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Surgery_Schwartz. 482BASIC CONSIDERATIONSPART IFigure 15-3. DNA replication. As the nucleotide A only pairs with T, and G with C, each strand of DNA can determine the nucleotide sequence in its complementary strand. In this way, double-helical DNA can be copied precisely.Table 15-1Historical events in genetics and molecular biologyYEARINVESTIGATOREVENT1865MendelLaws of genetics established1869MiescherDNA isolated1905GarrodHuman inborn errors of metabolism1913SturtevantLinear map of genes1927MullerX-rays cause inheritable genetic damage1928GriffithTransformation discovered1941Beadle and Tatum“One gene, one enzyme” concept1944Avery, MacLeod, McCartyDNA as material of heredity1950McKlintockExistence of transposons confirmed1953Watson and CrickDouble-helical structure of DNA1957Benzer and KornbergRecombination and DNA polymerase1966Nirenberg, Khorana, HolleyGenetic code determined1970Temin and BaltimoreReverse transcriptase1972Cohen, Boyer, BergRecombinant DNA technology1975SouthernTransfer of DNA


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Surgery_Schwartz. and DNA polymerase1966Nirenberg, Khorana, HolleyGenetic code determined1970Temin and BaltimoreReverse transcriptase1972Cohen, Boyer, BergRecombinant DNA technology1975SouthernTransfer of DNA fragments from sizing gel to nitrocellulose (Southern blot)1977Sanger, Maxim, GilbertDNA sequencing methods1982—GenBank database established1985MullisPolymerase chain reaction1986—Automated DNA sequencing1989CollinsCystic fibrosis gene identified by positional cloning and linkage analysis1990—Human Genome Project initiated1997Roslin InstituteMammalian cloning (Dolly)2001IHGSC and Celera GenomicsDraft versions of human genome sequence published2003—Human Genome Project completedIHGSC = International Human Genome Sequencing Consortium.GTGGCCTAAACAGGCCTTTACAGGCCTTTAGTGGCCTAAACAGGCCTTTAGTGGCCTAAAS strandS strandNew S strandNew S strandTemplate S strandTemplate S strand5'3'3'5'5'5'5'5'3'3'3'Parent DNA double helix (shown flat):3'DNA is a template for its own duplicationBrunicardi_Ch15_p0479-p0510.indd


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Surgery_Schwartz. strandNew S strandNew S strandTemplate S strandTemplate S strand5'3'3'5'5'5'5'5'3'3'3'Parent DNA double helix (shown flat):3'DNA is a template for its own duplicationBrunicardi_Ch15_p0479-p0510.indd 48218/02/19 11:12 AM 483MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15NucleusCytoplasmDNARNAtranscriptmRNAmRNAProteinActiveproteinmRNAturnoverProteinturnoverTranscriptionRNAtransportTranscriptionalcontrolPosttranscriptionalcontrolTranslationalcontrolPosttranslationalcontrolNuclear envelopeRNAdegradationProteindegradationPosttranslationalmodificationTranslationRNAprocessingFigure 15-4. Four major steps in the control of eukaryotic gene expression. Transcriptional and posttranscriptional control determine the level of messenger RNA (mRNA) that is available to make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several


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Surgery_Schwartz. make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several steps.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 prokary-otic system precedes that of the more complex transcription and posttranscriptional regulation of eukaryotic genes.Transcription in Bacteria Initiation of transcription in pro-karyotes begins with the recognition of DNA sequences by RNA polymerase. First, the bacterial RNA polymerase cata-lyzes 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 pro-teins called σ


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Surgery_Schwartz. cata-lyzes 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 pro-teins 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.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


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Surgery_Schwartz. 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 nega-tive 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 bac-teria 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 ini-tiation of transcription, the


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Surgery_Schwartz. 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 ini-tiation 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 poly-merase with regulatory proteins.Transcription in Eukaryotes 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 pre-cursor to


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Surgery_Schwartz. 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 pre-cursor 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. Transcrip-tion 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.Eukaryotic gene transcription also involves the recogni-tion and binding of RNA polymerase to the promoter DNA. However, the interaction between the


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Surgery_Schwartz. undergo uncoupled transcription and translation.Eukaryotic gene transcription also involves the recogni-tion 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.Translation. 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


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Surgery_Schwartz. 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. How-ever, 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 Brunicardi_Ch15_p0479-p0510.indd 48318/02/19 11:12 AM 484BASIC CONSIDERATIONSPART ITable 15-2The genetic codeSECOND BASE IN CODON U C A G First Base in CodonUUUUPhe[F]UCUSer[S]UAUTyr[Y]UGUCys[C]UThird Base in


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Surgery_Schwartz. 48318/02/19 11:12 AM 484BASIC CONSIDERATIONSPART ITable 15-2The genetic codeSECOND BASE IN CODON U C A G First Base in CodonUUUUPhe[F]UCUSer[S]UAUTyr[Y]UGUCys[C]UThird Base in Codon UUCPhe[F]UCCSer[S]UACTyr[Y]UGCCys[C]C UUALeu[L]UCASer[S]UAASTOP—UGASTOP—A UUGLeu[L]UCGSer[S]UAGSTOP—UGGTrp[W]G CCUULeu[L]CCUPro[P]CAUHis[H]CGUArg[R]U CUCLeu[L]CCCPro[P]CACHis[H]CGCArg[R]C CUALeu[L]CCAPro[P]CAAGln[Q]CGAArg[R]A CUGLeu[L]CCGPro[P]CAGGln[Q]CGGArg[R]G AAUUIle[I]ACUThr[T]AAUAsn[N]AGUSer[S]U AUCIle[I]ACCThr[T]AACAsn[N]AGCSer[S]C AUAIle[I]ACAThr[T]AAALys[K]AGAArg[R]A AUGMet[M]ACGThr[T]AAGLys[K]AGGArg[R]G GGUUVal[V]GCUAla[A]GAUAsp[D]GGUGly[G]U GUCVal[V]GCCAla[A]GACAsp[D]GGCGly[G]C GUAVal[V]GCAAla[A]GAAGlu[E]GGAGly[G]A GUGVal[V]GCGAla[A]GAGGlu[E]GGGGly[G]G A = adenine; C = cytosine; G = guanine; U = uracil; Ala = alanine; Arg = arginine; Asn = asparagine; Asp = aspartic acid; Cys = cysteine; Glu = glutamic acid; Gln = glutamine; Gly = glycine; His = histidine;


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Surgery_Schwartz. C = cytosine; G = guanine; U = uracil; Ala = alanine; Arg = arginine; Asn = asparagine; Asp = aspartic acid; Cys = cysteine; Glu = glutamic acid; Gln = glutamine; Gly = glycine; His = histidine; Ile = isoleucine; Leu = leucine; Lys = lysine; Met = methionine; Phe = phenylalanine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Tyr = tyrosine; Val = valine. Letter in [ ] indicates single letter code for amino acid.arranged sequences of four bases that are transferred to the lin-ear 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


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Surgery_Schwartz. 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


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Surgery_Schwartz. 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 step-wise 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.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


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Surgery_Schwartz. 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 mem-branes. 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 deter-mined by their structure (Fig. 15-5).Regulation of Gene Expression. The human organism is made up of a myriad of different cell types that, despite their vastly different characteristics, contain the same genetic mate-rial. This cellular diversity is controlled by the genome and is 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


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Surgery_Schwartz. 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 tran-script (RNA processing control) and selection of completed Brunicardi_Ch15_p0479-p0510.indd 48418/02/19 11:12 AM 485MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Figure 15-5. Maturation of a functional protein. Although the lin-ear amino acid sequence of a protein often is shown, the function of a protein also is controlled by its correctly folded three-dimensional


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Surgery_Schwartz. 15-5. Maturation of a functional protein. Although the lin-ear amino acid sequence of a protein often is shown, the function of a protein also is controlled by its correctly folded three-dimensional structure. In addition, many proteins also have covalent posttransla-tional modifications such as phosphorylation or noncovalent bind-ing to a small molecule or a protein.Unfolded inactive proteinFolded inactive protein Mature inactive proteinBinding proteinPosttranslationalmodification(e.g., phosphorylation)PCofactor bindingmRNAs for nuclear export (RNA transport control) represent additional potential regulatory steps. The mRNAs in the cyto-plasm can be selectively translated by ribosomes (translational control) or selectively stabilized or degraded (mRNA degrada-tion control). Finally, the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional level,


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Surgery_Schwartz. the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional 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 differ-ent 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 lon-ger


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Surgery_Schwartz. 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 lon-ger 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 Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP


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Surgery_Schwartz. The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP and polymerase II holoenzyme; TBP = TATA-binding protein and associated factors; TF = hypothetical transcription factor; TFBS = transcription factor binding site; ball-shaped structures = nucleosomes. Coactivator or corepressor is a factor linking the TF with the Pol II complex.TFCoactivator orCorepressorPol IIHoloenzymeTBPTBPTATATFBSbetween transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regu-lated 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 homeodo-main motif, the zinc finger, the leucine


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Surgery_Schwartz. of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.Human GenomeGenome is a collective term for all genes present in one organ-ism. 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 envi-ronmental factors.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


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Surgery_Schwartz. factors.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 muta-tions to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This break-through 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 21st century, work will begin to incorporate the information


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Surgery_Schwartz. is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st 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 suscep-tibility. 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 3Brunicardi_Ch15_p0479-p0510.indd 48518/02/19 11:12 AM 486BASIC CONSIDERATIONSPART Ilight on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new gen-eration 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


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Surgery_Schwartz. the development of a new gen-eration 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 tradi-tional 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.Proteomics refers to the study of the structure and expression of proteins as well as the interactions among pro-teins encoded by a human genome (see Fig. 15-1).9 A num-ber of Internet-based repositories for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of


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Surgery_Schwartz. for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of similarities, identifi-cation of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profil-ing 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


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Surgery_Schwartz. 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.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 biomark-ers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many


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Surgery_Schwartz. and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many different cell types at dif-ferent 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 synthe-sis, 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


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Surgery_Schwartz. 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 Figure 15-7. The cell cycle and its control system. M is the mito-sis phase, when the nucleus and the cytoplasm divide; S is the phase when DNA is duplicated; G1 is the gap between M and S; G2 is the gap between S and M. A complex of cyclin and cyclin-dependent kinase (CDK) controls specific events of each phase. Without cyclin, CDK is inactive. Different cyclin/CDK complexes are shown around the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle,


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Surgery_Schwartz. cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells 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 activi-ties 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 transi-tion. 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.The cell cycle is connected with signal transduction path-ways as well as gene expression.


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Surgery_Schwartz. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular sig-nals, 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 greenor 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


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Surgery_Schwartz. 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 dam-aged, 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 Brunicardi_Ch15_p0479-p0510.indd 48618/02/19 11:12 AM 487MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the


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Surgery_Schwartz. AND PRECISION SURGERYCHAPTER 15improper 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 malig-nant transformation.In addition to cell cycle control, cells use genetically pro-grammed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the mainte-nance of tissue homeostasis (Fig. 15-8).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 fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing


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Surgery_Schwartz. as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apop-tosis: 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. Similar 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 transforma-tion or other diseases.Signal Transduction PathwaysGene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors,


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Surgery_Schwartz. temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling 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, reti-noids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by NucleusDeath signal(e.g., TNF or Fas)DeathreceptorPlasmamembraneActivation ofcaspase cascadeCytochrome creleaseDeathreceptorsignalingpathwayMitochondrionNormal target cellApoptotic target cellFigure 15-8. A simplified view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way.


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Surgery_Schwartz. view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way. Intracellular death pathway indicates the release of cytochrome c from mitochon-dria, which also triggers the activation of the caspase cascade. During apoptosis, cells undergo DNA fragmentation and nuclear and cell membrane breakdown and are eventually digested by other cells.Ligand(e.g., growth factor)Cell-surfacereceptorPlasmamembraneNucleusGeneexpressionLigand(e.g., hormone)Signaling cascadeIntracellularreceptorFigure 15-9. Cell-surface and intracellular receptor pathways. Extracellular signaling pathway: Most growth factors and other hydrophilic signaling molecules are unable to move across the plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream


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Surgery_Schwartz. plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream signals in the cell. Intracellular signaling pathway: Hormones or other diffusible molecules enter the cell and bind to the intracel-lular receptor in the cytoplasm or in the nucleus. Either extracel-lular or intracellular signals often reach the nucleus to control gene expression.Brunicardi_Ch15_p0479-p0510.indd 48718/02/19 11:12 AM 488BASIC CONSIDERATIONSPART Idiffusion to bind to a specific target protein within the cyto-plasm (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 inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and


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Surgery_Schwartz. biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a com-mon 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


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Surgery_Schwartz. 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.Advances in biology in the last two decades have dramati-cally expanded the view on how cells are wired with signal-ing pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell gener-ally 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 acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a


Surgery_Schwartz

Surgery_Schwartz. acti-vate multiple receptorand non–receptor-mediated signal trans-duction 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 associa-tions of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the under-standing of signaling networks will require methods of inves-tigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount


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Surgery_Schwartz. approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and pro-teomic data mining will require the development of new model-ing methodologies within the emerging disciplines of medical mathematics and physics.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 recep-tor 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


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Surgery_Schwartz. 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 sig-nals, leading to biologic actions.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, protein-ases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus,


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Surgery_Schwartz. inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory 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 recep-tors 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


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Surgery_Schwartz. 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.Insulin Pathway and Diabetes.13 The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease. 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


Surgery_Schwartz

Surgery_Schwartz. 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 sig-naling 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).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


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Surgery_Schwartz. 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 insu-lin 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. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER


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Surgery_Schwartz. components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15PlasmamembraneNucleusInsulinreceptor(InsR)GeneexpressionMAPKcascadeLipid & glucosemetabolismCellsurvivalIRSInsulinAdaptorPI3KFigure 15-10. Insulin-signaling pathway. Insulin is a peptide growth factor that binds to and activates the heterotetrameric recep-tor complex (InsR). InsR possesses protein tyrosine kinase activity and is able to phosphorylate the downstream insulin receptor sub-strate (IRS). Phosphorylated IRS serves as a scaffold and controls the activation of multiple downstream pathways for gene expres-sion, cell survival, and glucose metabolism. Inactivation of the insulin pathway can lead to type 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of


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Surgery_Schwartz. 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, 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.Transforming Growth Factor-a (TGF-a) Pathway and Cancers.14 Growth factor signaling controls cell growth, differ-entiation, and apoptosis. 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


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Surgery_Schwartz. 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.The TGF-β superfamily comprises a large number of struc-turally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The com-plex 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 differen-tiation. For example,


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Surgery_Schwartz. 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 differen-tiation. For example, TGF-β strongly induces the transcription PlasmamembraneTGF b receptorGeneexpressionNucleusSMADTGF bAnti-proliferationFigure 15-11. TGF-β signaling pathway. The TGF-β family has at least 29 members encoded in the human genome. They are also peptide growth factors. Each member binds to a heterotetrameric complex consisting of a distinct set of type I and type II recep-tors. TGF-β receptors are protein serine/threonine kinases and can phosphorylate the downstream substrates called SMAD proteins. Phosphorylated SMADs are directly transported into the nucleus, where they bind to the DNA and regulate gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer,


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Surgery_Schwartz. gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer, leading to the uncontrolled proliferation of cancer cells.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 dura-tion 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 mecha-nism for cells to ensure controlled proliferation.Resistance to TGF-β’s anticancer action is one hall-mark 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


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Surgery_Schwartz. 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. Alterna-tively, 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 con-stitutively inactivate tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers,


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Surgery_Schwartz. tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic 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 Brunicardi_Ch15_p0479-p0510.indd 48918/02/19 11:12 AM 490BASIC CONSIDERATIONSPART Iand the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregu-lation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.Gene Therapy and Molecular Drugs in CancerModern 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


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Surgery_Schwartz. 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 func-tions 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 molecu-lar medicine includes gene therapy and molecular drugs that target genes or gene products within human cells.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 character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have


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Surgery_Schwartz. spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern nor-mal 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 progres-sive 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 sub-tle 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.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a


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Surgery_Schwartz. leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a dis-ease 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 muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by


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Surgery_Schwartz. and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppres-sors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.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. Meta-static cancer cells that enter the bloodstream can reach virtu-ally all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with


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Surgery_Schwartz. the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with multiple genetic mutations. Through repeated alterations in the genome, mutant epithelial cells are able to develop into a cluster of cells (called a tumor clone) that proliferates in an uncontrollable fashion. Further changes in the tumor cells can transform the tumor cells into a population of cells that can enter the blood vessels and repopulate in a new location.Tumor cells escape fromblood vessel and proliferateto form metastatic tumorsBlood vesselTumor cells break looseand enter bloodstreamUncontrolled cellproliferationCell proliferationCell proliferationCell with multiplemutationsCell with two mutationsMutant epithelial cellNormal epithelial cellmetastasis 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


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Surgery_Schwartz. two mutationsMutant epithelial cellNormal epithelial cellmetastasis 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.The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge Brunicardi_Ch15_p0479-p0510.indd 49018/02/19 11:12 AM 491MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15acquired 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. Alterna-tively,


Surgery_Schwartz

Surgery_Schwartz. 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. Alterna-tively, several new biologicand gene-based therapies are aimed at enhancing the body’s natural defenses against invading can-cers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, geneti-cally engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.Immunotherapy. 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


Surgery_Schwartz

Surgery_Schwartz. 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 local-izing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is


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Surgery_Schwartz. cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor 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 pre-venting 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


Surgery_Schwartz

Surgery_Schwartz. 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 antitu-mor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell


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Surgery_Schwartz. can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell growth and replica-tion. 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 abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only


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Surgery_Schwartz. resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion 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 abnormali-ties caused by the leukemia in a majority of patients, achiev-ing 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.Gene Therapy. Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lym-phocytes (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.


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Surgery_Schwartz. or lym-phocytes (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 molec-ular 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.Inactive(In the absence of ATP)Overly activeUncontrolledcell proliferationBlockedcell proliferationBlocked activityATPSubstrateBcr-AblkinasePO4TyrSTI571SubstrateBcr-AblkinaseTyrSubstrateBcr-AblkinaseTyrFigure 15-13. Mechanism of STI571 as a molecular drug. Bcr-Abl is an overly activated oncogene product resulting from a specific genetic abnormality generated by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate


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Surgery_Schwartz. by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate substrates, which in turn promote cell proliferation. STI571 is a small molecule that competes with the ATP-bind-ing site and thus blocks the transfer of phos-phoryl group to substrate. PO4 = phosphate; Tyr = tyrosine.Brunicardi_Ch15_p0479-p0510.indd 49118/02/19 11:12 AM 492BASIC CONSIDERATIONSPART ISeveral 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 enhance-ment 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). In situ gene therapy also offers a better distribution of the


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Surgery_Schwartz. of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). 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 sub-stances 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


Surgery_Schwartz

Surgery_Schwartz. 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 respon-sible 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.It is noteworthy that the use of multiple therapeutic meth-ods 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. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A


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Surgery_Schwartz. few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant 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 che-motherapeutic 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 combina-tion chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human


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Surgery_Schwartz. effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neuro-logic 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 abil-ity 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 (hESCs) are derived from early preim-plantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem


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Surgery_Schwartz. embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem cellDifferentiationTerminallydifferentiatedcellFigure 15-14. Stem cells. A stem cell is capable of self-renewal (unlimited cell cycle) and differentiation (becoming nondividing cells with specialized functions). Differentiating stem cells often undergo additional cell divisions before they become fully mature cells that carry out specific tissue functions.dish—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. There are two pluripotent states associ-ated with hESCs, one of which is the classic culture with bFGF (basic fibroblast growth factor) and knock out replacer (KSR), termed as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium


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Surgery_Schwartz. as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium in addition to bFGF.Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can gen-erate 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. Another example is mesenchymal stem cells (MSCs), which is initially identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp,


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Surgery_Schwartz. identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp, skeletal muscle, tendons, and synovial, etc., and are reported to obtain the ability to differen-tiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Due to their characteristics of easy acquisition (from adi-pose tissue, for example), strong ex vivo proliferation, immune-modulatory function, and ability to migrate to damaged tissue, MSCs have been utilized in regenerative medicine.Stem cells can be grown in culture and be induced to dif-ferentiate 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 differentia-tion in response to environmental cues. It is believed that dis-covery of the signals


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Surgery_Schwartz. cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery 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 Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM


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Surgery_Schwartz. 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 Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM 493MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15potentially limitless source of patient-specific stem cells for tis-sue engineering and regenerative medicine.The Atomic Theory of Disease22As early as the 5th century b.c., the ancient Greek Democritus first proposed that matter is composed of indivisible particles called “atoms.” In the 17th and 18th centuries, Isaac Newton described the expansion of gases as rushed atoms into empty space. The existence of atoms was doubted until the discovery of subatomic particles in the 20th century, which demonstrated that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the


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Surgery_Schwartz. that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the discovery of the X-ray. The most advanced and well-applied atomic technologies include accurate imaging such as X-ray computed tomography (CT) scan, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). Additional applica-tions include radiation oncology, which utilizes ionizing parti-cles to treat malignant diseases by inducing double-strand DNA breaks resulting in programed cell death.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 21st century. 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


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Surgery_Schwartz. atom is now the anatomy of the 21st century. 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 sub-atomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composi-tion 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 ulti-mate protein structure. This can lead to subtle changes in func-tion or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymor-phisms 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


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Surgery_Schwartz. level, there could potentially be polymor-phisms 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 dif-fer 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. This is where the basic human variations originate because an atomic polymerism would result in particular genetic change. Remarkably, somatic point mutations of KRAS and P53 are caused by a single proton shift in cytosine, which is known as tautomerization of cyto-sine. In turn, the tautomerized cytosine binds with adenine, and not guanine, thus leading to point mutations in KRAS and P53, which are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of


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Surgery_Schwartz. are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemo-globinemia, 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. Met-hemoglobinemia 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 oxi-dize hemoglobin. Although there is less than 1% of methemo-globin 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


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Surgery_Schwartz. 1% of methemo-globin 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.Another example would be exposure to external energy such as radiation that leads to instability of nuclear genome. In Chernobyl and Fukushima, radiolabeled food was metabolized and incorporated into body cells and decay to emit gamma radi-ation, causing DNA damage. This radiation damage occurs pri-marily at a subatomic level from a radiobiologic point of view with a direct or indirect ionization of atoms. The clinical results depend on the tissue characteristics and the equilibrium between the damage applied to normal and diseased tissues.TECHNOLOGIES OF MOLECULAR AND CELL BIOLOGYDNA CloningSince the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic


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Surgery_Schwartz. the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic and microbiologic techniques to manipu-late DNA.23 Pure pieces of any DNA can be inserted into bac-teriophage DNA or other carrier DNA such as plasmids to produce recombinant DNA in bacteria. In this way, DNA can be reconstructed, amplified, and used to manipulate the functions of individual cells or even organisms. This technology, often referred to as DNA cloning, is the basis of all other DNA analy-sis methods. It is only with the awesome power of recombinant DNA technology that the completion of the Human Genome Project was possible. It also has led to the identification of the entire gene complements of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into


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Surgery_Schwartz. of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into bacterial or mammalian cells or tissues24,25 (Fig. 15-15). This represents a very basic technique that is widely used in almost all areas of biomedical research. DNA vectors often are called plasmids, which are extrachromosomal molecules of DNA that vary in size and can replicate and be transmitted from bacterial cell to cell. Plasmids can be propagated either in the cytoplasm or after insertion, as part of the bacterial chromosome in Escherichia coli. The process of molecular cloning involves several steps of manipulation of DNA. First, the vector plasmid DNA is cleaved with a restriction enzyme to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through


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Surgery_Schwartz. to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through the so-called Gateway Technology that allows for the rapid and efficient transfer of DNA fragments between different cloning vectors while maintaining reading frame and orientation, with-out the use of restriction endonucleases and DNA ligase. The technology, which is based on the site-specific recombination system of bacteriophage l, is simple, fast, robust, and automat-able and thus compatible for high-throughput DNA cloning.Finally, the ligation product or the Gateway reaction prod-uct is introduced into competent host bacteria; this procedure is called transformation, which can be done by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM


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Surgery_Schwartz. by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM 494BASIC CONSIDERATIONSPART IFigure 15-15. Generation of recombinant DNA. The vector is a circular DNA molecule that is capable of replicating in Escherichia coli cells. Insert DNA (often your favorite gene) is ligated to the vector after ends of both DNA are properly treated with restriction enzymes. Ligated DNA (i.e., the recombinant plasmid DNA) is then transformed into E. coli cells, where it replicates to produce recombinant progenies. E. coli cells carrying the recombinant plasmid can be propagated to yield large quantities of plasmid DNA.InsertDNA of interestDigest withrestriction enzymeVectorLigationRecombinantplasmidIntroduceinto E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation


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Surgery_Schwartz. E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation of recombinants; for example, it must be enzymatically treated to prevent self-ligation. Host bacteria must be made sufficiently competent to permit the entry of recombinant plasmids into cells. The selection of desired recombinant plasmid-bearing E. coli normally is achieved by the property of drug resistance conferred by the plasmid vectors. The plasmids encoding mark-ers provide specific resistance to (i.e., the ability to grow in the presence of) antibiotics such as ampicillin, kanamycin, and tetracycline. The foreign component in the plasmid vector can be a mammalian expression cassette, which can direct expres-sion of foreign genes in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and


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Surgery_Schwartz. in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and knockout mice.Detection of Nucleic Acids and ProteinsSouthern Blot Hybridization. Southern blotting refers to the technique of transferring DNA fragments from an electropho-resis gel to a membrane support and the subsequent analysis of the fragments by hybridization with a radioactively or chemi-luminescently labeled probe (Fig. 15-16).26 Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. Southern blotting is composed of several steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an


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Surgery_Schwartz. steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an agarose gel by electrophoresis with appropriate DNA size markers, called the DNA ladder. The DNA gel is stained with a dye, usually ethidium bromide, and photographed with a ruler laid alongside the gel so that band positions can later be identified on the membrane. The DNA gel then is treated so the DNA fragments are denatured (i.e., strand separation). The DNA then is transferred onto a nitrocellulose membrane by capillary diffusion or under electricity. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a radioactively or chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the


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Surgery_Schwartz. chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The later applica-tion of Southern blotting hybridization to the study of restriction fragment length polymorphisms opened up new possibilities DNA is digested withrestriction enzymes.DNA fragments are denaturedand separated by gelelectrophoresis.DNA fragments are transferredto a membrane filter.The filter is hybridized with a radioactive DNA probe.DNA fragment that is hybridized to the radioactive DNA is detected by autoradiography.Radioactive probeFigure 15-16. Southern blotting. Restriction enzymatic fragments of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic


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Surgery_Schwartz. of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic diseases.Northern Blot Hybridization. Northern blotting refers to the technique of size fractionation of RNA in a gel and the transfer-ring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to deter-mine the size and abundance of the target RNA. In principle, Brunicardi_Ch15_p0479-p0510.indd 49418/02/19 11:12 AM 495MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although


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Surgery_Schwartz. DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although reverse-transcriptase PCR has been used in many applications (described in the next sec-tion, “Polymerase Chain Reaction”), Northern analysis is the only method that provides information regarding mRNA size and has remained a standard method for detection and quantita-tion of mRNA. The process of Northern hybridization involves several steps, as does Southern hybridization, including elec-trophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization to a radio-actively labeled DNA probe. Data from hybridization allow Etc.Double-strandedDNAHeat toseparatestrandsHybridization of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded


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Surgery_Schwartz. of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded DNA to beamplifiedDNA oligonucleotideprimersSeparate DNA strandsand annual primerDNAsynthesisSeparate DNA strandsand annual primerDNAsynthesisFirst cycleProducing two double-strandedDNA moleculesSecond cycleProducing four double-strandedDNA moleculesThird cycleProducing eight double-strandedDNA moleculesABFigure 15-17. Amplification of DNA using the polymerase chain reaction (PCR) technique. Knowledge of the DNA sequence to be ampli-fied is used to design two synthetic DNA oligonucleotides, each complementary to the sequence on one strand of the DNA double helix at opposite ends of the region to be amplified. These oligonucleotides serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA,


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Surgery_Schwartz. serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA, and each cycle of the reaction begins with a brief heat treatment to separate the two strands (Step 1). After strand separation, cooling of the DNA in the presence of a large excess of the two primer DNA oligonucleotides allows these primers to hybridize to complementary sequences in the two DNA strands (Step 2). This mixture is then incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA is synthesized, starting from the two primers (Step 3). The entire cycle is then begun again by a heat treatment to separate the newly synthesized DNA strands. B. As the procedure is performed over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the


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Surgery_Schwartz. over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. Of the DNA put into the original reaction, only the sequence bracketed by the two primers is amplified because there are no primers attached anywhere else. In the example illustrated in B, three cycles of reaction produce 16 DNA chains, eight of which (boxed in brown) are the same length as and correspond exactly to one or the other strand of the original bracketed sequence shown at the far left; the other strands contain extra DNA downstream of the original sequence, which is replicated in the first few cycles. After three more cycles, 240 of the 256 DNA chains cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at


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Surgery_Schwartz. cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at the same time, provide information related to the presence, size, and integrity of discrete mRNA species. Thus, Northern blot analy-sis, also termed RNA gel blot analysis, commonly is used in molecular biology studies relating to gene expression.Polymerase Chain Reaction. PCR is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the tar-get DNA (Fig. 15-17).27 One cycle of PCR reaction involves template denaturation, primer annealing, and the extension of Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve


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Surgery_Schwartz. Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve as a template in the next, the number of target DNA copies nearly doubles at each cycle. Thus, a repeated series of cycles result in the exponential accumulation of a specific fragment in which the termini are sharply defined by the 5′ ends of the primers. The introduction of the thermostable DNA polymerase (e.g., Taq polymerase) transforms the PCR into a simple and robust reaction. The reaction components (e.g., template, primers, Taq polymerase, 2′-deoxynucleoside 5′-triphosphates, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperatures within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and


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Surgery_Schwartz. within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and Mg2+ concen-tration, as well as the temperature cycling profile). Modifying various PCR parameters to optimize the specificity of amplifi-cation yields more homogenous products, even in rare template reactions.The emergence of the PCR technique has dramatically altered the approach to both fundamental and applied bio-logic problems. The capability of amplifying a specific DNA fragment from a gene or the whole genome greatly advances the study of the gene and its function. It is simple, yet robust, speedy, and most of all, flexible. As a recombinant DNA tool, it underlies almost all of molecular biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression,


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Surgery_Schwartz. biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression, making it a fun-damental cornerstone of genetic and molecular analysis.Immunoblotting and Immunoprecipitation. Analyses of proteins are primarily carried out by antibody-directed immu-nologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a popu-lation of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool. Using specific antibod-ies, microscopic analysis called immunofluorescence and immu-nohistochemistry is possible for the subcellular localization and expression of proteins in cells or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis