 EGF Receptors and Signaling W hen epidermal growth factor (or EGF) binds to receptors on the plasma membrane, the receptor molecules structurally rearrange and dimerize. EGF receptors are in a class of receptor tyrosine kinases, and the dimerization of these receptors results in the reciprocal activation of the two kinase domains. Once a tyrosine kinase is activated, it phosphorylates the C-terminal cytoplasmic tail of the other receptor at multiple tyrosine residues. For many receptors, this transphosphorylation is reciprocal, with each receptor molecule phosphorylating the other. As we will see, the phosphate groups decorating the c-terminal tail enable the receptor to activate a series of downstream signaling events. Through a series of intermediary signaling proteins, the activated receptor turns on a protein called Ras. Ras acts like a binary switch, and EGF signaling triggers Ras to convert from its GDP-bound inactive state to its GTP-bound, actively signaling state. If mutated into a constitutively active form, Ras becomes oncogenic. It has long been known that the activation of the EGF receptor has very similar effects on the cell as the activation of a Ras protein. This suggested the possibility of signaling between them. The discovery of SH2 domains provided important clues of how this signaling proceeds. These domains, which are parts of larger proteins, enable proteins to recognize and bind phosphotryrosine residues displayed by yet other proteins. The SH2 group allows recognition of two chemical structures: first, the phosphotyrosine itself; and second, the adjacently located amino acid residues. There are many phosphotyrosines attached to an activated growth factor receptor, and each of these receives its unique identity from its neighboring amino acid residues. Such phosphate groups decorate the C-terminal tail of a growth factor receptor. Accordingly, a molecule like Shc, which has an SH2 group, can recognize and bind a phosphotyrosine group on the C-terminal tail of the EGF receptor. In the case of Shc, the phosphotyrosine is followed by three other amino acids, yielding the sequence: tyrosine (Y), leucine (L), isoleucine (I), and proline (P). Like Shc, this theme recurs in a number of other proteins, which also contain their own specialized SH2 domains. Each recognizes a particular phosphotyrosine followed by three distinct amino acid residues. Because certain phosphotyrosines attract multiple SH2 groups, the entire array of SH2 containing proteins that can become associated with a receptor is quite elaborate and can be depicted like this. Each of these associated proteins has its own distinct biochemical function, which is carried out by other domains linked to its SH2 domain. For example, JAK2 is a tyrosine kinase that becomes activated after it binds to the receptor tail via its SH2 group. PLC-g is an enzyme that cleaves phospholipids in the plasma membrane. And Grb2 is a member of an important class of adaptor proteins whose sole function is to build a bridge between the receptor and a third protein. Focusing on Grb2, we can see that SH2 groups are not the only way by which proteins can recognize and bind to one another. For example, SH3 groups recognize and bind proline-rich sequences on other proteins. In the case of Grb2, its two SH3 groups recognize a third protein called SOS. Once SOS becomes bound indirectly to the receptor, it is brought in close proximity to Ras proteins tethered to the plasma membrane. SOS acts as a guanine nucleotide exchange factor. Such proteins are capable of inducing RAS to release its bound GDP, allowing GTP to jump aboard. These two forms of Ras represent different states of activity. The GDP-bound form is inactive and the GTP-bound form is active in signaling. The period of active signaling is quite short since other proteins interact with Ras and induce it to hydrolize its bound GTP. 17
  18. 18. 18 Media Guide for The Biology of Cancer, Second Edition While Ras is in its active state, it is able to interact with a variety of partner proteins that serve, in turn, to activate downstream signaling pathways. For example, Ras can bind to the serine threonine kinase called Raf. When Raf interacts with Ras, it becomes an active signal-emitting kinase. An immediate downstream target of Raf is called MEK. Once MEK becomes activated through phosphorylation, it also becomes an active serine threonine kinase. Its most important substrate is called Erk. Once activated, Erk proceeds to phosphorylate a diverse group of proteins that favor cell proliferation. Another important event initiated by Ras is the PI3 kinase cascade. Once again, a kinase is being activated, but in this case the object of phosphorylation is not a protein, but instead a phospholipid embedded in the plasma membrane. The phospholipid molecule is constructed from two long hydrocarbon tails embedded in the plasma membrane, a glycerol, and an inositol sugar-like molecule. In the case of PI3K, its immediate substrate is a phosphotidyl inositol to which two phosphates have been previously attached. PI3K extends this process by adding yet another phosphate to the inositol ring. This now creates a site to which a number of cytoplasmic proteins can attach. These proteins use another specialized domain to recognize and bind the triply phosphorylated inositol. The domain is called a PH domain. One of the most important molecules that contains a PH domain is a kinase called Akt or PkB. Once Akt binds, it becomes phosphorylated and then becomes functionally activated. Once activated, Akt is released and can travel through the cytoplasm to activate multiple substrates. The ability of Akt to phosphorylate these other proteins, thereby altering them, has multiple effects on the cell. For example, by phosphorylating Bad, it reduces the likelihood of apoptosis. By phosphorylating mTOR, it facilitates the physical growth of the cell. And by phosphorylating GSK-3beta, it stimulates cell proliferation. Storyboard and Animation: Sumanas, Inc. (
  19. 19. Media Guide for The Biology of Cancer, Second Edition 19 Movie 7.1 Intestinal Crypt T he lining of the small intestine, like the lining of most of the gut, is a single-layered epithelium. Depending upon location in the gut, the epithelial cells, usually called enterocytes, help absorb nutrients from the lumen of the gut or absorb water from the intestinal contents. In the small intestine, the surface of the lining of the gut is increased enormously by thousands of villi that protrude into the lumen. Here we see a single villus and its internal architecture. The surface of the villus is covered by a single layer of enterocytes, which extend down into the crypt below. Stem cells at the bottom of the crypt, located between paneth cells, divide and make copies of themselves, and also make transit-amplifying cells. The transit amplifying cells proliferate rapidly and move up the walls of the crypt. As the cells migrate upwards they begin to differentiate into goblet cells and enterocytes. While the cells are moving up the sides of the villus, they carry out the essential functions of the small intestine, notably absorption of nutrients. When the differentiated cells reach the tip of the villus, they undergo apoptosis and are shed into the lumen of the small intestine. The entire process of out-migration and cell death is completed in just three to four days. The process of out-migration and rapid cell replacement is a defense mechanism against the development of colon cancer, since almost all epithelial cells, including those that have accidentally sustained mutations, are shed within days of their formation. Therefore, the only mutations that can lead to the development of a cancer are those that are retained in the crypt. This dictates that such mutations must block the outmigration of mutant cells from the crypt. The outmigration of transit amplifying cells from the bottom of the crypt depends on the protein called adenomatous polyposis coli, or simply APC. In the absence of functional APC, this continuous outmigration is blocked, leading to the accumulation of transit amplifying cells in the crypt. In this animation of an experiment, APC loss is achieved through an induced gene inactivation, which appears to mimic the mutation that initiates most gastrointestinal tumors. APC is usually required to inactivate the intracellular protein called β-catenin; the inactivation of β-catenin permits the differentiation of the transit amplifying cells and their continued outmigration from the base of the crypt. In the absence of APC function, β-catenin accumulates within the transit amplifying cells, which blocks both their outmigration and differentiation. The accumulated transit amplifying cells do not themselves form a carcinoma. However, they and their descendants can now accumulate additional mutations that will drive such cells progressively to become full-fledged carcinoma cells. Animation by Digizyme, Inc. ( Models, Animation, Surfacing, Composite: Eric Keller Storyboard and Art Direction: Gael McGill © 2009 by Hans Clevers Hans Clevers Hubrecht Institute
  20. 20. 20 Media Guide for The Biology of Cancer, Second Edition Movie 8.1 Animal Cell Division D ifferential interference contrast microscopy is used here to visualize mitotic events in a lung cell grown in tissue culture. Individual chromosomes become visible as the replicated chromatin starts to condense. The two chromatids in each chromosome remain paired as the chromosomes become aligned on the metaphase plate. The chromatids then separate and get pulled by the mitotic spindle into the two nascent daughter cells. The chromatin decondenses as the two new nuclei form and cytokinesis continues to constrict the remaining cytoplasmic bridge until the two daughter cells become separated. Video reproduced from: The Journal of Cell Biology 122:859–875, 1993. © The Rockefeller University Press Edward D. (Ted) Salmon and Victoria Skeen University of North Carolina at Chapel Hill Robert Skibbens Lehigh University Movie 8.2 CDK2 L ike other kinases, Cyclin-dependent kinases, or Cdks for short, are crucial regulatory proteins in the cell cycle. When activated, Cdks transfer phosphate groups from ATP to serine and threonine side chains on targeted substrate proteins. When inactive, the active site of Cdks is sterically obstructed by a loop, often referred to as the activation loop. As their name suggests, cyclin-dependent kinases are activated by cyclins. Cyclin binding to Cdk pulls the activation loop away from the active site and exposes the bound ATP, allowing it access to target proteins. Thus, a Cdk can phosphorylate target proteins only when it is in a cyclin–Cdk complex. A third protein called a Cdk-activating kinase is required for full activation of a Cdk. This activating kinase adds a phosphate group to a crucial threonine in the activation loop, thereby completing the activation of the cyclin–CDK complex. Oligopeptide domains of substrate proteins bind to the active site of the cyclin–Cdk complex so that the target serine or threonine side chains of the substrate are precisely positioned with respect to the gamma phosphate of the bound ATP. Cdk inhibitor proteins, or CKIs, help regulate the rise and fall of cyclin–Cdk activity. Some inhibitors—like the one shown here—bind directly at the kinase active site and block kinase activity by interfering with ATP binding. Other inhibitors—which are not shown here—bind near the active site and interfere with substrate binding. Molecular modeling and animation: Timothy Driscoll Original script: Peter Walter, Howard Hughes Medical Institute, University of California at San Francisco
  21. 21. Media Guide for The Biology of Cancer, Second Edition Movie 9.1 p53 Structure p53 is a tumor suppressor protein that prevents cells from dividing inappropriately. Loss of p53 function is associated with many forms of cancer. In this image, p53 polypeptide is shown binding to DNA, reflecting p53’s ability to act as a transcription factor. In fact, while not shown here, p53 is normally a tetramer and acts to induce expression of some genes and repress expression of others. p53 has a beta barrel adjacent to its DNA-binding domain. The p53–DNA interface is complex. It involves several loops and a helix that extends from the β barrel core. Residues from one loop and the helix bind in the DNA major groove. Arginine 248 from another loop makes extensive contacts with the DNA backbone and, indirectly through water molecules, with bases in the minor groove. Alterations in arginine 248 and other residues involved in DNA binding are commonly found in the p53 proteins present in human tumors. Loop 2 does not bind to DNA directly but is essential for correctly positioning arginine 248 on the DNA. Three cysteines and a histidine from both loop 2 and loop 3 cooperate to sequester a zinc ion, forming the rigid heart of a zinc-finger motif. Mutations that affect the zinc finger represent other examples of how DNA binding by p53 is compromised in human tumor cells. Molecular modeling and animation: Timothy Driscoll Original script: Peter Walter, Howard Hughes Medical Institute, University of California at San Francisco 21
  22. 22. 22 Media Guide for The Biology of Cancer, Second Edition 9.2 Apoptosis A poptosis, a form of programmed cell death, has been induced in these cultured cells. Cell death is characterized by blebbing of the plasma membrane and fragmentation of the nuclei. Suddenly, cells weaken attachment to the substratum that they have been growing on and shrivel up without lysing. In the following movies we observe the process at higher magnification.
The mechanism of apoptosis involves many tightly controlled steps, three of which are demonstrated here by different visualization techniques. One initial event is the sudden release of cytochrome c from mitochondria into the cytosol. This event has been visualized here using fluorescently labeled cytochrome c. Initially the greenish/yellow staining is restricted to a reticular pattern, which then suddenly disperses as the mitochondria release their content proteins into the cytosol. At a later step, the lipid asymmetry of the plasma membrane breaks down. In normal cells, phosphatidyl serine is found only on the cytosolic side of the plasma membrane; but when cells undergo apoptosis, it becomes exposed on the outside of the cell. This event has been visualized here by adding a red fluorescent protein to the media, which specifically binds phosphatidyl serine head groups as they become exposed. In an intact organism, exposure of phosphatidyl serine on the cell surface labels the dead cell and its remnants so that they are rapidly consumed by other cells, such as macrophages. Finally—although apoptosing cells don’t lyse—their plasma membranes do become permeable to small molecules. This event has been visualized here by adding a dye to the media that fluoresces blue when it can enter cells and bind to DNA. All three of these events can be observed in the same group of cells. These epithelial cells express green fluorescent cadherin. They are grown at low density, so that isolated cells can be observed. Initially, labeled cadherin is diffusely distributed over the whole cell surface. As cells crawl around and touch each other, cadherin becomes concentrated as it forms the adhesion junctions that link adjacent cells.
 Eventually, as the cell density increases further, the cells become completely surrounded by neighbors and form a tightly packed sheet of epithelial cells. Part I: © 2007 The Sakura Motion Picture Company. All rights reserved. Used with permission. Part II: © 2001 J.C. Goldstein and D.R. Green. All rights reserved. Used with permission. Part I: Shigekazu Nagata Kyoto University Part II: Joshua C. Goldstein The Genomics Institute of the Novartis Research Foundation Douglas R. Green St. Jude Children’s Research Institute
  23. 23. Media Guide for The Biology of Cancer, Second Edition Movie 10.1 Telomere Replication T he ends of linear chromosomes pose unique problems during DNA replication. Because DNA polymerases can only elongate from a free 3ʹ hydroxyl group, the replication machinery builds the lagging strand by a backstitching mechanism. RNA primers provide 3ʹ-hydroxyl groups at regular intervals along the lagging strand template. Whereas the leading strand elongates continuously in the 5ʹ-to-3ʹ direction all the way to the end of the template, the lagging strand stops short of the end. Even if a final RNA primer were built at the very end of the chromosome, the lagging strand would not be complete. The final primer would provide a 3ʹ-OH group to synthesize DNA, but the primers would later need to be removed. The 3ʹ-hydroxyl groups on adjacent DNA fragments provide starting places for replacing the RNA with DNA. However, at the end of the chromosome there is no 3ʹ-OH group available to prime DNA synthesis. Because of this inability to replicate the ends, chromosomes would progressively shorten during each replication cycle. This “end-replication” problem is solved by the enzyme telomerase. The ends of chromosomes contain a G-rich series of repeats called a telomere. Telomerase recognizes the tip of an existing repeat sequence. Using an RNA template within the enzyme, telomerase elongates the parental strand in the 5ʹ-to-3ʹ direction, and adds additional repeats as it moves down the parental strand. The lagging strand is then completed by DNA polymerase alpha, which carries a DNA primase as one of its subunits. In this way, the original information at the ends of linear chromosomes is completely copied in the new DNA. Storyboard and Animation: Sumanas, Inc. ( 23
  24. 24. 24 Media Guide for The Biology of Cancer, Second Edition Movie 12.1 DNA Repair Mechanisms F aults in DNA molecules can be harmful; so all organisms have DNA repair mechanisms that can correct most of them. Mutations (that is, changes in DNA sequence) can appear in DNA either as mistakes in DNA replication, which results in a mismatched nucleotide pair, or by the chemical or physical action of a mutagen. Other agents, known as clastogens, can cause breaks in DNA molecules that may, in turn, lead to mutations. Most cells possess four different types of DNA repair systems. Direct repair and excision repair systems fix DNA molecules carrying nucleotides damaged by mutagens. Mismatch repair corrects mismatched, but otherwise normal, nucleotides that result from errors in replication. Nonhomologous end-joining (NHEJ) is used to mend double-strand breaks in DNA. There is also a fifth repair system called homology-directed repair, but it will not be covered here. Direct repair is the simplest repair process and acts directly on damaged nucleotides, converting them back to their original structure. Only a few types of mutagen damage can be repaired directly. In this example, guanine acquires an alkyl group, which disrupts its normal pairing with cytosine. In a future round of DNA replication, the alkylated guanine would pair with thymine, thereby propagating a DNA mutation. To fix this chemical damage, an enzyme transfers the alkyl group to itself, restoring the base to normal. Many organisms have enzymes that perform this function, such as the Ada enzyme in E.coli and MGMT in humans. In contrast to this simple, single-step repair, most types of DNA damage require several steps to fix. They can be repaired only by removing the damaged nucleotides and then filling in the gap. This process is called excision repair. Base excision repair is a type of excision repair mechanism and is used to repair relatively minor damage. The damaged base is excised from a nucleotide by a specific DNA glycosylase, which first flips the damaged base out of the helix and then cuts the β-N-glycosidic bond between the base and sugar, leaving a baseless site, called an AP site. An AP endonuclease cuts the phosphodiester bond on the 5ʹ side of the AP site, and then the other 3ʹ side is cut, either by the endonuclease itself or by a phosphodiesterase. The resulting gap is filled in by a DNA polymerase and sealed by a DNA ligase. To correct more extensive types of damage, such as those that cause helix distortions, cells use another type of excision repair, called nucleotide excision repair. The process is different from base excision repair in that it begins with the removal of an entire block of nucleotides rather than a base from a single nucleotide. A characteristic example is the short-patch process used by E.coli. A complex of proteins, called the UvrAB trimer, scans DNA for damage. UvrA dissociates once the site has been found and plays no further part in the repair process. UvrC now binds, forming an UvrBC dimer that cuts the polynucleotide on either side of the damaged site, resulting in a 12-nucleotide excision. UvrB cuts a phosphodiester bond (often the 5th downstream of the damaged site) and then UvrC cuts another bond (often the 8th upstream of the damaged site). The excised segment is then removed by DNA helicase II, which breaks the hydrogen bonding holding the damaged segment in place. UvrC also detaches at this stage, but UvrB remains in place and bridges the gap produced by the excision. The gap is filled by DNA polymerase I. DNA ligase forms the last phosphodiester bond. In humans, a similar but more complex repair machinery fixes this type of damage. The direct, base excision, and nucleotide excision repair mechanisms all act by searching for and correcting abnormal chemical structures in DNA. However, they cannot correct mismatches resulting from errors in DNA replication, because the mismatched nucleotides are not abnormal in any way. A special type of excision repair, called mismatch repair, is used instead. Rather than detect an abnormal nucleotide, the mismatch repair system detects the absence of proper base pairing between the
  25. 25. Media Guide for The Biology of Cancer, Second Edition parent and daughter strands. Before the mismatch repair system can excise part of the daughter strand and fill in the gap, it must first distinguish between the parent and daughter strands. In E. coli, the two strands are distinguished by their differing levels of methylation. The newly synthesized daughter strand is not methylated, but the older parent strand is methylated at GATC, CCAGG, and CCTGG sequences. In the long-patch mismatch repair system of E. coli, a protein called MutS recognizes the mismatched nucleotides while another protein, called MutH, distinguishes the two strands by binding to nearby unmethylated 5ʹ-GATC-3ʹ sequences—that is, by binding to the daughter strand. MutH cuts the unmethylated DNA at the methylation site. Starting at this cut site, a DNA helicase detaches a segment of the single strand. The detached singlestranded region is degraded by an exonuclease that follows the helicase and continues beyond the mismatch site. The gap is then filled in by DNA polymerase I and DNA ligase. Note that eukaryotes don’t have heavily methylated DNA and likely use a different mechanism to distinguish between the parent and daughter strands. A double-stranded break in DNA is potentially devastating to a cell. Such breaks can be generated by exposure to ionizing radiation and some mutagens, and occasionally during DNA replication. They are repaired either by a process called nonhomologous end joining or homologous recombination, not illustrated here. In nonhomologous end-joining, a pair of proteins called Ku bind to broken DNA ends. The individual Ku proteins also have an affinity for one another, which brings them and the two broken ends of the DNA molecule into proximity. The DNA fragments are joined back together by a DNA ligase. Experimental studies indicate that restoring normal DNA structure is difficult to achieve. Often, the nucleotides flanking the double-stranded break are lost, resulting in loss of normal DNA sequence. Alternatively, if two chromosomes happen to be broken, a misrepair resulting in hybrid structures occurs relatively frequently. Hence, this type of repair is often prone to errors, in contrast to base excision repair, or mismatch repair, which restore normal DNA sequence with far greater fidelity. Storyboard and Animation: Sumanas, Inc. ( 25
  26. 26. 26 Media Guide for The Biology of Cancer, Second Edition 13.1 Mechanisms Enabling Angiogenesis A s a normal part of growth and development, the body must generate new blood vessels to oxygenate the tissues. In a process called angiogenesis, new vessels sprout from existing ones. In this movie, we see endothelial cells sprouting to form new branches from the aorta of a zebrafish embryo. Each sprout is initially formed by one or a few endothelial cells. Angiogenesis can be initiated in a number of different ways, such as when cells do not receive enough oxygen, often due to inadequate access to the circulation. In response, the cells emit molecules that stimulate angiogenesis, notably vascular endothelial growth factor, or VEGF. When VEGF molecules reach a nearby blood vessel, the vessel’s endothelial cells become motile and form a “tip cell” that produces long extensions called filopodia, which guide the development of a new vessel toward the cells emitting VEGF. As the tip cell moves toward its angiogenic stimulus, other endothelial cells behind it form a stalk. These stalk cells then start to hollow-out and form a tube. When tip cells emerging from different blood vessels meet, they merge, and blood can now begin to flow through the new vessel. As the young vessel matures, its endothelial cells recruit pericytes, which closely resemble smooth muscle cells, to the vessel walls. The pericytes help to stabilize vessel structure. Angiogenesis is also critical to the development and proliferation of tumors. From hypoxia or the actions of oncogenes, tumor cells often emit elevated levels of VEGF. Increased VEGF causes nearby blood vessels to produce excessive numbers of tip cells, often resulting in malformed capillaries. The capillaries found in tumors are ten times more permeable than normal capillaries, a condition directly attributable to the excessive levels of VEGF. Because blood cannot flow efficiently through malformed vessels, the tumor cells may remain hypoxic and continue to emit VEGF, perpetuating the formation of additional malformed vessels. Significantly, it is believed that the poorly assembled vessel walls permit escaped tumor cells to enter the bloodstream more readily, where they can travel to other locations in the body and start secondary tumors. Peter Carmeliet Vesalius Research Center, Catholic University of Leuven
  27. 27. Media Guide for The Biology of Cancer, Second Edition 27 13.2 Interactions of Innate Immune Cells with a Mammary Tumor T his time-lapse microscopic video of the tissue of a live mouse shows cells of the immune system patrolling groups of mammary carcinoma cells. This tumor was caused by the mouse mammary tumor virus polyoma middle T transgene. The tumor cells are stained in blue, while cells that have properties of macrophages and dendritic cells are labeled in green. Macrophages are known to play diverse, if not opposing, roles in tumor pathogenesis. One class of macrophages, called M1 macrophages, facilitate the attack on the tumor by the immune system. A second major class of macrophages, often called M2 macrophages, facilitate inflammatory responses and can actually accelerate tumorigenesis and malignant progression. As is apparent here, the cells of the immune system actively patrol the clusters of mammary tumor cells, ostensibly to acquire antigens for presentation to the immune system, and perhaps to release cytokines that may affect subsequent responses of the immune system to the presence of the tumor. Mikaela Egeblad and Zena Werb University of California, San Francisco Movie 14.1 Adhesion Junctions T hese epithelial cells express green fluorescent cadherin. They are grown at low density, so that isolated cells can be observed. Initially, labeled cadherin is diffusely distributed over the whole cell surface. As cells crawl around and touch each other, cadherin becomes concentrated as it forms the adhesion junctions that link adjacent cells. Eventually, as the cell density increases further, the cells become completely surrounded by neighbors and form a tightly packed sheet of epithelial cells. Music: Christopher Thorpe Stephen J. Smith Stanford University School of Medicine Cynthia Adams Finch University of Health Sciences and Chicago Medical School Yih-Tai Chen Cellomics, Inc. W. James Nelson Stanford University School of Medicine
  28. 28. 28 Media Guide for The Biology of Cancer, Second Edition Movie 14.2 Mechanisms of Brain Metastasis Formation M etastasis, or the formation of malignant growth at a site of the body far removed from the location of the primary tumor, is responsible for 90% of deaths from cancer. In this image we see multiple breast cancer metastases to the brain. Multiphoton laser scanning microscopy, together with image processing software, can be used to image metastases to the brain of a live mouse. A transparent window permits visualization of the various steps of the brain metastatic cascade that we will observe in this movie. The brain metastatic cascade can be broken down into four steps: (1) Initial arrest of circulating tumor cells in narrow microvessels; (2) escape of arrested cells from the microvessel into the brain parenchyma, the process of extravasation; (3) the establishment of the recently extravasated cancer cell in a location close to a blood vessel, referred to as the perivascular position; (4) and finally, two alternative routes of metastasis formation—either cooptive growth, in which cancer cells grow along existing blood vessels, or angiogenic growth, in which cancer cells actively induce the formation of new blood vessels to nourish them. In the examples that follow, melanoma cells took on cooptive growth, whereas lung cancer cells were found to take on angiogenic growth. Here we see the first step involving the initial arrest of a cancer cell, labeled red, in a capillary, labeled green. A second cell is later seen trapped above in an even smaller vessel. The second step in the brain metastatic cascade is the extravasation of cancer cells from the lumen of the capillary. Cancer cells use several alternative strategies to extravasate. In this movie, we see a cluster of cancer cells extravasating, one after the other, out of vessel number one into the brain parenchyma. The third step involves the establishment of perivascular positions, which seem to ensure the initial survival of the extravasated cells. This movie shows a tomographic image of cancer cells in perivascular positions. Nonetheless, assumption of a perivascular position does not on its own guarantee long-term survival, as seen in these images. Although this micrometastasis grows in a perivascular position for the first nine days, it is completely gone by Day 14. The fourth and final step in the brain metastatic cascade involves two alternative strategies—cooptive or angiogenic growth—depending on the type of cancer cell that has metastasized. As mentioned previously, lung cancer cells initiate angiogenic growth in order to nourish themselves. In this series of images, we see how the potent angiogenic powers of lung carcinoma cells are able to provoke the florid outgrowth of microvessels that sustain the active growth of the tumor. An alternative strategy for growth is exhibited by these melanoma cells, which have disseminated to the brain and assumed a perivascular position. Here we see how their expansion has depended on the cooptive strategy involving growth along the outside of blood vessels. The multiphoton microscopy used to create these movies demonstrates the extraordinary advances in the powers of modern imaging technology to study cancer in living tissue. Animation: Sumanas, Inc. ( Frank Winkler Neurology Clinic and National Center for Tumor Diseases & German Cancer Research Center (DKFZ), University of Heidelberg
  29. 29. Media Guide for The Biology of Cancer, Second Edition 29 Movie 14.3 Visualization of Cancer II: Metastasis M etastasis has generally been a difficult process to visualize. In this model, highresolution magnetic resonance imaging, or MRI, has been combined with tomography techniques to visualize the metastasis of breast cancer cells to the brain of a mouse. Before the injection of cancer cells into the circulation of the mouse, the cancer cells were labeled with a “dilutable” label, that is, one that loses half of its intensity each time a cell divides. This dilutable label is seen here in red. Following initial injection at Day 1, many hundreds of labeled cells are apparent in the brain. In the succeeding days, the number of these cells that retain full label, and therefore have not divided, is diminished. At the beginning of the third week, small metastatic growths suddenly begin to appear in green. These green signals represent actively growing cancer cells. These green metastases continue to grow in size while the individual nongrowing, single-cell metastases continue to disappear. Among other lessons, this experiment illustrates that hundreds if not thousands of micrometastatic single cells may be seeded initially in a target organ by cells arriving from the primary tumor via the circulation. However, only a minute fraction of the initially seeded cells succeed in spawning metastatic colonies. Movie 15.1 The Immune Response A n immune response involves events that unfold both locally, at the site of an infection, and at more distant sites, such as nearby lymph nodes. We can see the integration of the different parts of the immune response if we follow the course of a typical infection. Most pathogens are kept outside of the body by epithelial barriers, such as the epidermis, and are crossed only when there is an injury or tissue damage. After an injury, bacteria cross the epidermis and establish an infection in the underlying tissue. Phagocytic cells in the tissues, such as macrophages and neutrophils, engulf the pathogen. Dendritic cells are also phagocytic and are activated by binding pathogens to leave the site of infection and migrate to a lymph node. The migrating dendritic cells enter the lymphatic vessels and are collected in a draining lymph node. In the lymph node, T cells are activated by antigen presented by the dendritic cells, and in turn activate B cells to secrete antibody. Effector T cells and antibody molecules return to the circulation. They leave the circulation again at the site of infection, where inflammatory mediators have induced changes in the blood vessel endothelium. CD4 T cells activate macrophages to become more cytotoxic, while antibody recruits complement to lyse bacteria directly and to opsonize them, enhancing their uptake by phagocytes. In the case of a viral infection, activated CD8 T cells would kill any infected cells present. Ann Chambers London Health Sciences Centre
  30. 30. 30 Media Guide for The Biology of Cancer, Second Edition Movie 15.2 Antigen Display and T-Cell Attack W hen human cells are infected by viruses, the host cell ribosomes are exploited by the virus to translate their viral mRNA into viral proteins. In the normal course of protein turnover in the cell, the viral protein, like many endogenous proteins, can be tagged by the attachment of a chain of Ubiquitin molecules, shown here in blue. The poly-ubiquitin tagged protein is then led to a proteasome. The protein chain is then fed into the proteasome, which cleaves it into oligopeptides. Peptidase enzymes present in the cytosol then digest them further. On the cytoplasmic surface of the endoplasmic reticulum, some of these peptide fragments will interact with TAP proteins, which are specialized peptide transporters that pump them into the lumen of the endoplasmic reticulum. Upon entering the lumen of the endoplasmic reticulum, the peptides encounter MHC class I molecules. The peptides may then bind tightly to the peptide-binding groove of the MHC molecules, a specialized part of the MHC molecule, shaped like the palm of a hand. A membranous vesicle containing the MHC class I–peptide complex is then pinched off the ER and dispatched to the inner surface of the plasma membrane. The vesicle then fuses with the plasma membrane, and thereby exposes the contents of its lumen at the cell surface. In this way, the MHC class I and bound peptide are displayed on the cell surface, making them available for surveillance by the immune system. Cytotoxic T Lymphocytes, or CTLs, are an important arm of the immune system. A CTL, shown here in pink, may display on its surface an antigen-recognizing protein, specifically, a T cell receptor. The T cell receptor may recognize the oligopeptide being displayed by the MHC class I receptor. The CTL also depends on a second cell surface molecule, called CD8, which recognizes and binds all MHC class I molecules. This recognition and binding activates the cytotoxic T cell. The activated T cell first uses perforin to punch holes in the surface of the targeted cell, and then it injects proapoptotic enzymes, called granzymes, into the cytoplasm. Activation of the apoptotic cascade by granzymes eventually results in the death of the targeted, virus-infected cell. Biointeractive, Howard Hughes Medical Institute ©2013 HHMI Bruce D. Walker Howard Hughes Medical Institute, Harvard Medical School
  31. 31. Media Guide for The Biology of Cancer, Second Edition 31 Movie 16.1 Drug Export by the Multi-Drug Resistance Pump T ransmembrane drug efflux pumps can remove chemotherapeutic drugs from inside a cancer cell, thereby reducing intracellular drug concentration, which generates resistance to the cytotoxic effects of the drug. Certain drug efflux pumps can extrude multiple types of drug molecules creating the state of multi-drug resistance, or MDR. P-glycoprotein (Pgp) is the most prevalent MDR transporter and it is expressed at elevated levels in many kinds of cancer cells, especially those that have survived a chemotherapeutic treatment. This space-filling model of P-glycoprotein shows a drug molecule (colored in pink) bound to the pump’s drug binding pocket (colored in silver). Such a drug molecule will have entered the pump and its drug-binding pocket from the cytosol through the open portal seen at the bottom. Energy is required to pump drugs out of the cell. P-glycoprotein is an ATP-dependent transmembrane protein, and ATP must bind to the interior nucleotide-binding domains in order to pump a drug or other toxins out of the cell. Switching to a ribbon representation of the pump, we can observe the dramatic conformational change in the pump caused by ATP binding. Drugs or other toxins enter the pump, and attach to the drug-binding pocket, which results in an ATP-dependent shift in the conformation of the entire pump. The drug is then extruded into to the extracellular space. Rotating the structure 90 degrees in the same plane, we can observe the pumping action, and the opening and closing of the cytosolic and extracellular ends of the pump. In a third view, we observe the pump through its channel, looking down from the extracellular side. Animation: Sumanas, Inc. ( Geoffrey Chang The Scripps Research Institute
  32. 32. 32 Media Guide for The Biology of Cancer, Second Edition Movie 16.2 PI3K P hosphatidylinositol-3 kinase, or PI3K, is a multidomain enzyme. The structure of PI3K, as determined by X-ray crystallography, is shown here as a ribbon diagram. Each domain of PI3K is associated with distinct functions and colored differently. The Ras-binding domain is shown in red; this domain enables Ras to directly activate the PI3K catalytic domain. The catalytic domain is colored purple. This space-filling model shows the same protein but more closely resembles the actual 3-dimensional structure. Zooming-in to the catalytic domain, we enter the catalytic cleft and observe a drug molecule that inhibits normal PI3K enzyme function. The inhibitor molecule blocks the catalytic site and prevents PI3K from accessing its usual ATP substrate. This subsequently prevents PI3K from phosphorylating phosphatidylinositol diphosphate and disrupts downstream signaling. This drug molecule, called GDC-001, is shown here as a stick figure . . . and now as a space-filling model. In this view, we can see the complementary three-dimensional structures of the drug molecule and the walls of the surrounding catalytic cleft. The specificity of binding between the drug molecule and the catalytic cleft is achieved, in part, through the formation of hydrogen bonds between the drug molecule and the side-chains of amino acid residues lining the wall of the cleft. Paul Workman and Rob L.M. van Montfort Cancer Research UK
  33. 33. Media Guide for The Biology of Cancer, Second Edition Mini-Lectures by Robert A. Weinberg T he author has recorded sixteen mini-lectures for students and instructors on a range of topics covered in the book. These are available in MP3 format and can be transferred to a mobile device, as well as enjoyed on your computer. They are located in the “Mini-Lectures” folder on the DVD or can be found on the Garland Science website. Below is a list of the Mini-Lectures, followed by transcripts of the recordings. Mini-Lecture Table of Contents: 01. Mini-Lecture: Mutations and the Origin of Cancer 02. Mini-Lecture: Epidemiology and Cancer 03. Mini-Lecture: Cancer and Reproduction 04. Mini-Lecture: Growth Factors 05. Mini-Lecture: Tumor Suppressor Genes 06. Mini-Lecture: p53 and Apoptosis 07. Mini-Lecture: Cell Senescence 08. Mini-Lecture: Cancer Diagnosis 09. Mini-Lecture: Cancer Stem Cells 10. Mini-Lecture: Inflammation and Cancer 11. Mini-Lecture: Heterotypic Cells 12. Mini-Lecture: Metastasis I 13. Mini-Lecture: Metastasis II 14. Mini-Lecture: Immunology and Cancer 15. Mini-Lecture: Cancer Therapies 16. Mini-Lecture: The Coming Cancer Epidemic 33
  34. 34. 34 Media Guide for The Biology of Cancer, Second Edition 01. Mini-Lecture: Mutations and the Origin of Cancer A n abiding theme in much of modern cancer research is the notion that many cancer-causing agents, carcinogens, act through their ability to enter into the body’s tissues, and to damage specific genes inside previously normal cells. In other words, that carcinogens can act as mutagens to mutate genes. And, in fact, we do know that a large number of carcinogenic agents are responsible for creating mutations inside cells. And through these mutations that they create, these carcinogens are able to elicit the disease of cancer. Stated differently, we know, without any doubt, that cancer cells invariably have mutated genomes. This raises the question of: what are the agents that provoke many human cancers? In fact, in the case of lung cancer there is a clear chain of causality. Cigarette smoke contains a large number of combustion products that are inhaled into the lungs, and these agents, these chemicals, are then converted via various metabolic enzymes into chemical compounds that are capable of interacting with and forming covalent bonds with the DNA. Such structurally altered DNA molecules then may be replicated and yield ultimately altered DNA sequences, which we would call mutant genes. This raises the question of whether this can be generalized, or whether there are other sources of human cancer. The fact of the matter is that lung cancer may be leading us astray because it may be the case that the great majority of human cancers are not traceable to specific mutagenic chemicals that enter into the body. Possibly it’s the case that many of the agents that are carcinogens don’t act as mutagens but rather act through other mechanisms to provoke cancer. For example, we know that certain kinds of chronic viral infections are able to cause cancer in certain specific tissues. In East Asia for example, we know that life-long chronic Hepatitis B virus can lead to an almost 100-fold increased risk of liver cancer. We know that the Hepatitis B virus is not directly mutating genes inside the liver; instead it’s creating a chronic inflammatory state that, in turn, seems to be responsible for the pathogenesis for the development of the liver cancers. In a variety of other tissues we realize as well that areas of chronic inflammation have a greatly increased risk of ultimately spawning cancers. Then there is the striking and provocative discovery that certain anti-inflammatory drugs, including even simple household aspirin, can be very effective if taken on a daily basis in reducing by 30 or 40 % the incidence of certain commonly occurring cancers, including for example, colon cancer and perhaps even breast cancer. These effects of inflammation might be integrated into our rapidly evolving understanding of what the real agents are that are responsible for triggering many kinds of human cancers. Simply focusing on mutagenic chemicals blinds us to these other agents, which may be more commonly involved in the etiology that is the causation of human cancers.
  35. 35. Media Guide for The Biology of Cancer, Second Edition 02. Mini-Lecture: Epidemiology and Cancer T he search for the origins of cancer has gone on for several centuries. Our first clues to what triggered cancer came in the late eighteenth century, with the discovery by the London physician Percival Pott, that men who, in their youths, had worked as chimney sweeps came down with an unusually high rate of cancer of the scrotum, a disease that was otherwise relatively rare. He speculated at the time that, in fact, this unusual cancer came from the fact that these individuals were exposed to a large amount of creosote and tars that were present in the floes of London chimneys. In fact, his discovery soon went to the Continent, where, within a decade, chimney sweeps made sure that after they swept chimneys they made sure they washed themselves within a day or two, unlike in England, where people washed themselves every week or so, whether or not they needed it. And so by the beginning decades of the nineteenth century there was a dramatic drop in the rate of scrotal cancer among the chimney sweeps of the Continent. This interesting tale represents the first documented instance where we can actually point to a mechanism of cancer that can be traceable ultimately to an external source, that is, some type of carcinogen entering into the body from the outside and provoking a disease, in this case a tumor, at a greatly elevated rate. Today, two centuries later, we take for granted that many kinds of cancers are actually caused by external sources, external agents, which enter into the body, damage our tissues in one way or another, and provoke elevated risks of cancer. In the end, much of our conviction about these external agents for causing cancer comes from the science of epidemiology that surveys tumor incidence and tumor mortality in a variety of populations and attempts to correlate the risk of developing one or another kind of cancer with a specific lifestyle. Of course, such correlations do not prove causality, but sometimes correlations are so striking that they virtually represent a proof of causality. For example, already in 1950 there were indications in the United States and Britain that people who smoked heavily, specifically men, had as much as a 20-fold increased risk of lung cancer. Of course, this did not prove that smoking actually causes lung cancer, but it already sounded an alarm; and in the decades that followed there became experimental proofs of the notion that some of the substances in tobacco smoke are actually carcinogenic, that is to say, they are actually cancer-causing. Today the science of epidemiology has developed in a number of different directions, but it is clear already from the science of epidemiology that a number of the cancers that we experience in the Western world are due in no small part to differences in lifestyle: differences in smoking habits, in exercise, in body mass, and, very importantly, in diet. Again, these are all correlations, but in some cases the correlations are so strong as to remove much doubt about causation. For example, in many parts of Africa the rate of colon cancer is 1/20th as much as it is in Europe and in the Western Hemisphere, specifically in the United States and Canada. These staggering differences cannot be attributed to genetic differences in the populations. That is to say, American blacks that derive largely from western Africa and have therefore largely a African genetic heritage have rates of colon cancer that are comparable to the white population, and this proves to us that these staggering differences in colon cancer incidence and mortality are not due to genetic susceptibility. Similarly, until recently the rate of different kinds of diet-induced cancers was drastically different between Japan and the United States. However, when Japanese migrated to Hawaii, within a generation their cancer rates closely approximated the rates of cancer that people of European origin developed in the Hawaiian Islands. Again, this provides fuel for the argument that these staggering differences in cancer rates are not due to genetic susceptibility but rather to differences in lifestyle, in this case, largely one imagines, diet. As a consequence, one can begin to assess what fraction of cancer in Western societies is due to differences in lifestyle and what differences are due to the surrounding environment. When dealing with environmental pollution and contamination of the food chain, most epidemiologists estimate that less than one percent of cancer rates are due to these sources. Instead, the great majority of the cancers in the Western world are due to either tobacco consumption or alternatively to various types of 35
  36. 36. 36 Media Guide for The Biology of Cancer, Second Edition diets. Tobacco consumption is responsible in the United States, for example, for about 30–40 % of cancer incidence. At the same time, one imagines an almost equal amount of causation to be attributable to various kinds of diet. In 2007, for example, the American Cancer Society estimated that as many as 90,000 of the more than 500,000 deaths due to cancer in that year were ascribable to the fact that individuals had a high body mass index, that is to say, they had a high weight ratio compared with the physical dimensions of their body. In that sense, body mass index, or obesity, is the second most important cause of cancer causation after tobacco usage. If we look at various types of commonly occurring cancers in the West, including, for example, colon cancer, prostate cancer, and breast cancer, we see that diet appears to play a very important role in the causation of these diseases. And if we were to add up all these various causes, one might imagine that as much as 70 % of cancer in the West could be avoided if people were to alter their lifestyle. This in fact is a stunning revelation because it indicates that to the extent that we anticipate massive reductions in cancer mortality over the next generation, the bulk of those reductions will come not from the development of dramatic new cures to treat existing tumors, but rather from changes in lifestyle that prevent the appearance of cancer in the first place. Discussions like these often provoke the question of how a person might be able to avoid the increased risk of cancer, and here one focuses on the assignable causes that have been identified to date. Tobacco smoke, either directly inhaled or secondhand smoke, is an obvious cause, being responsible for about 35 % of cancer and obviously avoidable in most cases. Diet seems to be an extraordinarily important cause of cancer. Increased body mass index, that is, increased obesity, is correlated directly with elevated incidence of a variety of different kinds of cancer, as many as a dozen in men and in women. And this obviously suggests the notion that it is good to stay slim and physically in good shape throughout one’s life, even though we don’t fully realize how slim physique actually reduces the risk of cancer incidence. Also, it is becoming increasingly apparent that certain elements of Western diet, such as large amounts of red meat, of high fat, and of meat cooked at high temperatures is also deleterious and is almost certainly responsible for the increased risks of colon cancer, and likely prostate cancer, that are observed in the West. Breast cancer incidence is also influenced by diet, but here one must also take into account the fact that the reproductive history of a woman is also an important risk factor. For example, a woman who has had children starting at a relatively early age, say the age of 20, is at a much lower risk of eventually developing breast cancer than a woman who has her first child at the age of 30 or 35, or a woman who never has had any children. In the end, another important and unavoidable factor comes from the genes that we inherit from our parents. This obviously does not fall under the purview of epidemiology, because here the playing field is tilted from the moment we are born, and here there is little we can do to avoid certain susceptibilities that we have inherited from our parents.
  37. 37. Media Guide for The Biology of Cancer, Second Edition 03. Mini-Lecture: Cancer and Reproduction B reast cancer is of great concern to many women. Last year in the United States, for example, about 40,000 women died of the disease and perhaps five times as many were actually diagnosed with the disease. With that said, this raises the question of: what are the causes of breast cancer? We know, for example, that the internal hormonal environment of a woman is a strong determinant of risk of breast cancer; it is important to realize that because of nutrition and reproductive practices, the internal hormonal environment of women in the United States, for example, has changed dramatically over the last hundred and fifty years. We know, for example, that the number of menstrual cycles through which a woman goes is approximately proportional to her ultimate cancer risk, breast cancer risk specifically. We know, for example, that the breast cancer risk of a woman is roughly proportional to the number of menstrual cycles she goes through in a lifetime. These cycles have changed dramatically over the past 150 years and therefore so has the internal hormonal environment of women. In the mid-nineteenth century, for example, a woman might at the age of sixteen begin cycling, at the age of eighteen become married and begin pregnancy, and between subsequent pregnancies, lactation, and breast feeding, might rarely if ever go through a full menstrual cycle for the next twenty or thirty years, and then she might go into menopause at the age of forty. These days a girl will begin menstrual cycling, in the United States at least, at the age of as early as eleven or twelve because of increased nutrition, and she may not become pregnant until the age of twenty-five or thirty and then have only one or two children. And because of improved nutrition she may continue to cycle until she’s in her late forties before she goes into menopause. What this means is something quite astounding. An average eighteen year old American girl may already have gone through as many menstrual cycles as her great, great grandmother went through in an entire lifetime, to give you one example of how the internal hormonal environment has changed dramatically. In addition we know that once a woman goes through a pregnancy, part of the breast tissue is converted to a state where it is no longer susceptible to generating breast cancers. And therefore, the earlier in life one goes through this state of bearing a child, sometimes called parody, the more protective pregnancy is. The longer one postpones parody, the greater the breast cancer risk; women who never have children have a correspondingly increased risk of breast cancer. In saying all these things one can see how reproductive practices and nutrition have had a dramatic affect on the risk of breast cancer and that these effects dwarf any that might arise from the environment, that is, for example, from environmental pollution, which seem miniscule in comparison to the profound changes in the hormonal milieu existing inside a woman’s body. 37