Berek and Novak's Gynecology 15th Ed.

6 Molecular Biology and Genetics

Oliver Dorigo

Otoniel Martínez-Maza

Jonathan S.Berek

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• The regulation and maintenance of normal tissue requires a balance between cell proliferation and programmed cell death, or apoptosis.

• The regulation of ovarian function occurs through autocrineparacrine, and endocrine mechanisms. Disruption of these autocrine and paracrine intraovarian pathways may be the basis of polycystic ovarian disease, disorders of ovulation, and ovarian neoplastic disease.

• Among the genes that participate in control of cell growth and function, proto-oncogenes and tumor suppressor genes are particularly important.

• Growth factors trigger intracellular biochemical signals by binding to cell membrane receptors. In general, these membrane-bound receptors are protein kinases that convert an extracellular signal into an intracellular signal. Many of the proteins that participate in the intracellular signal transduction system are encoded by proto-oncogenes that are divided into subgroups based on their cellular location or enzymatic function.

• Oncogenes comprise a family of genes that result from gain of function mutations of their normal counterparts, proto-oncogenes. The normal function of proto-oncogenes is to stimulate proliferation in a controlled context. Activation of oncogenes can lead to stimulation of cell proliferation and development of a malignant phenotype.

• Tumor suppressor genes are involved in the development of most cancers and are usually inactivated in a two-step process in which both copies of the tumor suppressor gene are mutated or inactivated by epigenetic mechanisms like methylation. The most commonly mutated tumor suppressor gene in human cancers is p53.

• T lymphocytes have a central role in the generation of immune responses by acting as helper cells in both humoral and cellular immune responses and by acting as effector cells in cellular responses. T cells can be distinguished from other types of lymphocytes by their cell surface phenotype, based on the pattern of expression of various molecules, as well as by differences in their biologic functions.

• There are two major subsets of mature T cells that are phenotypically and functionally distinct: T-helper/inducer cells, which express the CD4 cell surface marker, and the T-suppressor/cytotoxic cells, which express the CD8 marker. TH1 and TH2 are two helper T-cell subpopulations that control the nature of an immune response by secreting a characteristic and mutually antagonistic set of cytokines: Clones of TH1 produce interleukin-2 (IL-2) and interferon-γ (IFN-γ), whereas TH2 clones produce IL-4, IL-5, IL-6, and IL-10.

Advances in molecular biology and genetics have improved our understanding of basic biologic concepts and disease development. The knowledge acquired with the completion of the human genome project, the data available through The Cancer Genome Atlas (TCGA), the development of novel diagnostic modalities, such as the microarray technology for the analysis of DNA and proteins, and the emergence of treatment strategies that target specific disease mechanisms all have an increasing impact on the specialty of obstetrics and gynecology.

Normal cells are characterized by discrete metabolic, biochemical, and physiologic mechanisms. Specific cell types differ with respect to their mainly genetically determined responses to external influences (Fig. 6.1). An external stimulus is converted to an intracellular signal, for example, via a cell membrane receptor. The intracellular signal is transferred to the nucleus and generates certain genetic responses that lead to changes in cellular function, differentiation, and proliferation. Although specific cell types and tissues exhibit unique functions and responses, many basic aspects of cell biology and genetics are common to all eukaryotic cells.

Figure 6.1 External stimuli affect the cell, which has a specific coordinated response.

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Cell Cycle

Normal Cell Cycle

Adult eukaryotic cells possess a well-balanced system of continuous production of DNA (transcription) and proteins (translation). Proteins are constantly degraded and replaced depending on the specific cellular requirements. Cells proceed through a sequence of phases called the cell cycle, during which the DNA is distributed to two daughter cells (mitosis) and subsequently duplicated (synthesis phase). This process is controlled at key checkpoints that monitor the status of a cell, for example, the amount of DNA present. The cell cycle is regulated by a small number of heterodimeric protein kinases that consist of a regulatory subunit (cyclin) and a catalytic subunit (cyclin-dependent kinase). Association of a cyclin with a cyclin-dependent kinase (CdkC) determines which proteins will be phosphorylated at a specific point during the cell cycle.

The cell cycle is divided into four major phases: M phase (mitosis), G1 phase (period between mitosis and initiation of DNA replication), S phase (DNA synthesis), and G2 phase (period between completion of DNA synthesis and mitosis) (Fig. 6.2). Postmitotic cells can “exit” the cell cycle into the so-called G0 phase and remain for days, weeks, or even a lifetime without further proliferation. The duration of the cell cycle may be highly variable, although most human cells complete the cell cycle within approximately 24 hours. During a typical cell cycle, mitosis lasts about 30 to 60 minutes, the G1 phase 7 to 10 hours, S phase 10 hours, and G2 phase 5 hours. With respect to the cell cycle, there are three subpopulations of cells:

Figure 6.2 The cell cycle.

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1. Terminally differentiated cells cannot re-enter the cell cycle.

2. Quiescent (G0cells can enter the cell cycle if appropriately stimulated.

3. Dividing cells are currently in the cell cycle.

Red blood cells, striated muscle cells, uterine smooth muscle cells, and nerve cells are terminally differentiated. Other cells, such as fibroblasts, exit from the G1 phase into the G0 phase and are considered to be out of the cell cycle. These cells enter the cell cycle following exposure to specific stimuli, such as growth factors and steroid hormones. Dividing cells are found in the gastrointestinal tract, the skin, and the cervix.

G1 Phase

In response to specific external stimuli, cells enter the cell cycle by moving from the G0 phase into the G1 phase. The processes during G1 phase lead to the synthesis of enzymes and regulatory proteins necessary for DNA synthesis during S phase and are mainly regulated by G1 cyclin-dependent kinase–cyclin complexes (G1CdkC). Complexes of G1CdkC induce degradation of the S phase inhibitors in late G1. Release of the S phase CdkC complex subsequently stimulates entry into the S phase. Variations in the duration of the G1 phase of the cell cycle, ranging from less than 8 hours to longer than 100 hours, account for the different generation times exhibited by different types of cells.

S Phase

The nuclear DNA content of the cell is duplicated during the S phase of the cell cycle. The S phase CdkC complex activates proteins of the DNA prereplication complexes that assemble on DNA replication origins during G1. The prereplication complex activates initiation of DNA replication and inhibits the assembly of new prereplication complexes. This inhibition ascertains that each chromosome is replicated only once during the S phase.

G2 Phase

RNA and protein synthesis occurs during the G2 phase of the cell cycle. The burst of biosynthetic activity provides the substrates and enzymes to meet the metabolic requirements of the two daughter cells. Another important event that occurs during the G2 phase of the cell cycle is the repair of errors of DNA replication that may have occurred during the S phase. Failure to detect and correct these genetic errors can result in a broad spectrum of adverse consequences for the organism and the individual cell (1). Defects in the DNA repair mechanism are associated with an increased incidence of cancer (2). Mitotic CdkC complexes are synthesized during the S and G2 phases, but are inactive until DNA synthesis is completed.

M Phase

Nuclear–chromosomal division occurs during the mitosis or M phase. During this phase, the cellular DNA is equally distributed to each of the daughter cells. Mitosis provides a diploid (2n) DNA complement to each somatic daughter cell. Following mitosis, eukaryotic mammalian cells contain diploid DNA, reflecting a karyotype that includes 44 somatic chromosomes and an XX or XY sex chromosome complement. Exceptions to the diploid cellular content include hepatocytes (4n) and the functional syncytium of the placenta.

Mitosis is divided into prophase, metaphase, anaphase, and telophase. Mitotic CdkC complexes induce chromosome condensation during the prophase, assembly of the mitotic spindle apparatus, and alignment of the chromosomes during the metaphase. Activation of the anaphase promoting complex (APC) leads to inactivation of the protein complexes that connect sister chromatids during metaphase, permitting the onset of anaphase. During anaphase, sister chromatids segregate to opposite spindle poles. The nuclear envelope breaks down into multiple small vesicles early in mitosis and reforms around the segregated chromosomes as they decondense during telophase. Cytokinesis is the process of division of the cytoplasm that segregates the endoplasmic reticulum and the Golgi apparatus during mitosis. After completion of mitosis, cells enter the G1 phase and either re-enter the cell cycle or remain in G0.

Ploidy

After meiosis, germ cells contain a haploid (1n) genetic complement. After fertilization, a 46,XX or 46,XY diploid DNA complement is restored. Restoration of the normal cellular DNA content is crucial to normal function. Abnormalities of cellular DNA content cause distinct phenotypic abnormalities, as exemplified by hydatidiform molar pregnancy (see Chapter 39). In a complete hydatidiform mole, an oocyte without any nuclear genetic material (e.g., an empty ovum) is fertilized by one sperm. The haploid genetic content of the fertilized ovum is then duplicated, and the diploid cellular DNA content is restored, resulting in a homozygous 46,XX gamete. Less often, a complete hydatidiform mole results from the fertilization of an empty ovum by two sperm, resulting in a heterozygous 46,XX or 46,XY gamete. In complete molar pregnancies, the nuclear DNA is usually paternally derived, embryonic structures do not develop, and trophoblast hyperplasia occurs. Rarely, complete moles are biparental. This karyotype seems to be found in patients with recurrent hydatiform moles and is associated with a higher risk of persistent trophoblastic disease.

A partial hydatiform mole follows the fertilization of a haploid ovum by two sperm, resulting in a 69,XXX, 69,XXY, or 69,XYY karyotype. A partial mole contains paternal and maternal DNA, and both embryonic and placental development occur. Both the 69,YYY karyotype and the 46,YY karyotype are incompatible with embryonic and placental development. These observations demonstrate the importance of maternal genetic material, in particular the X chromosome, in normal embryonic and placental development.

In addition to total cellular DNA content, the chromosome number is an important determinant of cellular function. Abnormalities of chromosome number, which often are caused by nondisjunction during meiosis, result in well-characterized clinical syndromes such as trisomy 21 (Down syndrome), trisomy 18, and trisomy 13.

Genetic Control of the Cell Cycle

Cellular proliferation must occur to balance normal cell loss and maintain tissue and organ integrity. This process requires the coordinated expression of many genes at discrete times during the cell cycle (3). In the absence of growth factors, cultured mammalian cells are arrested in the G0 phase. With the addition of growth factors, these quiescent cells pass through the so-called restriction point 14 to 16 hours later and enter the S phase 6 to 8 hours thereafter. The restriction point or G1/S boundary marks the point at which a cell commits to proliferation. A second checkpoint is the G2/M boundary, which marks the point at which repair of any DNA damage must be completed (47). To successfully complete the cell cycle, a number of cell division cycle (cdc) genes are activated.

Cell Division Cycle Genes

Among the factors that regulate the cell cycle checkpoints, proteins encoded by the cdc2 family of genes and the cyclin proteins appear to play particularly important roles (8,9). Growth factor–stimulated mammalian cells express early-response or delayed-response genes, depending on the chronological sequence of the appearance of specific RNAs. The early- and delayed-response genes act as nuclear transcription factors and stimulate the expression of a cascade of other genes. Early-response genes such as c-Jun and c-Fos enhance the transcription of delayed-response genes such as E2Fs. E2F transcription factors are required for the expression of various cell cycle genes and are functionally regulated by the retinoblastoma (Rb) protein. Binding of Rb to E2F converts E2F from a transcriptional activator to a repressor of transcription. Phosphorylation of Rb inhibits its repressing function and permits E2F-mediated activation of genes required for entry into the S phase. Cdk4-cyclin D, Cdk6-cyclin D, and Cdk2-cyclin E complexes cause phosphorylation of Rb, which remains phosphorylated throughout the S, G2, and M phases of the cell cycle. After completion of mitosis, a decline of the level of Cdk-cyclins leads to dephosphorylation of Rb by phosphatases and, consequently, an inhibition of E2F in the early G1 phase.

Cdks are being evaluated as targets for cancer treatments because they are frequently overactive in cancer disease and Cdk-inhibiting proteins are dysfunctional. The Cdk4 inhibitor P1446A-05, for example, specifically inhibits Cdk4-mediated G1-S phase transition, arresting cell cycling and inhibiting cancer cell growth (10). SNS-032 selectively binds to Cdk2, -7, and -9, preventing their phosphorylation and activation and subsequently preventing cell proliferation.

As cells approach the G1-S phase transition, synthesis of cyclin A is initiated. The Cdk2-cyclin A complex can trigger initiation of DNA synthesis by supporting the prereplication complex. The p34 cdc2protein and specific cyclins form a complex heterodimer referred to as mitosis-promoting factor (MPF), which catalyzes protein phosphorylation and drives the cell into mitosis. Cdk1 assembles with cyclin A and cyclin B in the G2 phase and promotes the activity of the MPF. Mitosis is initiated by activation of the cdc gene at the G2-M checkpoint (11,12). Once the G2-M checkpoint is passed, the cell undergoes mitosis. In the presence of abnormally replicated chromosomes, progression past the G2-M checkpoint does not occur.

The p53 tumor suppressor gene participates in cell cycle control. Cells exposed to radiation therapy exhibit an S-phase arrest that is accompanied by increased expression of p53. This delay permits the repair of radiation-induced DNA damage. In the presence of p53 mutations, the S-phase arrest that normally follows radiation therapy does not occur (13,14). The wild type p53 gene can be inactivated by the human papillomavirus E6 protein, preventing S-phase arrest in response to DNA damage (15).

Apoptosis

The regulation and maintenance of normal tissue requires a balance between cell proliferation and programmed cell death, or apoptosis. When proliferation exceeds programmed cell death, the result is hyperplasia. When programmed cell death exceeds proliferation, the result is atrophy. Programmed cell death is a crucial concomitant of normal embryologic development. This mechanism accounts for deletion of the interdigital webs, palatal fusion, and development of the intestinal mucosa (1618). Programmed cell death is also an important phenomenon in normal physiology (19). The reduction in the number of endometrial cells following alterations in steroid hormone levels during the menstrual cycle is, in part, a consequence of programmed cell death (20,21). In response to androgens, granulosa cells undergo programmed cell death (e.g., follicular atresia) (22).

Programmed cell death, or apoptosis, is an energy-dependent, active process that is initiated by the expression of specific genes. This process is distinct from cell necrosis, although both mechanisms result in a reduction in total cell number. In programmed cell death, cells shrink and undergo phagocytosis. Conversely, groups of cells expand and lyse when undergoing cell necrosis. The process is energy independent and results from noxious stimuli. Programmed cell death is triggered by a variety of factors, including intracellular signals and exogenous stimuli such as radiation exposure, chemotherapy, and hormones. Cells undergoing programmed cell death may be identified on the basis of histologic, biochemical, and molecular biologic changes. Histologically, apoptotic cells exhibit cellular condensation and fragmentation of the nucleus. Biochemical correlates of impending programmed cell death include an increase in transglutaminase expression and fluxes in intracellular calcium concentration (23).

Programmed cell death emerged as an important factor in the growth of neoplasms. Historically, neoplastic growth was characterized by uncontrolled cellular proliferation that resulted in a progressive increase in tumor burden. It is recognized that the increase in tumor burden associated with progressive disease reflects an imbalance between cell proliferation and cell death. Cancer cells fail to respond to the normal signals to stop proliferating, and they may fail to recognize the physiologic signals that trigger programmed cell death.

Modulation of Cell Growth and Function

The normal cell exhibits an orchestrated response to the changing extracellular environment. The three groups of substances that signal these extracellular changes are steroid hormones, growth factors, and cytokines. The capability to respond to these stimuli requires a cell surface recognition system, intracellular signal transduction, and nuclear responses for the expression of specific genes in a coordinated fashion. Among the genes that participate in control of cell growth and function, proto-oncogenes and tumor suppressor genes are particularly important. More than 100 proto-oncogene products that contribute to growth regulation have been identified (24) (Table 6.1). As a group, proto-oncogenes exert positive effects upon cellular proliferation. In contrast, tumor suppressor genes exert inhibitory regulatory effects on cellular proliferation (Table 6.2).

Steroid Hormones

Steroid hormones play a crucial role in reproductive biology and in general physiology. Among the various functions, steroid hormones influence pregnancy, cardiovascular function, bone metabolism, and an individual’s sense of well-being. The action of steroid hormones is mediated via extracellular signals to the nucleus to affect a physiologic response.

Table 6.1 Proto-Oncogenes

Proto-oncogenes

Gene Product/Function

 

Growth factors

 

 Fibroblast growth factor

 fgf-5

 

 Sis

 Platelet-derived growth factor beta

 hst, int-2

 
 

Transmembrane receptors

 erb-B

 Epidermal growth factor (EGF) receptor

 HER-2/neu

 EGF-related receptor

 Fms

 Colony-stimulating factor (CSF) receptor

 Kit

 Stem cell receptor

 Trk

 Nerve growth factor receptor

 

Inner-membrane receptor

 bcl-2

 

 Ha-ras, N-ras, N-ras

 

 fgr, lck, src, yes

 
 

Cytoplasmic messengers

 Crk

 

 cot, plm-1, mos, raf/mil

 
 

Nuclear DNA binding proteins

 erb-B1

 

 jun, ets-1, ets-2,fos, gil 1,rel, ski, vav

 

 lyl-1, maf, myb, myc, L- myc, N-myc, evi-1

 

Table 6.2 Tumor Suppressor Genes

 p53

Mutated in as many as 50% of solid tumors

 Rb

Deletions and mutations predispose to retinoblastoma

 PTEN

Dual specificity phosphatase that represses PI3-kinase/Akt pathway activation with negative effect on cell growth

 P16INK4a

Binds to cylin-CDK4 complex inhibiting cell cycle progression

 FHIT

Fragile histidine triad gene with tumor suppressor function via unknown mechanisms

 WT1

Mutations are correlated with Wilms' tumor

 NF1

Neurofibromatosis gene

 APC

Associated with colon cancer development in patients with familial adenomatous

Estrogens exert a variety of effects on growth and development of different tissues. The effects of estrogens are mediated via estrogen receptors (ER), intracellular proteins that function as ligand-activated transcription factors and belong to the nuclear receptor superfamily (25). Two mammalian ERs have been identified: ERα and ERβ. The structure of both receptors is similar and consists of six domains named A through F from the N- to C-terminus, encoded by 8 to 9 exons (26). Domains A and B are located at the N-terminus and contain an agonist-independent transcriptional activation domain (activation function 1, or AF-1). The C domain is a highly conserved central DNA-binding domain composed of two zinc fingers through which ER interacts with the major groove and the phosphate backbone of the DNA helix. The C-terminus of the protein contains domains E and F and functions as ligand-binding domain (LBD–domain E) and AF-2–domain F (Fig. 6.3).

Activation of transcription via the ER is a multistep process. The initial step requires activation of the ER via various mechanisms (Fig. 6.4). For example, estrogens such as 17β-estradiol can diffuse into the cell and bind to the LBD of the ER. Upon ligand binding, the ER undergoes conformational changes followed by a dissociation of various bound proteins, mainly heat shock proteins 90 and 70 (Hsp90 and Hsp70). Activation of the ER also requires phosphorylation by several protein kinases, including casein kinase II, protein kinase A, and components of the Ras/Mapk (mitogen-activated protein kinase) pathway (26). Four phosphorylation sites of the ER are clustered in the NH2 terminus with the AF-1 region.

The activated ER elicits a number of different genomic as well as nongenomic effects on intracellular signaling pathways. The classical steroid signaling pathway involves binding of the activated estrogen receptor to an estrogen responsive element (ERE) on the genome as homodimers and subsequent stimulation of transcription (27,28). The minimal consensus sequence for the ERE is a 13 bp palindromic inverted repeat (IR) and is defined as 5′-GGTCAnnnTGACC-3′. Genes that are regulated by activated ERs include early gene responses such as c-myc, c-fos, and d-jun, and genes encoding for growth factors such as insulin growth factor (IGF-1 and IGF-2), epidermal growth factor (EGF), transforming growth factor-α, and colony-stimulating factor (CSF-1).

Figure 6.3 Structure of the two mammalian estrogen receptors. ERα (595 amino acids) and ERβ (530 amino acids) consist of six domains (A–F from the N- to C-terminus). Domains A and B at the N-terminus contain an agonist-independent transcriptional activation domain (activation function 1, or AF-1). The C domain is the central DNA-binding sequence (DBD). Domains E and F function as ligand binding domain (LBD) and activation function 2 (AF-2). Also shown is the structure of the ER ligand 17β-estradiol.

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Figure 6.4 Activation of estrogen receptor mediated transcription. Intracellular estrogen receptor signaling is mediated via different pathways. A: 17β-estrodiol diffuses through the cell membrane and binds to cytoplasmic ER. The ER is subsequently phosphorylated, undergoes dimerization, and binds to the estrogen response element (ERE) on the promoter of an estrogen responsive gene. B: Estrogen ligand binds to membrane-bound ER and activates the mitogen-activated protein kinase (MAPK) pathways that support ER-mediated transcription. C: Binding of cytokines such as insulinlike growth factor (IGF) or epidermal growth factor to their membrane receptor can cause activation of protein kinases like PKA, which subsequently activates ER by phosphorylation.

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In addition to the described genomic effects of estrogens, there is growing evidence for nongenomic effects of estrogens on intracellular signal transduction pathways. These effects include rapid activation of the adenylate cyclase, which results in cyclic adenosine monophosphate (cAMP)–dependent activation of protein kinase A (PKA) (29). Estrogens can stimulate the MAPK pathway and rapidly activate the Erk1/Erk2 proteins.

Various ligands with different affinities to the ER were developed and are called selective estrogen receptor modulators (SERMs). Tamoxifen, for example, is a mixed agonist/ antagonist for ERα, but it is a pure antagonist for ERβ. The ERβ receptor is ubiquitously expressed in hormone-responsive tissues, whereas the expression of ERα fluctuates in response to the hormonal milieu. The cellular and tissue effects of an estrogenic compound appear to reflect a dynamic interplay between the actions of these estrogen receptor isoforms. These observations underscore the complexity of estrogen interactions with both normal and neoplastic tissue. Mutations of hormone receptors and their functional consequences illustrate their important contributions to normal physiology. For example, absence of ERα in a male human was reported (30). The clinical sequelae attributed to this mutation include incomplete epiphyseal closure, increased bone turnover, tall stature, and impaired glucose tolerance. The androgen insensitivity syndrome is caused by mutations of the androgen receptor (31). Mutations of the receptors for growth hormone and thyroid-stimulating hormone result in a spectrum of phenotypic alterations. Mutations of hormone receptors may also contribute to the progression of neoplastic disease and resistance to hormone therapy (32,33).

Growth Factors

Growth factors are polypeptides that are produced by a variety of cell types and exhibit a wide range of overlapping biochemical actions. Growth factors bind to high-affinity cell membrane receptors and trigger complex positive and negative signaling pathways that regulate cell proliferation and differentiation (34). In general, growth factors exert positive or negative effects upon the cell cycle by influencing gene expression related to events that occur at the G1-S cell cycle boundary (35).

Because of their short half-life in the extracellular space, growth factors act over limited distances through autocrine or paracrine mechanisms. In the autocrine loop, the growth factor acts on the cell that produced it. The paracrine mechanism of growth control involves the effect of growth factors on another cell in proximity. Growth factors that play an important role in female reproductive physiology are listed in Table 6.3. The biologic response of a cell to a specific growth factor depends on a variety of factors, including the cell type, the cellular microenvironment, and the cell cycle status.

Table 6.3 Growth Factors that Play Important Roles in Female Reproductive Physiology

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The regulation of ovarian function occurs through autocrine, paracrine, and endocrine mechanisms (3642). The growth and differentiation of ovarian cells are particularly influenced by the insulinlike growth factors (IGF) (Fig. 6.5). IGFs amplify the actions of gonadotropin hormones on autocrine and paracrine growth factors found in the ovary. IGF-1 acts on granulosa cells to cause an increase in cAMP, progesterone, oxytocin, proteoglycans, and inhibin. On theca cells, IGF-1 causes an increase in androgen production. Theca cells produce tumor necrosis factor-α (TNF-α) and EGF, which are regulated by follicle-stimulating hormone (FSH). Epidermal growth factor acts on granulosa cells to stimulate mitogenesis. Follicular fluid contains IGF-1, IGF-2, TNF-α, TNF-β, and EGF. Disruption of these autocrine and paracrine intraovarian pathways may be the basis of polycystic ovarian disease, disorders of ovulation, and ovarian neoplastic disease.

Figure 6.5 The regulation of ovarian function occurs through autocrine, paracrine, and endocrine mechanisms.

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Transforming growth factor-β (TGF-β) activates intracytoplasmic serine threonine kinases and inhibits cells in the late G1 phase of the cell cycle (42). It appears to play an important role in embryonic remodeling. Mullerian-inhibiting substance (MIS), which is responsible for regression of the mullerian duct, is structurally and functionally related to TGF-β (43). TGF-α is an EGF homologue that binds to the EGF receptor and acts as an autocrine factor in normal cells. As with EGF, TGF-α promotes entry of G0 cells into the G1 phase of the cell cycle. The role of growth factors in endometrial growth and function was the subject of several reviews (3742). Similar to the ovary, autocrine, paracrine, and endocrine mechanisms of control also occur in endometrial tissue.

Intracellular Signal Transduction

Growth factors trigger intracellular biochemical signals by binding to cell membrane receptors. In general, these membrane-bound receptors are protein kinases that convert an extracellular signal into an intracellular signal. The interaction between growth factor ligand and its receptor results in receptor dimerization, autophosphorylation, and tyrosine kinase activation. Activated receptors in turn phosphorylate substrates in the cytoplasm and trigger the intracellular signal transduction system (Fig. 6.6). The intracellular signal transduction system relies on serine threonine kinases, src-related kinases, and G proteins. Intracellular signals activate nuclear factors that regulate gene expression. Many of the proteins that participate in the intracellular signal transduction system are encoded by proto-oncogenes that are divided into subgroups based on their cellular location or enzymatic function(44,45) (Fig. 6.7).

Figure 6.6 Pathways of intracellular signal transduction.

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Figure 6.7 Proto-oncogenes are divided into subgroups based on their cellular location or enzymatic function.

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The raf and mos proto-oncogenes encode proteins with serine threonine kinase activity. These kinases integrate signals originating at the cell membrane with those that are forwarded to the nucleus (46,47). Protein kinase C (PKC) is an important component of a second messenger system that exhibits serine threonine kinase activity. This enzyme plays a central role in phosphorylation, which is a general mechanism for activating and deactivating proteins. It also plays an important role in cell metabolism and division (48).

The Scr family of tyrosine kinases is related to PKC and includes protein products encoded by the scryesfgrhcklynfynlckalt, and fps/fes proto-oncogenes. These proteins bind to the inner cell membrane surface.

The G proteins are guanyl nucleotide-binding proteins. The heterotrimeric, or large G proteins, link receptor activation with effector proteins such as adenylcyclase, which activates the cAMP-dependent, kinase-signaling cascade (49). The monomeric or small G proteins, encoded by the ras proto-oncogene family, are designated p21 and are particularly important regulators of mitogenic signals. The p21 Rasprotein exhibits guanyl triphosphate (GTP) binding and GTPase activity. Hydrolysis of GTP to guanyl diphosphate (GDP) terminates p21 Ras activity. The p21 Ras protein influences the production of deoxyguanosine (dG) and inositol phosphate (IP) 3, arachidonic acid production, and IP turnover.

The phosphoinositide 3 (PI3) kinase can be activated by various growth factors like platelet-derived growth factor (PDGF) or IGF results. Activation of PI3 kinase results in an increase of intracellular, membrane-bound lipids, phosphatidylinositol-(3,4)-diphosphate (PIP2), and phosphatidylinositol-(3,4,5)-triphosphate (PIP3). The Akt protein is subsequently phosphorylated by PIP3-dependent kinases (PDK) for full activation. Activated Akt is released from the membrane and elicits downstream effects that lead to an increase in cell proliferation, prevention of apoptosis, invasiveness, drug resistance, and neoangiogenesis (50). The Pten (phosphatase and chicken tensin homologue deleted on chromosome 10) protein is an important factor in the PI3 kinase pathway, because it counteracts the activation of Akt by dephosphorylating PIP3. Cells with mutated tumor suppressor gene Pten and lack of functional Ptenexpression display an increased proliferation rate and decreased apoptosis, possibly supporting the development of a malignant phenotype. Pten frequently is mutated in endometrioid adenocarcinoma. Furthermore, lack of functional Pten expression was described in endometriosis.

The mammalian target of rapamycin (mTOR) is regulated by the PI3 kinase pathway. mTOR is a serine/threonine protein kinase that regulates a variety of cellular processes, including proliferation, motility, and translation (51). mTOR integrates the input from various upstream pathways, including insulin and growth factors like IGF proteins. The mTOR pathway provides important survival signals for cancer cells and therefore was one focus of targeted drug development (52). For example, rapamycin inhibits mTOR by associating with its intracellular receptor FKBP12. Derivates of rapamycin like everolimus(RAD001) and temsirolimus (CCI779) showed promising results in clinical trials (53).

mTOR functions as the catalytic subunit of two different protein complexes. Among the proteins associated with mTOR complex 1 (mTORC1) is mTOR, the regulatory associated protein of mTOR (Raptor), and PRAS40. This complex functions as a nutrient and energy sensor and controls protein synthesis (54). While mTORC1 is activated by insulin, growth factors, amino acids and oxidative stress, low nutrient levels, reductive stress, and growth factor deprivation inhibit its activity.

In contrast, the mTOR complex 2 (mTORC2) contains, among others, mTOR, the rapamycin-insensitive protein Rictor, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1). mTORC2 regulates the cytoskeleton and phosphorylates Akt (55). Its regulation is complex, but involves insulin, growth factors, serum, and nutrient levels.

Expression of Genes and Proteins

Regulation of genetic transcription and replication is crucial to the normal function of the daughter cells, the tissues and ultimately the organism. Transmission of external signals to the nucleus by way of the intracellular signal transduction cascade culminates in the transcription of specific genes and translation of the mRNA into proteins that ultimately affect the structure, function, and proliferation of the cell.

The human genome project resulted in the determination of the sequence of DNA of the entire human genome (56). With the completion of this project, it appears that the human haploid genome contains 23,000 protein coding genes. Sequencing the human genome is a major scientific achievement that opens the door for more detailed studies of structural and functional genomics. Structural genomics involves the study of three-dimensional structures of proteins based on their amino acid sequences. Functional genomics provides a way to correlate structure and function. Proteomics involves the identification and cataloging of all proteins used by a cell, and cytomics involves the study of cellular dynamics, including intracellular system regulation and response to external stimuli. The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other noncoding RNA produced in one or a population of cells. The transcriptome varies with external environmental conditions and reflects the actively expressed genes. The metabolome describes a set of small-molecule metabolites, including hormones and signaling molecules, that are found in a single organism. Similar to the transcriptome and proteome, the metabolome is subject to rapid changes (57). The kinome of an organism describes a set of protein kinases, enzymes that are crucial for phosphorylation reactions.

Cancer Genetics

Cancer is a genetic disease that results from a series of mutations in various cancer genes. Uncontrolled cell growth occurs because of accumulation of somatic mutations or the inheritance of one or more mutations through the germline, followed by additional somatic mutations. The mutation in genes that are directly involved in normal cellular growth and proliferation can lead to the development of uncontrolled growth, invasion, and metastasis.

According to the Knudson hypothesis, which was first described in children with hereditary retinoblastoma, two hits or mutations within the genome of a cell are required for a malignant phenotype to develop (58). In hereditary cancers, the first hit is present in the genome of every cell. Only one additional hit is necessary, therefore, to disrupt the correct function of the second cancer gene allele. In contrast, sporadic cancers develop in cells without hereditary mutations in the cancer predisposing alleles. In this case, both hits must occur in a single somatic cell to disrupt both cancer gene alleles (Fig. 6.8).

Figure 6.8 Hereditary and sporadic cancer development based on the Knudson “two-hit” genetic model. All cells harbor one mutant tumor suppressor gene allele in hereditary cancer. The loss of the second allele results in the malignant phenotype. Sporadic cancers develop in cells with normal genome, therefore requiring both alleles to be inactivated (two hits).

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Most adult solid tumors require 5 to 10 rate-limiting mutations to acquire the malignant phenotype. Among these mutations, some are responsible for causing the cancer phenotype, whereas others might be considered bystander mutations, as with, for example, the amplification of genes that are adjacent to an oncogene. The most compelling evidence for the mutagenic tumor development process is that the age-specific incidence rates for most human epithelial tumors increase at roughly the fourth to eighth power of elapsed time.

Gatekeepers and Caretakers

Cancer susceptibility genes are divided into “gatekeepers” and “caretakers” (59). Gatekeeper genes control cellular proliferation and are divided into oncogenes and tumor suppressor genes. In general, oncogenes stimulate cell growth and proliferation, and tumor suppressor genes reduce the rate of cell proliferation or induce apoptosis. Gatekeepers prevent the development of tumors by inhibiting growth or promoting cell death. Examples of gatekeeper genes include the tumor suppressor gene p53 and the retinoblastoma gene.

Caretaker genes preserve the integrity of the genome and are involved in DNA repair (stability genes). The inactivation of caretakers increases the likelihood of persistent mutations in gatekeeper genes and other cancer-related genes. The DNA mismatch repair genes, MLH1, MSH2, and MSH6, are examples of caretaker genes.

Hereditary Cancer

Most cancers are caused by spontaneous somatic mutations. However, a small percentage of cancers arise on a heritable genomic background. About 12% of all ovarian cancers and about 5% of endometrial cancers are considered to be hereditary (60,61). Germline mutations require additional mutations at one or more loci for tumorigenesis to occur. These mutations occur via different mechanisms, for example, via environmental factors such as ionizing radiation or mutations of stability genes. Characteristics of hereditary cancers include diagnosis at a relatively early age and a family history of cancer, usually of a specific cancer syndrome, in two or more relatives. Hereditary cancer syndromes associated with gynecologic tumors are summarized in Table 6.4.

Table 6.4 Hereditary Cancer Syndromes Associated with Gynecologic Tumors

Hereditary Syndrome

Gene Mutation

Tumor Phenotype

Li-Fraumeni syndrome

TP53, CHEK2

Breast cancer, soft tissues sarcoma, adrenal cortical carcinoma, brain tumors

Cowden syndrome, Bannayan-Zonana syndrome

PTEN

Breast cancer, hamartoma, glioma, endometrial cancer

Hereditary breast and ovarian cancer

BRCA1, BRCA2

Cancer of breast, ovary, fallopian tube

Hereditary nonpolyposis colorectal cancer (HNPCC)

MLH1, MSH2, MSH3, MSH6, PMS2

Cancer of colon, endometrium, ovary, stomach, small bowel, urinary tract

Multiple endocrine neoplasia type I

Menin

Cancer of thyroid, pancreas and pituitary, ovarian carcinoid

Multiple endocrine neoplasia type II

RET

Cancer of thyroid and parathyroid, pheochromocytoma, ovarian carcinoid

Peutz-Jeghers syndrome

STK11

Gastrointestinal hamartomatous polyps, tumors of the stomach, duodenum, colon, ovarian sex cord tumor with annular tubules (SCTAT)

Various cancer-causing genetic and epigenetic mechanisms are described. On the genomic level, gain of function gene mutations can lead to a conversion of proto-oncogenes into oncogenes, and loss of function gene mutations can inactivate tumor suppressor genes. Epigenetic changes include DNA methylation, which can cause inactivation of tumor suppressor gene expression by preventing the correct function of the associated promoter sequence. Collectively, these genetic and epigenetic changes are responsible for the development of cancer characterized by the ability of cells to invade and metastasize, grow independently of growth factor support, and escape from antitumor immune responses.

Oncogenes

Oncogenes comprise a family of genes that result from gain of function mutations of their normal counterparts, proto-oncogenes. The normal function of proto-oncogenes is to stimulate proliferation in a controlled context. Activation of oncogenes can lead to stimulation of cell proliferation and development of a malignant phenotype. Oncogenes were initially discovered through retroviral tumorigenesis. Viral infection of mammalian cells can result in integration of the viral sequences into the proto-oncogene sequence of the host cell. The integrated viral promoter activates transcription from the surrounding DNA sequences, including the proto-oncogene. Enhanced transcription of the proto-oncogene sequences results in the overexpression of growth factors, growth factor receptors, and signal transduction proteins, which results in stimulation of cell proliferation. One of the most important group of viral oncogenes is the family of ras genes, which include c-H(Harvey)-rasc-K(Kirsten)-ras, and N(Neuroblastoma)-ras.

Tumor Suppressor Genes

Tumor suppressor genes are involved in the development of most cancers and are usually inactivated in a two-step process in which both copies of the tumor suppressor gene are mutated or inactivated by epigenetic mechanisms like methylation (62). The most commonly mutated tumor suppressor gene in human cancers is p53 (63). The p53 protein regulates transcription of other genes involved in cell cycle arrest such as p21. Up-regulation of p53 expression is induced by DNA damage and contributes to cell cycle arrest, allowing DNA repair to occur. p53 also plays an important role in the initiation of apoptosis. The most common mechanism of inactivation of p53 differs from the classic two-hit model. In most cases, missense mutations that change a single amino acid in the DNA binding domain of p53 results in overexpression of nonfunctional p53 protein in the nucleus of the cell.

The identification of tumor suppressor genes was facilitated by positional cloning strategies. The main approaches are cytogenetic studies to identify chromosomal alterations in tumor specimens, DNA linkage techniques to localize genes involved in inherited predisposition to cancer, and examination for loss of heterozygosity or allelic alterations among studies in sporadic tumors. Comparative genomic in situ hybridization (CGH) allows fluorescence identification of chromosome gain and loss in human cancers within a similar experiment.

Stability Genes

The third class of cancer genes is “stability genes,” which promotes tumorigenesis in a way different from tumor suppressor genes or amplified oncogenes. The main function of stability genes is the preservation of the correct DNA sequence during DNA replication (caretaker function) (64). Mistakes that are made during normal DNA replication or induced by exposure to mutagens can be repaired by a variety of mechanisms that involve mismatch repair genes, nuclear-type excision repair genes, and base excision repair genes. The inactivation of stability genes potentially leads to a higher mutation rate in all genes. However, only mutations in oncogenes and tumor suppressor genes influence cell proliferation and confer a selective growth advantage to the mutant cell. Similar to tumor suppressor genes, both alleles of stability genes must be activated to cause loss of function.

Genetic Aberrations

Gene replication, transcription, and translation are imperfect processes, and the fidelity is less than 100%. Genetic errors may result in abnormal structure and function of genes and proteins. Genomic alterations such as gene amplification, point mutations, and deletions or rearrangements were identified in premalignant, malignant, and benign neoplasms of the female genital tract (65) (Fig. 6.9).

Figure 6.9 Genes can be amplified or undergo mutation, deletion, or rearrangement.

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Amplification

Amplification refers to an increase in the copy number of a gene. Amplification results in enhanced gene expression by increasing the amount of template DNA that is available for transcription. Proto-oncogene amplification is a relatively common event in malignancies of the female genital tract. The HER2/neu proto-oncogene, also known as c-erbB-2 and HER2, encodes a 185 kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity. It belongs to a family of transmembrane receptor genes that includes the epidermal growth factor receptors (erbB-1), erbB-3, and erbB-4HER2/neuinteracts with a variety of different cellular proteins that increase cell proliferation. Overexpression of HER2/neu was demonstrated in about 30% of breast cancers, 20% of advanced ovarian cancers, and as many as 50% of endometrial cancers (66). High tissue expression of HER2/neu is correlated with a decreased overall survival, particularly in patients with endometrial cancer.

Point Mutations

Point mutations of a gene may remain without any consequence for the expression and function of the protein (gene polymorphism). However, point mutations can alter a codon sequence and subsequently disrupt the normal function of a gene product. The ras gene family is an example of oncogene-encoded proteins that disrupt the intracellular signal transduction system following point mutations. Transforming Ras proteins contain point mutations in critical codons (i.e., codons 11, 125961) with decrease of GTPase activity and subsequent expression of constitutively active Ras. Point mutations of the p53 gene are the most common genetic mutations described in solid tumors. These mutations occur at preferential “hot spots” that coincide with the most highly conserved regions of the gene. The p53tumor suppressor gene encodes for a phosphoprotein that is detectable in the nucleus of normal cells. When DNA damage occurs, p53 can arrest cell cycle progression to allow the DNA to be repaired or undergo apoptosis. The lack of function of normal p53 within a cancer cell results in a loss of control of cell proliferation with inefficient DNA repair and genetic instability. Mutations of the p53 gene occur in approximately 50% of advanced ovarian cancers and 30% to 40% of endometrial cancers but are uncommon in cervical cancer.

Point mutations in the BRCA1 and BRCA2 genes can alter the activity of these genes and predispose to the development of breast and ovarian cancer (67). The frequency of BRCA1 and BRCA2mutations in the general population in the United States is estimated at 1:250. Specific founder mutations were reported for various ethnic groups. For example, two BRCA1 mutations (185delAG and 5382insC) and one BRCA2 mutation (6174delT) are found in 2.5% of Ashkenazi Jews of Central and Eastern European descent. Additional founder mutations were described in other ethnic groups, including from the Netherlands (BRCA1, 2804delAA and several large deletion mutations), Iceland (BRCA2, 995del5), and Sweden (BRCA1, 3171ins5).

The BRCA proteins are involved in DNA repair. If DNA is damaged, for example, by ionizing radiation or chemotherapy, the BRCA2 protein binds to the RAD51 protein, which is central for the repair of double-stranded breaks via homologous recombination. BRCA2 regulates the availability and activity of RAD51 in this key reaction. Phosphorylation of the BRCA2/RAD51 complex allows RAD51 to bind to the site of DNA damage and, in conjunction with several other proteins, mediates repair of DNA by homologous recombination. BRCA1 functions within a complex network of protein–protein interactions, mediating DNA repair by homologous recombination and regulating transcription via the BRCA1-associated surveillance complex (BASC).

Deletions and Rearrangements

Deletions and rearrangements reflect gross changes in the DNA template that may result in the synthesis of a markedly altered protein product. Somatic mutations may involve chromosomal translocations that result in chimeric transcripts with juxtaposition of one gene to the regulatory region of another gene. This mutation type is most commonly reported in leukemias, lymphomas, and mesenchymal tumors. The Philadelphia chromosome in chronic myeloid leukemia, for example, is the result of a reciprocal translocation between one chromosome 9 and one chromosome 22. The DNA sequence removed from chromosome 9 contains the proto-oncogene c-ABL and inserts into the BCR gene sequence on chromosome 22 (Philadelphia chromosome). The resulting chimeric BCR-ABL gene product functions as a constitutively active tyrosine kinase and stimulates cellular proliferation by such mechanisms as an increase of growth factors.

Single-nucleotide polymorphism (SNP) describes a variation in the DNA sequence (68). Single nucleotides in the genome differ between paired chromosomes in either one individual or between two individuals. For example, the sequences TGACTA and TCACTA contain one single change in the second nucleotide from guanine (G) to cytosine (C). This results in a G and C allele for this particular gene sequence. SNPs can occur within coding or noncoding sequences of genes or in the intergenic regions. SNPs might not change the amino acid sequence of the protein that is produced (synonymous SNP) or produce a different peptide (nonsynonymous SNPs). If SNPs are located in noncoding regions, various other processes like gene splicing or transcription factor binding might be affected.

The frequency of SNPs in a given population is provided by the minor allele frequency. This frequency differs between ethnic groups and geographic locations. SNPs were associated with various human diseases including cancer disease. They also influence the effect of drug treatment and responses to pathogens and chemicals (69). SNPs are important for the comparison between genomes of different populations for examples providing information about the susceptibility of certain population to develop specific cancers (70).

The Cancer Genome Atlas Project

In 2006 the National Cancer Institute and the National Human Genome Research Institute initiated the Cancer Genome Atlas (TCGA) project. The goal of the project is to provide a comprehensive genomic characterization and sequence analysis of cancer diseases. The initial phase included glioblastoma multiforme, lung, and ovarian cancer (71,72). Many more tumor types will be added to the analysis.

The TCGA is taking advantage of high-throughput genome analysis techniques, including gene expression profiling, SNP genotyping, copy number variation profiling, genome wide methylation profiling, microRNA profiling, and exon sequencing (73). These data are accessible for researchers via the TCGA Web page (74).

Immunology

The immune system plays an essential part in host defense mechanisms, in particular the response to infections and neoplastic transformation. Our increased understanding of immune system regulation provides opportunities for the development of novel immunotherapeutic and immunodiagnostic approaches.

Immunologic Mechanisms

The human immune system has the potential to respond to abnormal or tumor cells in various ways. Some of these immune responses occur in an innate or antigen-nonspecific manner, whereas others are adaptive or antigen specific. Adaptive responses are specific to a given antigen. The establishment of a memory response allows a more rapid and vigorous response to the same antigen in future encounters. Various innate and adaptive immune mechanisms are involved in responses to tumors, including cytotoxicity directed to tumor cells mediated by cytotoxic T cells, natural killer (NK) cells, macrophages, and antibody-dependent cytotoxicity mediated by complementation activation (75).

Adaptive or specific immune responses include humoral and cellular responses. Humoral immune responses refer to the production of antibodies. Antibodies are bifunctional molecules composed of a variable region with specific antigen-binding sites, combined with a constant region that directs the biologic activities of the antibody, such as binding to phagocytic cells or activation of complement. Cellular immune responses are antigen-specific immune responses mediated directly by activated immune cells rather than by the production of antibodies. The distinction between humoral and cellular responses is historical and originates from the experimental observation that humoral immune function can be transferred by serum, whereas cellular immune function requires the transfer of cells. Most immune responses include both humoral and cellular components. Several types of cells, including cells from both the myeloid and lymphoid lineages, make up the immune system. Specific humoral and cellular immune responses to foreign antigens involve the coordinated action of populations of lymphocytes operating in concert with one another and with phagocytic cells (macrophages). These cellular interactions include both direct cognate interactions involving cell-to-cell contact and cellular interactions involving the secretion of and response to cytokines or lymphokines. Lymphoid cells are found in lymphoid tissues, such as lymph nodes or spleen, or in the peripheral circulation. The cells that make up the immune system originate from stem cells in the bone marrow.

B Cells, Hormonal Immunity, and Monoclonal Antibodies

B lymphocytes synthesize and secrete antibodies. Mature, antigen-responsive B cells develop from pre-B cells (committed B-cell progenitors) and differentiate to become plasma cells, which produce large quantities of antibodies. Pre-B cells originate from bone marrow stem cells in adults after rearrangement of immunoglobulin genes from their germ cell configuration. Mature B cells express cell surface immunoglobulin molecules that function as receptors for antigen.

Upon interaction with antigen, mature B cells respond to become antibody-producing cells. The process requires the presence of appropriate cell–cell stimulatory signals and cytokines. Monoclonal antibodies are directed against a specific antigenic determinant. In contrast, polyclonal antibodies detect multiple epitopes that might be presented by just one or a panel of proteins. The in vitro production of monoclonal antibodies, pioneered by Kohler and Milstein in the 1970s, has become an invaluable diagnostic and therapeutic tool, particularly for the management of malignancies (76). The tumor antigen CA125, for example, was detected in a screen of antibodies generated against ovarian cancer cell lines. A radioimmunoassay is widely used to measure CA125 in the serum of patients with ovarian cancer and guide treatment decisions. Therapeutic approaches utilized immunotoxin-conjugated monoclonal antibodies directed to human ovarian adenocarcinoma antigens. These antibodies induce tumor cell killing and can prolong survival in mice implanted with a human ovarian cancer cell line. However, some obstacles limit the clinical use of monoclonal antibodies, including tumor cell antigenic heterogeneity, modulation of tumor-associated antigens, and cross-reactivity of normal host and tumor-associated antigens. No unique tumor-specific antigens were identified. All tumor antigens have to be considered as tumor-related antigens because they are expressed on the malignant as well as the nonmalignant tissues. Because most monoclonal antibodies are murine, the host’s immune system can recognize and respond to these foreign mouse proteins. The use of the genetically engineered monoclonal antibodies composed of human-constant regions with specific antigen-reactive murine variable regions can result in reduced antigenicity.

T Lymphocytes and Cellular Immunity

T lymphocytes have a central role in the generation of immune responses by acting as helper cells in both humoral and cellular immune responses and by acting as effector cells in cellular responses. T-cell precursors originate in bone marrow and move to the thymus, where they mature into functional T cells. During their thymic maturation, T cells that can recognize antigen in the context of the major histocompatibility complex (MHC) molecules are selected, while self-responding T cells are removed (75).

T cells can be distinguished from other types of lymphocytes by their cell surface phenotype, based on the pattern of expression of various molecules, and by differences in their biologic functions.All mature T cells express certain cell surface molecules, such as the cluster determinant 3 (CD3) molecular complex and the T-cell antigen receptor, which is found in close association with the CD3 complex. T cells recognize antigen through the cell surface T-cell antigen receptor (TCR). The structure and organization of this molecule are similar to those of antibody molecules, which are the B-cell receptors for the antigen. During T-cell development, the T-cell receptor gene undergoes gene arrangements similar to those seen in B cells, but there are important differences between the antigen receptors on B cells and T cells. The T-cell receptor is not secreted, and its structure is somewhat different from that of antibody molecules. The way in which the B-cell and T-cell receptors interact with antigens is quite different. T cells can respond to antigens only when these antigens are presented in association with MHC molecules on antigen-presenting cells. Effective antigen presentation involves the processing of antigen into small fragments of peptide within the antigen-presenting cell and the subsequent presentation of these fragments of antigen in association with MHC molecules expressed on the surface of the antigen-presenting cell. T cells can respond to antigens only when presented in this manner, unlike B cells, which can bind antigens directly, without processing and presentation by antigen-presenting cells (75).

There are two major subsets of mature T cells that are phenotypically and functionally distinct: T-helper/inducer cells, which express the CD4 cell surface marker, and the T-cytotoxic cells, which express the CD8 marker.The expression of these markers is acquired during the passage of T cells through the thymus. CD4 T cells can provide help to B cells, resulting in the production of antibodies by B cells, and interact with antigen presented by antigen-presenting cells in association with MHC class II molecules. CD4 T cells can act as helper cells for other T cells. CD8 T cells include cells that are cytotoxic (cells that can kill target cells bearing appropriate antigens), and they interact with antigen presented on target cells in association with MHC class I molecules. These T cells can inhibit the biologic functions of B cells or other T cells (75). Although the primary biologic role of cytotoxic T cells(CTLs) seems to be lysis of virus-infected autologous cells, cytotoxic immune T cells can mediate the lysis of tumor cells directly. Presumably, CTLs recognize antigens associated with MHC class I molecules on tumor cells through their antigen-specific T-cell receptor, setting off a series of events that ultimately results in the lysis of the target cell.

Monocytes and Macrophages

Monocytes and macrophages, which are myeloid cells, have important roles in both innate and adaptive immune responses; macrophages play a key part in the generation of immune responses. T cells do not respond to foreign antigens unless those antigens are processed and presented by antigen-presenting cells. Macrophages (and B cells and dendritic cells) express MHC class II molecules and are effective antigen-presenting cells for CD4 T cells.Helper-inducer (CD4) T cells that bear a T-cell receptor of appropriate antigen and self-specificity are activated by this antigen-presenting cell to provide help (various factors—lymphokines—that induce the activation of other lymphocytes). In addition to their role as antigen-presenting cells, macrophages play an important part in innate responses by ingesting and killing microorganisms. Activated macrophages, besides their many other functional capabilities, can act as cytotoxic, antitumor killer cells.

Natural Killer Cells

Natural killer cells are effector cells in an innate type of immune response: the nonspecific killing of tumor cells and virus-infected cells. Therefore, NK activity represents an innate form of immunity that does not require an adaptive, memory response for optimal biologic function, but the antitumor activity can be increased by exposure to several agents, particularly cytokines such as interleukin-2 (IL-2). Characteristically, NK cells have a large granular lymphocyte morphology. NK cells display a pattern of cell surface markers that differs from those characteristic of T or B cells. NK cells can express a receptor for the crystallizable fragment (Fc) portion of antibodies, and other NK-associated markers. NK cells appear to be functionally and phenotypically heterogeneous, when compared with T or B cells. The cells that can carry out antibody-dependent cellular cytotoxicity, or antibody-targeted cytotoxicity, are NK-like cells. Antibody-dependent cellular cytotoxicity by NK-like cells resulted in the lysis of tumor cells in vitro. The mechanisms of this tumor cell killing are not clearly understood, although close cellular contact between the effector cell and the target cell seems to be required.

Table 6.5 Sources, Target Cells, and Biological Activities of Cytokines Involved in Immune Responses

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Cytokines, Lymphokines, and Immune Mediators

Many events in the generation of immune responses (as well as during the effector phase of immune responses) require or are enhanced by cytokines, which are soluble mediator molecules (Table 6.5) (7792). Cytokines are pleiotropic in that they have multiple biologic functions that depend on the type of target cell or its maturational state. Cytokines are heterogeneous in the sense that most cytokines share little structural or amino acid homology. Cytokines are called monokines if they are derived from monocytes, lymphokines if they are derived from lymphocytes, interleukins if they exert their actions on leukocytes, or interferons (IFNs) if they have antiviral effects. They are produced by a wide variety of cell types and seem to have important roles in many biologic responses outside the immune response, such as inflammation or hematopoiesis. They may also be involved in the pathophysiology of a wide range of diseases and show great potential as therapeutic agents in immunotherapy for cancer. Although cytokines are a heterogeneous group of proteins, they share some characteristics. For instance, most cytokines are low- to intermediate-molecular weight (1060 kd) glycosylated-secreted proteins. They are involved in immunity and inflammation, are produced transiently and locally (they act in an autocrine and paracrine rather than an endocrine manner), are extremely potent in small concentrations, and interact with high-affinity cellular receptors that are specific for each cytokine. The cell surface binding of cytokines by specific receptors results in signal transduction followed by changes in gene expression and, ultimately, by changes in cellular proliferation or altered cell behavior, or both. Their biologic actions overlap, and exposure of responsive cells to multiple cytokines can result in synergistic or antagonistic biologic effects.

T-cell subsets characterized by the secretion of distinct patterns of cytokines were identified. TH1 and TH2 are two helper T-cell subpopulations that control the nature of an immune response by secreting a characteristic and mutually antagonistic set of cytokines: Clones of TH1 produce IL-2 and IFN-γ, whereas TH2 clones produce IL-4, IL-5, and IL-10 (86). A similar dichotomy between TH1- and TH2-type responses was reported in humans (87,88). Human IL-10 inhibits the production of IFN-γ and other cytokines by human peripheral blood mononuclear cells and by suppressing the release of cytokines (IL-1, IL-6, IL-8, and TNF-α) by activated monocytes (8992). IL-10 down-regulates class II MHC expression on monocytes, resulting in a strong reduction in the antigen-presenting capacity of these cells (92). Together, these observations support the concept that IL-10 has an important role as an immune-inhibitory cytokine. Additional T-cell subsets were identified, including Th17 cells and regulatory T cells (Treg). Th17 cells are a distinct, pro-inflammatory T-cell subset, which is functionally characterized by mediating protection against extracellular bacteria and by its pathogenic role in autoimmune disorders (9398). Th17 cells characteristically produce IL-17, CXCL13 (a B-cell stimulatory chemokine), IL-6, and TNF-α, in contrast to Th2 cells, which characteristically produce IL-4, IL-5, IL-9 and IL-13, or Th1 cells, which produce IFN-γ (Fig. 6.2). Treg cells constitute another subset of CD4-positive T cells that participates in the maintenance of immunologic self-tolerance by actively suppressing the activation and expansion of self-reactive lymphocytes. Treg cells are characterized by the expression of CD25 (the IL-2 receptor chain) and the transcription factor FoxP3 (99,100). Treg cell activity is thought to be important in preventing the development of autoimmune diseases. Removal of Treg may enhance immune responses against infectious agents or cancer. Although much remains to be learned about the role of Treg activity in antitumor immunity, it is clear that such cells may play a role in modulating host responses to cancer.

Because epithelial cancers of the ovary usually remain confined to the peritoneal cavity, even in the advanced stages of the disease, it was suggested that the growth of ovarian cancer intraperitoneally could be related to a local deficiency of antitumor immune effector mechanisms (102,103). Studies showed that ascitic fluid from patients with ovarian cancer contained increased concentrations of IL-10 (102). Various other cytokines are seen in ascitic fluid obtained from women with ovarian cancer, including IL-6, IL-10, TNF-α, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (103). A similar pattern was seen in serum samples from women with ovarian cancer with elevations of IL-6 and IL-10.

TNF-α is a cytokine that can be directly cytotoxic for tumor cells, can increase immune cell–mediated cellular cytotoxicity, and can activate macrophages and induce secretion of monokines. Other biologic activities of TNF-α include the induction of cachexia, inflammation, and fever; it is an important mediator of endotoxic shock.

Cytokines in Cancer Therapy

Cytokines are extraordinarily pleiotropic with a bewildering array of biologic activities, including some outside the immune system (55,56,77,83). Because some cytokines have direct or indirect antitumor and immune-enhancing effects, several of these factors are used in the experimental treatment of cancer.

The precise roles of cytokines in antitumor responses have not been elucidated. Cytokines can exert antitumor effects by many different direct or indirect activities. It is possible that a single cytokine could increase tumor growth directly by acting as a growth factor while at the same time increasing immune responses directed toward the tumor. The potential of cytokines to increase antitumor immune responses was tested in experimental adoptive immunotherapy by exposing the patient’s peripheral blood cells or tumor-infiltrating lymphocytes to cytokines such as IL-2 in vitro, thus generating activated cells with antitumor effects that can be given back to the patient (104106). Some cytokines can exert direct antitumor effects. Tumor necrosis factor can induce cell death in sensitive tumor cells.

The effects of cytokines on patients with cancer might be modulated by soluble receptors or blocking factors. For instance, blocking factors for TNF and for lymphotoxin were found in ascitic fluid from patients with ovarian cancer (106). Such factors could inhibit the cytolytic effects of TNF or lymphotoxin and should be taken into account in the design of clinical trials of intraperitoneal infusion of these cytokines.

Cytokines have growth-increasing effects on tumor cells in addition to inducing antitumor effects. They can act as autocrine or paracrine growth factors for human tumor cells, including those of nonlymphoid origin. For instance, IL-6 (which is produced by various types of human tumor cells) can act as a growth factor for human myeloma, Kaposi’s sarcoma, renal carcinoma, and epithelial ovarian cancer cells (7783).

Cytokines are of great potential value in the treatment of cancer, but because of their multiple—even conflicting—biologic effects, a thorough understanding of cytokine biology is essential for their successful use (104117).

Factors that Trigger Neoplasia

Cell biology is characterized by considerable redundancy and functional overlap, so a defect in one mechanism does not invariably jeopardize the function of the cell. When a sufficient number of abnormalities in structure and function occur, normal cell activity is jeopardized, and uncontrolled cell growth or cell death results. Either end point may result from accumulated genetic mutations over time. Factors are identified that enhance the likelihood of genetic mutations, jeopardize normal cell biology, and may increase the risk of cancer.

Increased Age

Increasing age is considered the single most important risk factor for the development of cancer (118). Cancer is diagnosed in as much as 50% of the population by 75 years of age (111). It was suggested that the increasing risk of cancer with age reflects the accumulation of critical genetic mutations over time, which ultimately culminates in neoplastic transformation. The basic premise of the multistep somatic mutation theory of carcinogenesis is that genetic or epigenetic alterations of numerous independent genes result in cancer. Factors that are associated with an increased likelihood of cancer include exposure to exogenous mutagens, altered host immune function, and certain inherited genetic syndromes and disorders.

Environmental Factors

A mutagen is a compound that results in a genetic mutation. A number of environmental pollutants act as mutagens when tested in vitro. Environmental mutagens usually produce specific types of mutations that can be differentiated from spontaneous mutations. As an example, activated hydrocarbons tend to produce GT transversions (119). A carcinogen is a compound that can produce cancer. It is important to recognize that all carcinogens are not mutagens and that all mutagens are not necessarily carcinogens.

Smoking

Cigarette smoking is perhaps the best known example of mutagen exposure that is associated with the development of lung cancer when the exposure is of sufficient duration and quantity in a susceptible individual. An association between cigarette smoking and cervical cancer has been recognized for decades. It was determined that the mutagens in cigarette smoke are selectively concentrated in cervical mucus (59). It was hypothesized that exposure of the proliferating epithelial cells of the transformation zone to cigarette smoke mutagens may increase the likelihood of DNA damage and subsequent cellular transformation.

Others observed that human papillomavirus (HPV) DNA is frequently inserted into the fragile histidine triad (FHIT) gene in cervical cancer specimens. The FHIT is an important tumor suppressor gene. Cigarette smoking might facilitate the incorporation of HPV DNA into the FHIT gene with subsequent disruption of correct tumor suppressor gene function.

Radiation

Radiation exposure can increase the risk of cancer. The overall risk of radiation-induced cancer is approximately 10% greater in women than in men (120). This difference is attributed to gender-specific cancers, including breast cancer. Radiation-induced cancer may be the result of sublethal DNA damage that is not repaired (120). Normally, radiation damage prompts an S-phase arrest so that DNA damage is repaired. This requires normal p53 gene function. If DNA repair fails, the damaged DNA is propagated to daughter cells following mitosis. If a sufficient number of critical genes are mutated, cellular transformation may result.

Immune Function

Systemic immune dysfunction was recognized as a risk factor for cancer for decades. The immunosuppressed renal transplant patient may have a 40-fold increased risk of cervical cancer (60). Patients infected with HIV who have a depressed CD4 cell count are reported to be at increased risk of cervical dysplasia and invasive disease (116). Individuals who underwent high-dose chemotherapy with stem cell support may be at increased risk of developing a variety of solid neoplasms. These examples illustrate the importance of immune function in host surveillance for transformed cells. Another example of altered immune function that may be related to the development of cervical dysplasia is the alteration in mucosal immune function that occurs in women who smoke cigarettes (60). The Langerhans cell population of the cervix is decreased in women who smoke. Langerhans cells are responsible for antigen processing. It is postulated that a reduction in these cells increases the likelihood of successful HPV infection of the cervix.

Diet

The role of diet in disease prevention and predisposition is widely recognized but poorly understood (116,121). Dietary fat intake is correlated with the risk of colon and breast cancer. Fiber is considered protective against colon cancer. With respect to the female reproductive system, epidemiologic studies provide conflicting results. Deficiencies of folic acid and vitamins A and C were associated with the development of cervical dysplasia and cervical cancer. Considerable research must be performed to clarify the impact of diet on cancer prevention and development.

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