2. Calcium and DAG are second messengers that mediate some responses initi- ated by signaling from G protein-coupled receptors (Figure 14–3). a. Activation of PLC by binding of a G protein α subunit activates the en- zyme. b. PLC hydrolyzes a membrane-bound inositol phospholipid, phosphatidyl- inositol 4,5-bisphosphate (PIP 2 ), into the active products IP 3 and DAG. c. DAG forms a binding site for protein kinase C (PKC) and thereby re- cruits it to the plasma membrane, which partially activates the enzyme. d. IP 3 binds to the endoplasmic reticulum to release Ca 2+ stores. e. Ca 2+ binds to PKC and further activates it. f. PKC phosphorylates multiple substrates to alter gene expression in the cell. TUMOR-PROMOTING PHORBOL ESTERS • Extracts from the croton plant (croton oil) are not themselves carcinogenic but enhance tumor forma- tion if administered after initial exposure to a carcinogen. Chapter 14: Cellular Signaling and Cancer Biology 205 N α GTP PLC P P P P P P PIP 2 IP 3 Ca 2 + DAG Endoplasmic reticulum Activated PKC Phosphorylation of cellular substrates Figure 14–3. Signaling through protein kinase C (PKC). Activated phospholipase C cleaves the inositol phospholipid PIP 2 to form both soluble (IP 3 ) and membrane- associated (DAG) second messengers. DAG recruits PKC to the membrane, where binding of calcium ions to PKC fully activates it. To accomplish this, IP 3 promotes a transient increase of intracellular Ca 2+ concentration by binding to a receptor on the endoplasmic reticulum, which opens a channel allowing release of stored cal- cium ions. PIP 2 , phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; PLC, phospholipase C; IP 3 , inositol trisphosphate. CLINICAL CORRELATION • The active agents in croton oil are phorbol esters, specifically 12-O-tetradecanoyl phorbol-13-acetate (TPA) or phorbol myristate acetate (PMA), which are structural analogs of DAG. • Both TPA and PMA enhance carcinogenesis by binding to the DAG binding site and activating PKC, which bypasses normal cell cycle regulation and stimulates cell division to produce its “tumor pro- moting” effect. III. Receptor Tyrosine Kinases A. Some cell-surface receptors transduce their signals by means of a kinase cascade initiated by their protein tyrosine kinase activity (Figure 14–4). 206 USMLE Road Map: Biochemistry N P P PGDP GDP GTP GTP Activated tyrosine kinase GRB2 SOS Raf Ras Ras Ligand Receptor Raf MEK Protein kinases Transcription factors Other proteins P ERK P 1 2 3 Figure 14–4. Receptor tyrosine kinase signaling mediated by the Ras-MEK-ERK pathway. Binding of a growth factor (ligand) to its cell-surface receptor promotes dimerization of the receptor with subsequent autophosphorylation mediated by ac- tivation of the intrinsic tyrosine kinase of the receptor’s cytoplasmic domain. Dock- ing of the adaptor GRB2-SOS complex promotes activation of Ras by GDP-GTP exchange. Ras recruits the first serine/threonine kinase of the signaling pathway, Raf. Raf then phosphorylates itself as well as the downstream kinase (MEK), which in turn phosphorylates ERK (also called MAP kinase). Activated ERK is capable of dis- tributing the signal by phosphorylation of multiple substrates leading to the cell’s pleiotropic response to the growth factor. Reactions of the kinase cascade are de- noted by the numbers in diamonds. 1. These signal transducers have a large extracellular domain with its ligand- binding site, a single transmembrane domain and an intracellular domain with intrinsic tyrosine kinase activity. 2. Ligand binding to the receptor’s extracellular domain activates signaling by causing the receptors to form dimers and cross-phosphorylate (autophospho- rylate) their intracellular domains on tyrosine sites. B. The signaling pathway downstream of the activated receptors is composed of a se- ries of kinases, a kinase cascade. 1. The phosphotyrosine sites on the receptor act as docking points for adap- tors and effectors, which couple the signal to the kinase cascade. 2. One of the major adaptors is the GRB2-SOS complex, which upon docking to the phosphorylated receptor, binds the small G protein Ras and activates it by GDP-GTP exchange in a manner analogous to the heterotrimeric G proteins. 3. Activated Ras recruits the first kinase in the cascade, Raf-1, to the plasma membrane, where it becomes active. 4. The signal is then transferred from one kinase to the other by sequential phos- phorylation and activation, ie, the kinase cascade. 5. The signal ultimately is sent into the nucleus, where transcription factors such as Elk-1 are activated by phosphorylation. CLINICAL APPLICATIONS OF MONOCLONAL ANTIBODIES THAT TARGET LIGANDS AND RECEPTORS • By 2005, 18 monoclonal antibodies had been approved for treatment of several diseases, especially for various cancers as well as infectious and inflammatory conditions, with many more under devel- opment. • Some of these agents are targeted to ligands or their receptors, and they work by preventing binding and subsequent signal transduction, as illustrated in the following examples. – HER2, a member of the EGF receptor family, drives growth of breast cancers that overexpress the re- ceptor. Trastuzumab, which binds HER2 and prevents receptor activation, has been shown to be ef- fective in reducing tumor growth and metastasis in such cases. – Interleukin-2 (IL-2) signaling is important in the immune response that can lead to rejection in solid organ transplantation. Basiliximab binds the α subunit of the IL-2 receptor to prevent IL-2 binding and provide an immunosuppressive effect to inhibit renal transplant rejection. – Tumor necrosis factor-␣ (TNF-␣) is a critical mediator of inflammation in autoimmune diseases like Crohn’s disease and rheumatoid arthritis. Infliximab binds TNF-α and prevents its binding to the TNF receptor for treatment of these diseases. – Many cancers depend on vascular endothelial growth factor (VEGF) for formation of a blood sup- ply to allow tumor growth and metastasis. Bevacizumab binds VEGF, which prevents its binding to the VEGF receptor and thereby inhibits tumor vascularization (angiogenesis) in combination therapy with 5-fluorouracil for treatment of metastatic cancers, particularly colorectal cancer. IV. The Nuclear Receptor Superfamily A. Many hormones diffuse into the cell and initiate signaling by binding to soluble intracellular receptors that act as transcription factors. 1. This mechanism is used by steroid hormones (Table 14–2), thyroid hormone, vitamin D 3 , and retinoic acid. a. These ligands for the nuclear receptor superfamily are capable of dissolving in water at low concentrations but are mainly lipophilic, capable of passing through the lipid bilayer into the cell by diffusion. Chapter 14: Cellular Signaling and Cancer Biology 207 N CLINICAL CORRELATION b. Some of these molecules require metabolism or modification to be able to bind their receptors. (1) Dihydrotestosterone is the preferred (high affinity) ligand for the an- drogen receptor and is formed by reduction of testosterone catalyzed by the enzyme steroid 5α-reductase. (2) The form of thyroid hormone active in binding its receptor is tri- iodothyronine (T 3 ) rather than thyroxine (T 4 ). 2. The receptors may be located in the nucleus or cytoplasm of the cell, but they are collectively called the “nuclear receptor superfamily” because the nucleus is their main site of action. 3. The receptors in this family have a similar overall structure with a ligand- binding domain specific for the hormone or vitamin, a DNA-binding domain, and a variable domain that differs among the receptors. B. Binding of ligand activates the receptor so that it can bind specific DNA se- quences in regulatory regions of target genes that have hormone-response ele- ments (HREs) (Figure 14–5). 1. After formation of the initial ligand-receptor complex, other partner proteins are recruited that complete the active complex. 2. Binding of a co-activator confers on the complex the ability to activate tran- scription when it binds to the target gene. 3. Conversely, transcription of a target gene may be inhibited by binding of a complex formed when a co-repressor binds to the ligand-receptor. 208 USMLE Road Map: Biochemistry N Table 14–2. Ligands of the nuclear receptor superfamily. Hormone or Ligand Family Name Major Ligands in Humans Glucocorticoids Cortisol Mineralocorticoids Aldosterone Progestins Progesterone Estrogens Estradiol Estriol Estrone Androgens Testosterone Dihydrotestosterone (DHT) Dehydroepiandrosterone (DHEA) Vitamin D compounds 1,25-Dihydroxycholecalciferol or 1,25-Dihydroxy vitamin D 3 Retinoids (vitamin A compounds) All-trans retinoic acid Thyroid hormones Thyroxine (T 4 ) Triiodothyronine (T 3 ) DISORDERS OF ANDROGEN ACTION PRODUCE FEMINIZATION IN MALES • Steroid 5␣-reductase deficiency is an autosomal recessive disorder that causes decreased conver- sion of testosterone to dihydrotestosterone and decreased androgen action that is particularly critical during sexual development. Chapter 14: Cellular Signaling and Cancer Biology 209 N HRE Receptor Activated hormone-receptor complex Coactivator Steroid hormone Cytoplasm Nucleus RNA Polymerase Gene transcription RESPONSE Figure 14–5. Regulation of gene transcription by members of the nuclear recep- tor superfamily. Binding of a steroid hormone to its receptor promotes a confor- mational change that causes dissociation of proteins that associate with the inactivated receptor, including several heat shock proteins. In this example, the re- ceptor is localized in the cytoplasm in its inactive state. In such a case, the acti- vated hormone-receptor complex undergoes a conformational change that exposes a nuclear localization signal. Within the nucleus, the receptor binds a coactivator protein and the complete complex mediates transcriptional activation of target genes having the appropriate hormone-response element (HRE). CLINICAL CORRELATION • External genitalia of men deficient in steroid 5α-reductase are female in character rather than male. • Several inherited disorders that produce defective androgen receptors (androgen resistance) also cause disruption of sexual development that may culminate in infertility or testicular feminization. • Testicular feminization is characterized by expression of a female external phenotype despite a nor- mal blood level of testosterone and standard male karyotype (46,XY). V. Overview of Cancer Biology A. Cancer is considered a genetic disease in that mutations of various genes cause disease by dysregulation of cellular mechanisms that control proliferation, sur- vival, and death. 1. Once a cell has become “transformed,” ie, capable of autonomous prolifera- tion through mutation of some of its genes, these characteristics are heritable from cell to cell. 2. Dominant, gain-of-function mutations that activate oncogenes confer a rapid-growth phenotype on cells. 3. Recessive, loss-of-function mutations that delete or inactivate tumor sup- pressor genes alleviate controls on cell proliferation and survival. 4. Activated oncogenes are rarely passed through the germline. 5. Mutated, inactivated tumor suppressor genes can be inherited through the germline from one person to another. a. These cancer susceptibility genes usually have an autosomal dominant ex- pression pattern. b. Examples of such conditions are the genes for familial colorectal cancer (eg, HNPCC or APC) and the familial breast cancer genes BRCA1 and BRCA2. B. Development of cancer or neoplastic transformation requires an accumulation of mutations in the same cell. 1. The first mutation in a tumor suppressor gene such as BRCA1 may be either inherited via the germline or sporadic (due to a random event in that person) and then the normal allele is somehow inactivated (see loss of heterozygosity below). 2. Multiple mutations that activate oncogenes or inactivate tumor suppressor genes accumulate due to progressive loss of DNA repair mechanisms and cell cycle control. 3. An important example of how a progression of somatic mutations leads to can- cer is in hereditary colorectal cancer (Figure 14–6). VI. Oncogenes and Tumor Suppressor Genes A. Oncogene activation by overexpression, mutation, or chromosomal rearrange- ment can lead to rapid proliferation of cells and cancer. 1. Oncogenes are the mutant, out-of-control versions of normal cellular genes, the proto-oncogenes, which regulate a variety of critical cellular processes such as signaling, cell cycle control, and transcription. 2. The mutations that have converted the proto-oncogenes to their oncogene forms are gain-of-function or activating mutations. RAS MUTATIONS OCCUR IN MANY HUMAN CANCERS • Over 30% of all human cancers have activating mutations of the gene encoding the small G protein Ras. 210 USMLE Road Map: Biochemistry N CLINICAL CORRELATION • Several missense mutations (ie, at codons 12, 13, or 61) render the mutant protein incapable of hy- drolyzing bound GTP to GDP. • These mutant forms of Ras thus persist in the ON state, which provides continuous activation of the ki- nase cascade downstream of Ras and stimulates the cell to keep dividing even in the absence of appro- priate signals from cell-surface receptors. 3. Tumor viruses carry activated versions of important cellular genes that regu- late cell cycle and transcription. a. The virus that causes Kaposi’s sarcoma, Kaposi’s sarcoma–associated her- pesvirus, induces transformation of infected cells by up-regulating expres- sion of the cellular form of the Kit oncogene, among others. b. Human papillomavirus (HPV) causes a variety of epithelial cancers, espe- cially of the alimentary canal and the cervix, by means of two associated oncogenes, E6 and E7. 4. Overexpression or deregulated expression of cell cycle-dependent transcription factors such as Myc and Fos may stimulate continued cell division. Chapter 14: Cellular Signaling and Cancer Biology 211 N Normal colon epithelial cell Loss of tumor suppressor gene APC Increased epithelial proliferation Activation of oncogene by mutation RAS Benign tumor (adenoma) Loss of tumor suppressor gene DCC Large adenoma Loss of tumor suppressor gene TP53 Aggressive, invasive tumor (carcinoma) Accumulation of many mutations Many genes Metastic tumors Figure 14–6. Accumulation of mutations leads to progressive development of familial colorectal cancer. Development of cancer does not require that these steps occur in the particular sequence shown. 5. Activation of an oncogene may occur by chromosomal rearrangement creat- ing a dysregulated fusion protein. THE PHILADELPHIA CHROMOSOME IN CHRONIC MYELOGENOUS LEUKEMIA • Cytogenetic analysis of patients with chronic myelogenous leukemia (CML) reveals an unusual translo- cation between chromosomes 9 and 22 termed the “Philadelphia chromosome.” • The translocation moves the c-ABL gene that encodes a tyrosine kinase from chromosome 9 to the breakpoint cluster region (BCR) of chromosome 22. • The resultant gene, BCR-ABL, encodes a constitutively active kinase that stimulates cell division and leads to the transformed phenotype of the cells. • Patients with CML experience weakness, fatigue, excessive sweating, low-grade fever, enlarged spleen, and elevated WBC count. • Imatinib, a drug that inhibits the kinase activity of the Bcr-Abl fusion protein, has been successfully used for treatment of CML. B. Loss or inactivation of tumor suppressor genes may lead to cancer. 1. Tumor suppressors are genes that encode a diverse array of proteins that con- trol cellular growth and death. 2. Loss or mutation that inactivates one copy of the gene can be tolerated because there is no functional deficit in the heterozygous condition. 3. Loss of heterozygosity (LOH) that deletes the only available functional copy of the gene can contribute to unregulated proliferation of those cells (Figure 14–7). 212 USMLE Road Map: Biochemistry N Loss of normal chromosome Loss and reduplication Somatic recombination or mitotic crossing over Independent mutation N Constitutional genotype M M M M M M M M Figure 14–7. Possible mech- anisms for loss of heterozy- gosity at a tumor suppressor locus. All these mechanisms have been observed in retinoblastoma involving the RB1 gene on chromosome 13. CLINICAL CORRELATION Chapter 14: Cellular Signaling and Cancer Biology 213 N LOH IN RETINOBLASTOMA • Retinoblastoma produces childhood neoplasms arising from neural precursor cells of the retina (retinoblasts) at an incidence of 1 in 20,000 live births. • The biochemical defect is mutation or loss of the tumor suppressor gene, RB1, encoding the protein pRb. – pRb binds to and inactivates members of the E2F transcription complex, which normally prevents cells from entering S phase of the cell cycle. – Loss of E2F regulation by pRb impairs cell cycle control, and unregulated proliferation (clonal ex- pansion) may lead to a tumor derived from that cell. • Most cases are inherited and multiple tumors arise bilaterally in heterozygotes when the normal RB1 allele undergoes mutation or loss due to LOH. • Retinoblastoma shows an apparently autosomal dominant phenotype due to the high probability of LOH during the ~10 6 cell divisions of retinoblasts and despite the recessive nature at the cellular level. 4. TP53 is an important tumor suppressor gene that encodes the p53 transcrip- tion factor that is up-regulated when the cellular DNA is damaged. a. High levels of p53 up-regulate transcription of the WAF1/CIP1 gene, whose protein product, p21, blocks entry into S phase of the cell cycle by a mecha- nism called checkpoint control. b. TP53 is the most commonly mutated gene in human cancer, occurring in over 50% of tumors examined. LI-FRAUMENI SYNDROME • Patients with Li-Fraumeni syndrome have increased susceptibility to a variety of cancers, including bone and soft-tissue sarcomas, breast tumors, brain cancers, leukemia, and adrenocortical carcinoma, all arising at an early age (often before 30 years). • The biochemical defect in families exhibiting this syndrome is a loss-of-function mutation of the tumor suppressor gene, TP53, encoding p53. • The incidence of Li-Fraumeni syndrome has not been calculated because it is so rare. • Inheritance is apparently autosomal dominant with high penetrance but with variable expression (family members may have a wide range of tumor types and ages of onset). VII. Apoptosis A. Apoptosis, or programmed cell death, is a complex, highly regulated process by which a cell self-destructs in an organized manner. 1. The mechanism of death in apoptosis contrasts with that occurring when a cell breaks open or lyses producing a necrosis. 2. Necrosis allows the contents of the cell to spill over the local area, causing an inflammatory response that leads to damage to nearby cells within the tissue. 3. By contrast, cells undergoing apoptosis do not lyse, so there is no associated in- flammatory response. B. Major changes that occur in the cell during apoptosis include the following: 1. Chromatin condensation. 2. Disintegration of the nuclear envelope. 3. Fragmentation of DNA between the nucleosomes. 4. Blebbing of the cell membrane. 5. Recruitment of macrophages, which ultimately engulf the dead cells. C. Both extrinsic and intrinsic pathways can lead to programmed cell death (Figure 14–8). CLINICAL CORRELATION CLINICAL CORRELATION [...]... domains/rafts, 40 Lipid metabolism See also Cholesterol metabolism clinical problems/solutions, 11 8–1 21 digestion and absorption of dietary fats, 103 fatty acid oxidation, 10 9–1 13 fatty acid synthesis, 10 6–1 09, 107 f, 108 f functions of fatty acids, 105 lipid malabsorption disorders, 104 lipoproteins/processing and transport, 10 4–1 05 Lipids, dietary, 54 Loss of heterozygosity (LOH), 212 Lung surfactant, 6 and respiratory... metabolism, 7 8–8 0 glycolysis, 70, 71f, 7 2–7 3 pentose phosphate pathway (PPP), 7 6–7 7, 77f regeneration of NAD+, 7 3–7 6, 75f, 76t Carbohydrates dietary, 5 3–5 4, 70 as membrane components, 42, 43f, 44 Carbonic acid-bicarbonate system, 4, 5f Cardiolipin, 37 Carnitine, 10 9–1 10 CPT-I/-II deficiency, 110 primary deficiency, 109 secondary deficiency to other conditions, 110 shuttle, 109 , 109 f N Index 219 Catabolism, 52... therapy for, 24 Familial breast cancer genes (BRCA1/2), 210 Familial colorectal cancer genes (HNPCC or FAP), 210, 211f Fanconi anemia, 160 Farnesylation inhibitors (as anti-cancer/antiparasitic agents), 17 4–1 75 Fatty acids, 6 oxidation, 10 9–1 13 synthesis, 10 6–1 09, 107 f, 108 f Fetal hemoglobin (HbF), 16 Folic acid deficiency, 142 Fragile X syndrome, 15 7–1 58 example of anticipation, 193 Fructose metabolism,... deficiency, 209 Sugars, 4 1–4 2, 43f, 44 Sulfur-containing amino acids, 9 Tay-Sachs disease, 18 6–1 87 TCA (tricarboxylic acid) cycle, 90 acetyl CoA biosynthesis, 9 0–9 1, 91f clinical problems/solutions, 9 9–1 02 electron transport chain, 9 6–9 9, 96f oxaloacetate synthesis from pyruvate, 9 5–9 6 PDH deficiency, 9 1–9 2 regulation of, 93f, 94 role in metabolic reactions, 9 4–9 5, 95f steps of, 9 2–9 3, 93f Telomerase activity,... functions, 139 purine biosynthesis, 13 9–1 42, 140f, 141f, 142f pyrimidine biosynthesis, 14 2–1 44, 143f Nucleic acid/structure and function chromosomal DNA structure, 15 2–1 54, 153f DNA repair, 159 functional overview, 15 1–1 52 mutations, 158 replication, 15 4–1 58, 155f RNA structure, 16 0–1 61 transcription, 16 1–1 64, 162f, 163f Nutritional needs and diet, 5 2–5 4 dietary carbohydrates, 5 3–5 4 dietary proteins, 53 metabolism... diabetes mellitus clinical problems/solutions, 6 6–6 9 diet and nutritional needs, 5 2–5 4 glucose homeostasis, 5 6–5 8, 57f metabolism (fasting state), 6 1–6 3, 62f metabolism (fed state), 5 8–6 1, 59f, 60f metabolism (starvation), 6 3–6 4, 64f regulation of metabolic pathways, 5 4–5 6, 55f N 222 Index Metabolic responses (long-term), 5 5–5 6 Methemoglobinemia, 1 7–1 8 Methylxanthines, 203 Micelles, 6 Michaelis-Menten... 21 5–2 17 neoplastic transformation, 210 oncogenes, 21 0–2 12 overview, 210 tumor suppressors, 21 2–2 13, 212f tumor viruses, 211 Carbohydrate metabolism See also G6PD deficiency; Lactic acidosis; Pyruvate kinase deficiency clinical problems/solutions, 8 7–8 9 digestion and absorption of dietary carbohydrates, 70 enzymes regulating glucose metabolism rate-limiting steps, 78, 78t glycogen metabolism, 7 8–8 0... 16 8–1 73, 171f, 172f mutations, 17 9–1 81, 180f oncogenes, 210 regulation of gene expression, 55, 17 6–1 78, 177f transcription, 16 1–1 64, 162f, 163f Genomic imprinting, 192 disorders (examples), 193 Genotype, 185 Glucagon, 56 mechanism of actin, 56 regulatin of blood glucose by, 5 6–6 4 Gluconeogenesis, 8 2–8 5 Glucose homeostasis, 5 6–5 8, 57f Glycerophospholipids (phosphoglycerides), 3 7–3 8, 38f distinguishing structures,... mechanisms, 2 7–2 8, 28f catalytic of reactions by, 2 6–2 7, 27f clinical problems/solutions, 3 4–3 6 classification, 2 5–2 6, 26t coenzymes and cofactors, 32, 33t covalent modification of, 5 4–5 5 in glucose metabolism (rate-limiting steps), 78, 78f inhibitors, 3 0–3 2 N 220 Index Enzymes (cont.) low-Km and ethanol sensitivity, 30 physiological roles of/ clinical problems and solutions, 3 4–3 6 snake venom, 2 8–2 9 as therapeutic... 206f signaling modes, 20 0–2 01 signaling pathway, 200 Cholera toxin, 204 Cholesterol, 39 gallstone disease, 11 6–1 17 metabolism, 11 5–1 16, 116f CK (creatine kinase), and heart attack/muscle damage diagnosis, 2 5–2 6 Coenzymes and cofactors, 32, 33t Collagen, protein structure and function, 1 3–1 4, 13f Competitive enzyme inhibitors, 3 0–3 1 Crohn’s disease and lipid malasorption disorders, 104 Cyclic AMP mechanisms . oxidation, 10 9–1 13 fatty acid synthesis, 10 6–1 09, 107 f, 108 f functions of fatty acids, 105 lipid malabsorption disorders, 104 lipoproteins/processing and transport, 10 4–1 05 Lipids, dietary, 54 Loss of. 10 9–1 10 CPT-I/-II deficiency, 110 primary deficiency, 109 secondary deficiency to other conditions, 110 shuttle, 109 , 109 f inhibitors of topoiso- merases, 15 6–1 57 Antibodies/immunoglobulins (Ig),. 17 4–1 75 Fatty acids, 6 oxidation, 10 9–1 13 synthesis, 10 6–1 09, 107 f, 108 f Fetal hemoglobin (HbF), 16 Folic acid deficiency, 142 Fragile X syndrome, 15 7–1 58 example of anticipation, 193 Fructose metabolism,