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DNA forms a right-handed, double-helical structure that is composed of two antiparallel strands of unbranched polymeric deoxyribonucleotides linked by phosphodiester bonds between the 5′ carbon of one deoxyribose moiety to the 3′ carbon of the next (Fig. 15-2). DNA is composed of four types of deoxyribonucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T). The nucleotides are joined together by phosphodiester bonds. In the double-helical structure deduced by Watson and Crick, the two strands of DNA are complementary to each other. Because of size, shape, and chemical composition, A always pairs with T, and C with G, through the formation of hydrogen bonds between complementary bases that stabilize the double helix.
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Recognition of the hereditary transmission of genetic information is attributed to the Austrian monk, Gregor Mendel. His seminal work, ignored upon publication until its rediscovery in 1900, established the laws of segregation and of independent assortment. These two principles established the existence of paired elementary units of heredity and defined the statistical laws that govern them.5 DNA was isolated in 1869, and a number of important observations of the inherited basis of certain diseases were made in the early part of the twentieth century. Although today it appears easy to understand how DNA replicates, before the 1950s, the idea of DNA as the primary genetic material was not appreciated. The modern era of molecular biology began in 1944 with the demonstration that DNA was the substance that carried genetic information. The first experimental evidence that DNA was genetic material came from simple transformation experiments conducted in the 1940s using Streptococcus pneumoniae. One strain of the bacteria could be converted into another by incubating it with DNA from the other, just as the treatment of the DNA with deoxyribonuclease would inactivate the transforming activity of the DNA. Similarly, in the early 1950s, before the discovery of the double-helical structure of DNA, the entry of viral DNA and not the protein into the host bacterium was believed to be necessary to initiate infection by the bacterial virus or bacteriophage. Key historical events concerning genetics are outlined in Table 15-1.
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For cells to pass on the genetic material (DNA) to each progeny, the amount of DNA must be doubled. Watson and Crick recognized that the complementary base-pair structure of DNA implied the existence of a template-like mechanism for the copying of genetic material.1 The transfer of DNA material from the mother cell to daughter cells takes place during somatic cell division (also called mitosis). Before a cell divides, DNA must be precisely duplicated. During replication, the two strands of DNA separate, and each strand creates a new complementary strand by precise base-pair matching (Fig. 15-3). The two, new, double-stranded DNAs carry the same genetic information, which can then be passed on to two daughter cells. Proofreading mechanisms ensure that the replication process occurs in a highly accurate manner. The fidelity of DNA replication is absolutely crucial to maintaining the integrity of the genome from generation to generation. However, mistakes can still occur during this process, resulting in mutations, which may lead to a change of the DNA’s encoded protein and, consequently, a change of the cell’s behavior. The reliable dependence of many features of modern organisms on subtle changes in genome is linked to Mendelian inheritance and also contributes to the processes of Darwinian evolution. In addition, massive changes, so-called genetic instability, can occur in the genome of somatic cells such as cancer cells.
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Living cells have the necessary machinery to enzymatically transcribe DNA into RNA and translate the mRNA into protein. This machinery accomplishes the two major steps required for gene expression in all organisms: transcription and translation (Fig. 15-4). However, gene regulation is far more complex, particularly in eukaryotic organisms. For example, many gene transcripts must be spliced to remove the intervening sequences. The sequences that are spliced off are called introns, which appear to be useless, but in fact may carry some regulatory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of mRNA, control of mRNA stability, and its nuclear export into cytoplasm (where it is assembled into ribosomes for translation). After mRNA is translated into protein, the levels and functions of the proteins can be further regulated posttranslationally. However, the following sections will mainly focus on gene regulation at transcriptional and translational levels.
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Transcription is the enzymatic process of RNA synthesis from DNA.6 In bacteria, a single RNA polymerase carries out all RNA synthesis, including that of mRNA, rRNA, and tRNA. Transcription often is coupled with translation in such a way that an mRNA molecule is completely accessible to ribosomes, and bacterial protein synthesis begins on an mRNA molecule even while it is still being synthesized. Therefore, a discussion of gene regulation with a look at the simpler prokaryotic system precedes that of the more complex transcription and posttranscriptional regulation of eukaryotic genes.
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Transcription in Bacteria
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Initiation of transcription in prokaryotes begins with the recognition of DNA sequences by RNA polymerase. First, the bacterial RNA polymerase catalyzes RNA synthesis through loose binding to any region in the double-stranded DNA and then through specific binding to the promoter region with the assistance of accessory proteins called σ factors (sigma factors). A promoter region is the DNA region upstream of the transcription initiation site. RNA polymerase binds tightly at the promoter sites and causes the double-stranded DNA structure to unwind. Consequently, few nucleotides can be base-paired with the DNA template to begin transcription. Once transcription begins, the σ factor is released. The growing RNA chain may begin to peel off as the chain elongates. This occurs in such a way that there are always about 10 to 12 nucleotides of the growing RNA chains that are base-paired with the DNA template.
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The bacterial promoter contains a region of about 40 bases that include two conserved elements called –35 region and –10 region. The numbering system begins at the initiation site, which is designated +1 position, and counts backward (in negative numbers) on the promoter and forward on the transcribed region. Although both regions on different promoters are not the same sequences, they are fairly conserved and very similar. This conservation provides the accurate and rapid initiation of transcription for most bacterial genes. It is also common in bacteria that one promoter serves to transcribe a series of clustered genes, called an operon. A single transcribed mRNA contains a series of coding regions, each of which is later independently translated. In this way, the protein products are synthesized in a coordinated manner. Most of the time, these proteins are involved in the same metabolic pathway, thus demonstrating that the control by one operon is an efficient system. After initiation of transcription, the polymerase moves along the DNA to elongate the chain of RNA, although at a certain point, it will stop. Each step of RNA synthesis, including initiation, elongation, and termination, will require the integral functions of RNA polymerase as well as the interactions of the polymerase with regulatory proteins.
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Transcription in Eukaryotes
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Transcription mechanisms in eukaryotes differ from those in prokaryotes. The unique features of eukaryotic transcription are as follows: (a) Three separate RNA polymerases are involved in eukaryotes: RNA polymerase I transcribes the precursor of 5.8S, 18S, and 28S rRNAs; RNA polymerase II synthesizes the precursors of mRNA as well as microRNA; and RNA polymerase III makes tRNAs and 5S rRNAs. (b) In eukaryotes, the initial transcript is often the precursor to final mRNAs, tRNAs, and rRNAs. The precursor is then modified and/or processed into its final functional form. RNA splicing is one type of processing to remove the noncoding introns (the region between coding exons) on an mRNA. (c) In contrast to bacterial DNA, eukaryotic DNA often is packaged with histone and nonhistone proteins into chromatins. Transcription will only occur when the chromatin structure changes in such a way that DNA is accessible to the polymerase. (d) RNA is made in the nucleus and transported into cytoplasm, where translation occurs. Therefore, unlike bacteria, eukaryotes undergo uncoupled transcription and translation.
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Eukaryotic gene transcription also involves the recognition and binding of RNA polymerase to the promoter DNA. However, the interaction between the polymerase and DNA is far more complex in eukaryotes than in prokaryotes. Because the majority of studies have been focused on the regulation and functions of proteins, this chapter primarily focuses on how protein-encoding mRNA is made by RNA polymerase II.
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DNA directs the synthesis of RNA; RNA in turn directs the synthesis of proteins. Proteins are variable-length polypeptide polymers composed of various combinations of 20 different amino acids and are the working molecules of the cell. The process of decoding information on mRNA to synthesize proteins is called translation (see Fig. 15-1). Translation takes place in ribosomes composed of rRNA and ribosomal proteins. The numerous discoveries made during the 1950s made it easy to understand how DNA replication and transcription involve base-pairing between DNA and DNA or DNA and RNA. However, at that time, it was still impossible to comprehend how mRNA transfers the information to the protein-synthesizing machinery. The genetic information on mRNA is composed of arranged sequences of four bases that are transferred to the linear arrangement of 20 amino acids on a protein. Amino acids are characterized by a central carbon unit linked to four side chains: an amino group (–NH2), a carboxy group (–COOH), a hydrogen, and a variable (–R) group. The amino acid chain is assembled via peptide bonds between the amino group of one amino acid and the carboxy group of the next. Because of this decoding, the information carried on mRNA relies on tRNA. Translation involves all three RNAs. The precise transfer of information from mRNA to protein is governed by genetic code, the set of rules by which codons are translated into an amino acid (Table 15-2). A codon, a triplet of three bases, codes for one amino acid. In this case, random combinations of the four bases form 4 × 4 × 4, or 64 codes. Because 64 codes are more than enough for 20 amino acids, most amino acids are coded by more than one codon. The start codon is AUG, which also corresponds to methionine; therefore, almost all proteins begin with this amino acid. The sequence of nucleotide triplets that follows the start codon signal is termed the reading frame. The codons on mRNA are sequentially recognized by tRNA adaptor proteins. Specific enzymes termed aminoacyl-tRNA synthetases link a specific amino acid to a specific tRNA. The translation of mRNA to protein requires the ribosomal complex to move stepwise along the mRNA until the initiator methionine sequence is identified. In concert with various protein initiator factors, the methionyl-tRNA is positioned on the mRNA and protein synthesis begins. Each new amino acid is added sequentially by the appropriate tRNA in conjunction with proteins called elongation factors. Protein synthesis proceeds in the amino-to-carboxy-terminus direction.
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The biologic versatility of proteins is astounding. Among many other functions, proteins serve as enzymes that catalyze critical biochemical reactions, carry signals to and from the extracellular environment, and mediate diverse signaling and regulatory functions in the intracellular environment. They also transport ions and various small molecules across plasma membranes. Proteins make up the key structural components of cells and the extracellular matrix and are responsible for cell motility. The unique functional properties of proteins are largely determined by their structure (Fig. 15-5).
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Regulation of Gene Expression
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The human organism is made up of a myriad of different cell types that, despite their vastly different characteristics, contain the same genetic material. This cellular diversity is controlled by the genome and accomplished by tight regulation of gene expression. This leads to the synthesis and accumulation of different complements of RNA and, ultimately, to the proteins found in different cell types. For example, muscle and bone express different genes or the same genes at different times. Moreover, the choice of which genes are expressed in a given cell at a given time depends on signals received from its environment. There are multiple levels at which gene expression can be controlled along the pathway from DNA to RNA to protein (see Fig. 15-4). Transcriptional control refers to the mechanism for regulating when and how often a gene is transcribed. Splicing of the primary RNA transcript (RNA processing control) and selection of completed mRNAs for nuclear export (RNA transport control) represent additional potential regulatory steps. The mRNAs in the cytoplasm can be selectively translated by ribosomes (translational control) or selectively stabilized or degraded (mRNA degradation control). Finally, the resulting proteins can undergo selective activation, inactivation, or compartmentalization (protein activity control).
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Because a large number of genes are regulated at the transcriptional level, regulation of gene transcripts (i.e., mRNA) often is referred to as gene regulation in a narrow definition. Each of the steps during transcription is properly regulated in eukaryotic cells. Because genes are differentially regulated from one another, one gene can be differentially regulated in different cell types or at different developmental stages. Therefore, gene regulation at the level of transcription is largely context dependent. However, there is a common scheme that applies to transcription at the molecular level (Fig. 15-6). Each gene promoter possesses unique sequences called TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes longer distances) are a number of regulatory sequences referred to as enhancers that are recognized by regulatory proteins called transcription factors. These transcription factors specifically bind to the enhancers, often in response to environmental or developmental cues, and cooperate with each other and with basal transcription factors to initiate transcription. Regulatory sequences that negatively regulate the initiation of transcription also are present on the promoter DNA. The transcription factors that bind to these sites are called repressors, in contrast to the activators that activate transcription. The molecular interactions between transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regulated and context-dependent. Specifically, the recruitment of transcription factors to the promoter DNA occurs in response to physiologic signals. A number of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodomain motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.
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Genome is a collective term for all genes present in one organism. The human genome contains DNA sequences of 3 billion base pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall, it is 99.9% identical in all people.7,8 Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to environmental factors.
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The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of genetic material in detail (see Fig. 15-1). The medical field is building on the knowledge, resources, and technologies emanating from the human genome to further the understanding of the relationship of the genes and their mutations to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.
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The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This breakthrough has allowed scientists the opportunity to gain remarkable insights into the lives of humans. Ultimately, the goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the twenty-first century, work will begin to incorporate the information embedded in the human genome sequence into surgical practices. By doing so, the genomic information can be used for diagnosing and predicting disease and disease susceptibility. Diagnostic tests can be designed to detect errant genes in patients suspected of having particular diseases or of being at risk for developing them. Furthermore, exploration into the function of each human gene is now possible, which will shed light on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new generation of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of information on gene sequence and protein structure function rather than the traditional trial-and-error method. Drugs targeted to specific sites in the body promise to have fewer side effects than many of today’s medicines. Finally, other applications of genomics will involve the transfer of genes to replace defective versions or the use of gene therapy to enhance normal functions such as immunity.
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Proteomics refers to the study of the structure and expression of proteins as well as the interactions among proteins encoded by a human genome (see Fig. 15-1).9 A number of Internet-based repositories for protein sequences exist, including Swiss-Prot (http://www.expasy.ch). These databases allow comparisons of newly identified proteins with previously characterized sequences to allow prediction of similarities, identification of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profiling include two-dimensional gel electrophoresis, time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization, and protein microarrays. Structural proteomics aims to describe the three-dimensional structure of proteins that is critical to understanding function. Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential of this field of study is vast, it is in its early stages.
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It is anticipated that a genomic and proteomic approach to human disease will lead to a new understanding of pathogenesis that will aid in the development of effective strategies for early diagnosis and treatment.10 For example, identification of altered protein expression in organs, cells, subcellular structures, or protein complexes may lead to development of new biomarkers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accelerate drug development, but also lead to new strategies to evaluate therapeutic efficacy and potential toxicity.9
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Cell Cycle and Apoptosis
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Every organism is composed of many different cell types at different developmental stages. Some cell types continue to grow, while some cells stop growing after a developmental stage or resume growth after a break. For example, embryonic stem cells grow continuously, while nerve cells and striated muscle cells stop dividing after maturation. Cell cycle is the process for every cell including DNA replication and protein synthesis, DNA segregation in half, and package DNA and protein in two newly formed cells to enable passage of identical genetic information from one parental cell to two daughter cells. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-7). After a full cycle, the daughter cells enter G1 again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes called cyclin-dependent kinases (CDKs). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activities and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late G1 cyclin E/CDK2 controls the G1-S transition. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.
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The cell cycle is connected with signal transduction pathways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular signals, the G1 and G2 phases are the primary periods when cells decide whether or not to move on to the next phase. During the G1 phase, cells receive green- or red-light signals, S phase entry or G1 arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become committed to entry of the cell cycle only toward the end of G1. Mitogenic signals stimulate the activity of early G1 CDKs (e.g., cyclin D/CDK4) that inhibit the activity of pRb protein and activate the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor suppressors. These antiproliferative signals also act in the G1 phase to stop cells’ progress into the S phase by inducing CKI production. For example, when DNA is damaged, cells will repair the damage before entering the S phase. Therefore, G1 contains one of the most important checkpoints for cell cycle progression. If the analogy is made that CDK is to a cell as an engine is to a car, then cyclins and CKI are the gas pedal and brake, respectively. Accelerated proliferation or improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the cyclin/CDK complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for malignant transformation.
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In addition to cell cycle control, cells use genetically programmed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the maintenance of tissue homeostasis (Fig. 15-8).
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Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth factor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apoptosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. Similarly to CDK in the cell cycle, activities and expression of caspases are well controlled by positive and negative regulators. The complex machinery of apoptosis must be tightly controlled. Perturbations of this process can cause neoplastic transformation or other diseases.
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Signal Transduction Pathways
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Gene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.11 A signaling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-9). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, retinoids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by diffusion to bind to a specific target protein within the cytoplasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein interactions and the transfer of high-energy phosphate groups, leading to various cellular end responses.
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Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a common feature of signal transduction pathways in cells.12 Signaling also involves catalytic activities of signaling molecules, such as protein kinases/phosphatases, that modify the structures of key signaling proteins. Upon binding and/or modification by upstream signaling molecules, downstream effectors undergo a conformational (allosteric) change and, consequently, a change in function. The signal that originates at the cell surface and is relayed by the cytoplasmic proteins often ultimately reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal alterations in signaling activities and capacities in otherwise normal cells can lead to diseases such as cancer.
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Advances in biology in the last two decades have dramatically expanded the view on how cells are wired with signaling pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell generally may react to a hormonal signal in a variety of ways: (a) by changing its metabolite or protein, (b) by generating an electric current, or (c) by contracting. Cells continually are subject to multiple input signals that simultaneously and sequentially activate multiple receptor- and non–receptor-mediated signal transduction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a result of genomic and proteomic techniques, the specific functions of the individual proteins, how they assemble, and the networks that control cellular behavior remain to be defined. An increased understanding of cell regulatory pathways—and how they are disrupted in disease—will likely reveal common themes based on protein interaction domains that direct associations of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the understanding of signaling networks will require methods of investigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewildering biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and proteomic data mining will require the development of new modeling methodologies within the emerging disciplines of medical mathematics and physics.
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Signaling pathways often are grouped according to the properties of signaling receptors. Many hydrophobic signaling molecules are able to diffuse across plasma membranes and directly reach specific cytoplasmic targets. Steroid hormones, thyroid hormones, retinoids, and vitamin D are examples that exert their activity upon binding to structurally related receptor proteins that are members of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact with transmembrane protein receptors that couple ligand binding to intracellular signals, leading to biologic actions.
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There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–coupled receptors (GPCRs), and enzyme-linked receptors. The superfamily of GPCRs is one of the largest families of proteins, representing over 800 genes of the human genome. Members of this superfamily share a characteristic seven-transmembrane configuration. The ligands for these receptors are diverse and include hormones, chemokines, neurotransmitters, proteinases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through heterotrimeric G proteins, which are guanine-nucleotide regulatory complexes. Thus the receptor serves as the receiver, the G protein serves as the transducer, and the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a cytosolic domain that either has intrinsic enzymatic activity or directly links with an enzyme. Structurally, these receptors usually have only one transmembrane-spanning domain. Of at least five forms of enzyme-linked receptors classified by the nature of the enzyme activity to which they are coupled, the growth factor receptors such as tyrosine kinase receptor or serine/threonine kinase receptors mediate diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth factor signaling pathways and their connection with human diseases.
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Insulin Pathway and Diabetes
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The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease.13 Insulin is a peptide hormone that is secreted by the β-cell of the pancreas. Insulin is required for the growth and metabolism of most mammalian cells, which contain cell-surface insulin receptors (InsR). Insulin binding to InsR activates the kinase activity of InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a central role in coordinating the signaling of insulin by activating distinct signaling pathways, the PI3K-Akt pathway and MAPK pathway, both of which possess multiple protein kinases that can control transcription, protein synthesis, and glycolysis (Fig. 15-10).
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The primary physiologic role of insulin is in glucose homeostasis, which is accomplished through the stimulation of glucose uptake into insulin-sensitive tissues such as fat and skeletal muscle. Defects in insulin synthesis/secretion and/or responsiveness are major causal factors in diabetes, one of the leading causes of death and disability in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic populations points to a strong genetic background for the disease. More than 90% of affected individuals have insulin resistance, which develops when the body is no longer able to respond correctly to insulin circulating in the blood. Although relatively little is known about the biochemical basis of this metabolic disorder, it is clear that the insulin-signaling pathways malfunction in this disease. It is also known that genetic mutations in the InsR or IRS cause type 2 diabetes, although which one is not certain. The majority of type 2 diabetes cases may result from defects in downstream-signaling components in the insulin-signaling pathway. Type 2 diabetes also is associated with declining β-cell function, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of insulin resistance is crucial for the development of new therapies for type 2 diabetes. Furthermore, apart from type 2 diabetes, insulin resistance is a central feature of several other common human disorders, including atherosclerosis and coronary artery disease, hypertension, and obesity.
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Transforming Growth Factor-β (TGF-β) Pathway and Cancers
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Growth factor signaling controls cell growth, differentiation, and apoptosis.14 Although insulin and many mitogenic growth factors promote cell proliferation, some growth factors and hormones inhibit cell proliferation. TGF-β is one of them. The balance between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role in maintaining tissue homeostasis.
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The TGF-β superfamily comprises a large number of structurally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The complex consists of transmembrane serine/threonine kinases. The receptor signals through activation of heterotrimeric complexes of intracellular effectors called SMADs (which are contracted from homologous Caenorhabditis elegans Sma and Drosophila Mad, two evolutionarily conserved genes for TGF-β signaling). Upon phosphorylation by the receptors, SMAD complexes translocate into the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differentiation. For example, TGF-β strongly induces the transcription of a gene called p15INK4B (a type of CKI) and, at the same time, reduces the expression of many oncogenes such as c-Myc. The outcome of the altered gene expression leads to the inhibition of cell cycle progression. Meanwhile, the strength and duration of TGF-β signaling is fine-tuned by a variety of positive or negative modulators, including protein phosphatases. Therefore, controlled activation of TGF-β signaling is an intrinsic mechanism for cells to ensure controlled proliferation.
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Resistance to TGF-β’s anticancer action is one hallmark of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some lose TGF-β responsiveness through downregulation or mutations of their TGF-β receptors. The cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. The locus encoding cell cycle inhibitor p15INK4B may be deleted. Alternatively, the immediate downstream target of its actions, cyclin-dependent kinase 4 (CDK4), may become unresponsive to the inhibitory actions of p15INK4B because of mutations that block p15INK4B binding. The resulting cyclin D/CDK4 complexes constitutively inactivate tumor suppressor pRb by hyperphosphorylation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pancreatic and colorectal cancers, 100% of cells derived from these cancers carry genetic defects in the TGF-β signaling pathway. Therefore, the antiproliferative pathway converging onto pRb and the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregulation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.
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Gene Therapy and Molecular Drugs in Cancer
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Modern advances in the use of molecular biology to manipulate genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the genome, the continuous understanding of how the genome functions will make it possible to tailor medicine on an individual basis. Although significant hurdles remain, the course toward therapeutic application of molecular biology already has been mapped out by many proof-of-principle studies in the literature. In this section, cancer is used as an example to elaborate some therapeutic applications of molecular biology. Modern molecular medicine includes gene therapy and molecular drugs that target genes or gene products that wire human cells.
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Cancer is a complex disease, involving uncontrolled growth and spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific characteristics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis. Many lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives. The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as subtle as point mutations and as obvious as changes in chromosome complement. A succession of genetic changes, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into cancer cells.
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Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a disease involving dynamic changes in the genome. The causes of cancer include genetic predisposition, environmental influences, infectious agents, and aging. These transform normal cells into cancerous ones by derailing a wide spectrum of regulatory pathways including signal transduction pathways, cell cycle machinery, or apoptotic pathways.15,16 The early notion that cancer was caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. There are two classes of cancer genes in which alteration has been identified in human and animal cancer cells: oncogenes, with dominant gain-of-function mutations, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppressors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.
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During the development of most types of human cancer, cancer cells can break away from primary tumor masses, invade adjacent tissues, and hence travel to distant sites where they form new colonies. This spreading process of tumor cells, called metastasis, is the cause of 90% of human cancer deaths. Metastatic cancer cells that enter the bloodstream can reach virtually all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone metastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and problems for patients.
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The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge acquired through hard work and discovery has made it possible for cancer treatment and prevention. As a result of explosive new discoveries, some modern treatments were developed. The success of these therapies, together with traditional treatments such as surgical procedures, is further underscored by the fact that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alternatively, several new biologic- and gene-based therapies are aimed at enhancing the body’s natural defenses against invading cancers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, genetically engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.
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The growth of the body is controlled by many natural signals through complex signaling pathways. Some of these natural agents have been used in cancer treatment and have been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be produced in the laboratory. These agents, as well as the synthetic agents that mimic the natural signals, are given to patients to influence the natural immune response agents either by directly altering the cancer cell growth or by acting indirectly to help healthy cells control the cancer. One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of localizing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an example of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth factor receptor HER-2, which is overexpressed in approximately 25% of breast cancers. HER-2–overexpressing tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER-2. Trastuzumab is designed to attack cancer cells that overexpress HER-2 by slowing or preventing the growth of these cells, resulting in increased survival of HER-2–positive breast cancer patients. Another significant example is the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer, which has been shown to mediate the durable regression of metastatic cancer. IL-2, a cytokine produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct impact on cancer cells, the impact of IL-2 on cancers in vivo derives from its ability to expand lymphocytes with antitumor activity. The expanded lymphocyte pool enables recognition of the antigen on cancer cells. Thus, the molecular identification of cancer antigens has opened new possibilities for the development of effective immunotherapies for patients with cancer. Clinical studies using immunization with peptides derived from cancer antigens have shown that high levels of lymphocytes with antitumor activity can be produced in cancer-bearing patients. Highly avid antitumor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.
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The primary function of anticancer chemicals is to block different steps involved in cell growth and replication. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells.18 STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-13). Bcr-Abl is an overly activated protein kinase resulting from a specific genetic abnormality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibition of Bcr-Abl kinase activity not only prevents cell growth of Bcr-Abl–transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormalities caused by the leukemia in a majority of patients, achieving a complete disappearance of the leukemic blood cells and the return of normal blood cells. Additionally, the drug appears to have some effect on other cancers including certain brain tumors and gastrointestinal (GI) stromal tumors, a very rare type of stomach cancer.
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Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lymphocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer. Although a rapid progression in the understanding of the molecular and clinical aspects of gene therapy has been witnessed in the past decade, gene therapy treatment has not yet been shown to be superior to standard treatments in humans.
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Several problems must be resolved to transform it into a clinically relevant form of therapy. The major issues that limit its translation to the clinic are improving the selectivity of tumor targeting, improving the delivery to the tumor, and the enhancement of the transduction rate of the cells of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). The in situ gene therapy also offers a better distribution of the vector virus throughout the tumor. Finally, a combination of gene therapy strategies will be more effective than the use of a single gene therapy system. An important aspect of effective gene therapy involves the choice of appropriate genes for manipulation. Genes that promote the production of messenger chemicals or other immune-active substances can be transferred into the patient’s cells. These include genes that inhibit cell cycle progression, induce apoptosis, enhance host immunity against cancer cells, block the ability of cancer cells to metastasize, and cause tumor cells to undergo suicide. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental and is being studied in clinical trials for many different types of cancer. The mapping of genes responsible for human cancer is likely to provide new targets for gene therapy in the future. The preliminary results of gene therapy for cancer are encouraging, and as advancements are made in the understanding of the molecular biology of human cancer, the future of this rapidly developing field holds great potential for treating cancer.
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It is noteworthy that the use of multiple therapeutic methods has proven more powerful than a single method. The use of chemotherapy after surgery to destroy the few remaining cancerous cells in the body is called adjuvant therapy. Adjuvant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A major discovery in chemotherapy is the advantage of multiple chemotherapeutic agents (known as combination or cocktail chemotherapy) over single agents. Some types of fast-growing leukemias and lymphomas (tumors involving the cells of the bone marrow and lymph nodes) responded extremely well to combination chemotherapy, and clinical trials led to gradual improvement of the drug combinations used. Many of these tumors can be cured today by combination chemotherapy. As cancer cells carry multiple genetic defects, the use of combination chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.
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Stem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enormous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neurologic degeneration, and congenital heart disease. Stem cells are endowed with two remarkable properties (Fig. 15-14). First, stem cells can proliferate in an undifferentiated but pluripotent state and, as a result, can self-renew. Second, they have the ability to differentiate into many specialized cell types. There are two groups of stem cells: embryonic stem (ES) cells and adult stem cells. Human ES cells are derived from early preimplantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can generate the cell types comprising a particular tissue in the body; therefore, they are considered multipotent. However, in some cases, they can transdifferentiate into cell types found in other tissues, called transdifferentiation. For example, hematopoietic stem cells are adult stem cells. They reside in bone marrow and are capable of generating all cell types of the blood and immune system.
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Stem cells can be grown in culture and be induced to differentiate into a particular cell type, either in vitro or in vivo. With the recent and continually increasing improvement in culturing stem cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentiation in response to environmental cues. It is believed that discovery of the signals that control self-renewal vs. differentiation will be extremely important for the therapeutic use of stem cells in treating disease. It is possible that success in the study of the changes in signal transduction pathways in stem cells will lead to the development of therapies to replace diseased or damaged cells in the body using stem cell derivatives. Recently, stem cell research has been transformed by the discovery from the Shinya Yamanaka group and the James Thomsen group, who have found that a simple genetic manipulation can reprogram adult differentiated cells back into pluripotent stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a potentially limitless source of patient-specific stem cells for tissue engineering and regenerative medicine.
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The Atomic Theory of Disease
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The staggering advances in anatomy, physiology, and molecular biology over the past centuries have led us to our current state in which the atom is now the anatomy of the twenty-first century.22 As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be bridging the subatomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composition of subatomic particles are the root cause of disease. The atomic theory of disease would include genetic alterations at the atomic/subatomic level that are akin to single nucleotide polymorphisms (SNPs), in which alleles for a gene differ on the exact nucleotide in a single location, which can change the ultimate protein structure. This can lead to subtle changes in function or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymorphisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of subatomic polymorphism, as they differ in the number of neutrons present in the atom. Differences in other particles may not change the mass of the atom, but may alter some of the characteristics of the atom.
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A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemoglobinemia, a disorder characterized by an overabundance of methemoglobin. Methemoglobin contains an oxidized form of iron (carrying an extra electron), as opposed to the reduced form in normal hemoglobin. This results in a shift in the oxygen-hemoglobin dissociation curve to the left, causing hypoxia. Methemoglobinemia can be congenital, due to a defect in an enzyme that normally reduces methemoglobin back to hemoglobin, or acquired, caused by breakdown products of drugs that can oxidize hemoglobin. Although there is less than 1% of methemoglobin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and death.