Cellular Homeostasis

The development and survival of multicellular organisms such as human beings is reliant on the appropriate regulation of growth, differentiation and death of individual cell types to maintain the integrity of the organism

While cells have evolved a complex set of control mechanisms to deter replication of damaged cells and to enable repair, if the growth control mechanisms become damaged, this can lead to the development of cancer. The majority of cells in an adult are not dividing. However, under certain conditions such as tissue repair and senescence, regulated growth and proliferation are promoted. Thus, as cells die, either by senescence or as a result of tissue damage, they must be replaced in a strictly regulated manner. Cellular homeostasis maintains organ integrity through controlled cell survival, proliferation and cell death, to ensure that healthy cells, unlike cancerous (transformed) cells, generally stop dividing when they contact neighboring cells.

Research in transformed cancerous cells has highlighted the important mechanisms that regulate cellular growth and cell division in normal cells

Through investigation of genetic alterations in cancer cells, it has been possible to identify a large number of genes that are critical for the regulation of normal cell proliferation. It is perhaps unsurprising to find that molecular mechanisms that favor cell survival and proliferation processes are commonly upregulated in human cancers. Mutated proliferative genes are called oncogenes (cancer-causing genes), the normal cellular counterparts of which are called proto-oncogenes. Proto-oncogenes are predominantly signal transducers that act to regulate normal cell growth and division; aberrant regulation of these processes leads to cellular transformation. Conversely, the proteins involved in suppressing proliferation or tumor suppressor genes are generally inhibited during oncogenesis, leading to uncontrolled proliferation. Exploring the role of these proteins under normal physiologic conditions can assist in the understanding of how they can be subverted when they become dysregulated during oncogenesis.

Individual cells multiply by duplicating their contents and then dividing into two daughter cells

Cell division is tightly controlled by a complex mechanism called cell cycle. The duration of cell cycle varies between organisms, as well as between cell types within a single organism. In mammals, for example it can last from minutes to years. However, in immortalized cell lines, which are widely used as experimental model systems, one round of cell cycle is typically completed within 24 hours.

In recent years, extensive research of the cell cycle has defined a number of key control points

Traditionally, the cell cycle is divided into several phases (Fig. 42.1). Mitosis (M phase) is the stage of cell division, which is usually completed within an hour. The remainder of the cell cycle, during which cells prepare for division and duplicate deoxyribonucleic acid (DNA), is referred to as interphase. Nuclear DNA replication occurs in the synthesis (S) phase of interphase. The period between M and S phase is called G1 phase, while the interval between S and M phase is called G2 phase. During G1 and G2 phases, cells undergo several checkpoints to ensure appropriate cellular growth and accurate DNA synthesis occurs prior to cell division, thus preventing the incorporation of mutated DNA into daughter cells. As mentioned above, the duration of an individual cell cycle varies enormously and most of this variability can be attributed to the different length of the G1 phase. This is due to the fact that some cells, which are not stimulated to duplicate their DNA, can enter into the specialized form of G1 phase, called G0 phase.

The G0 phase is a form of the resting state, or quiescence, in which cells reside until they receive appropriate signals: for example, from growth factors, urging them to re-enter and progress through the cell cycle

In mammals, the time required for a cell to transit from the beginning of S phase through mitosis is typically 12–24 hours, irrespective of the duration of G1 phase. Thus, the majority of variation in proliferation rates observed between different cell types is due to the amount of time spent in the G0/G1 phase. In conditions that favor cell growth, the total ribonucleic acid (RNA) and protein content of the cell increases continuously, except in M phase when the chromosomes are too condensed to enable transcription to occur.

Of note, the majority of cells in the human body have irreversibly withdrawn from the cell cycle into either a terminally differentiated state (neurons, myocytes or surface epithelial cells of skin and mucosa), or they are in the reversible quiescent G0 phase (stem cells, glial cells, hepatocytes or thyroid follicular cells). Only a minority of cells are normally actively cycling and these cells are located mainly in the stem/transit compartments of self-renewing tissues, which include the bone marrow and epithelia.

Cells of a multicellular organism have to receive positive signals in order to grow and divide

Many of these signals are in the form of polypeptide hormones (e.g. insulin), growth factors (e.g. epidermal growth factor, EGF) or cytokines (e.g. interleukins IL-1 to IL-36). These growth factors bind to specific cell surface receptors, initiating an intricate network of intracellular signaling cascades that counteract negative regulatory controls present in resting cells to block cell cycle progression and division.

In most cell types, proliferation is controlled by signals generated from a specific combination of growth factors rather than stimulation by a single growth factor

In this way, a relatively small number of growth factors can selectively regulate the proliferation of many cell types. In addition, some of the factors may induce cell growth without providing a signal for division. Indeed, neurons in the G0 phase of cell cycle grow very large without dividing. Moreover, although proliferating cells can stop growing when they are deprived of growth factors, they continue their progress through the cell cycle until they reach the point in the G1 phase at which they can enter G0 phase (the resting state) or undergo senescence.

Growth factors bind to their specific cell surface receptors. There are about 50 known growth factors

Growth factors bind to their specific cell surface receptors expressed on effector cells, which are generally transmembrane proteins with a growth factor (or ligand) binding domain and cytoplasmic protein tyrosine kinase (PTK) domain. There are about 50 known growth factors, of which platelet-derived growth factor (PDGF) was the first identified. The growth and proliferative responses to PDGF are prototypic for many growth factors, and these responses include:

Growth factors initiate selective signaling cascades

Individual growth factors activate distinct cohorts of signaling molecules and transcription factors that in turn induce a unique repertoire of gene expression. In this way specific growth factors initiate characteristic differential responses that have a unique impact on cell behavior.

Growth factors initiate selective signaling cascades by binding to their receptors

The binding to a receptor causes receptor dimerization or oligomerization and activation of the intracellular tyrosine kinase domain, which in turn mediates receptor transphosphorylation at specific amino acids within the cytoplasmic domain. The phosphorylated region of the receptor is then able to act as a ‘docking site’ for the binding of specific proteins, thus enabling protein–protein interactions (Fig. 42.2). This in turn leads to the recruitment and activation of additional signaling molecules such as enzymes and ‘adapter’ molecules, which mediate the propagation of the intracellular signaling cascade from the surface of plasma membrane inside the cell. Transphosphorylation of receptor cytoplasmic domains creates a scaffold for binding of signal transduction elements such as phospholipase Cγ (PLC-γ), GTPase-activating proteins (GAPs), nonreceptor PTKs (Src, Fyn Abl), phosphotyrosine phosphatases (PTPases) and adapter molecules (Shc or Grb2), via their phosphotyrosine recognition domains.

Upon ligand binding, the EGF receptor (EGFR) leads to the activation of signals through phosphatidylintositol-, Ras/Raf/MAPK- and the PI3K/Akt/mTor-mediated signaling cascades

The EGF receptor (EGFR) can be bound and activated by a number of ligands including EGF and transforming growth factor-α (TGF-α). Ligation of the EGFR leads to the recruitment and activation of Src family of PTKs, which catalyze phosphorylation of PLC-γ, leading to the activation of this enzyme. Activated PLC-γ then catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate the intracellular second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ stimulates release of Ca²⁺ from intracellular stores (mainly ER) and DAG activates an important signal transducer family of proteins, the protein kinase C (PKC) family. Ligation of the EGFR also induces activation of another lipid modifying enzyme, phosphatidylinositol 3-kinase (PI3K). This enzyme mediates phosphorylation of PIP₂, generating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which contributes to the activation of certain members of the PKC family (Fig. 42.3). Moreover, PIP₃ can activate another kinase referred to as PIP₃-dependent kinase (PDK1) or serve as docking site for proteins that contain so-called pleckstrin homology (PH) domains.

Signaling cascade involving Ras GTPase is important in regulating cell division

A quarter of all tumors have constitutively active mutations in the signaling component Ras, which is critical for the transmission of proliferation and differentiation signals from the extracellular receptors to the nucleus. Ras is constitutively bound to the plasma membrane via a post-translational modification involving the addition of a lipophilic farnesyl group. Ligation of the EGFR recruits Ras by its binding to the adaptor protein Grb2. Ras is a GTPase, which cycles between an active GTP-bound and inactive GDP-bound form (Fig. 42.4). Its intrinsic catalytic activity is low, and is enhanced by binding to a GTPase-activating protein (GAP). GDP/GTP exchange is promoted by binding to a guanine nucleotide exchange factor, called Sos, which returns Ras into an active state. One of the main functions of active Ras is to act as an allosteric regulator of the mitogen-activated protein kinase (MAPK) signaling cascade. Ras transduces signals from EGFR via activation of two intermediary kinases, Raf and MEK kinase. MEK is a MAPK-type kinase that activates MAPK by mediating dual-specificity phosphorylation of tyrosine and threonine residues within its TEY activation motif. MAPK exists in two isoforms, extracellular-signal regulated kinase (ERK) 1 and 2. Upon activation, they translocate into the nucleus and phosphorylate (on serine and threonine) key transcription factors involved in the regulation of transcription of genes that regulate DNA synthesis and cell division.

mTORC-1 and mTORC-2 complexes integrate mitogen and nutrient signals

In addition to acting on selective members of the PKC family, PI3K is also responsible for activating PDK1, which in turn activates Akt. This enzyme mediates the activity of mammalian target of rapamycin (mTor), a serine/threonine protein kinase. mTor can participate in two distinct signaling complexes mTORC-1 and mTORC-2, integrating mitogen and nutrient signals to promote cell survival, growth and proliferation. PI3K/Akt activation by receptor ligation leads to phosphorylation of the tuberous sclerosis protein 1/2 (TSC1/2), resulting in mTORC-1 activation. The best-characterized downstream effectors of mTORC-1 are translational regulators within the protein synthesis pathway, initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and ribosomal S6 kinase 1 (S6K1), which are stimulated by mTORC1-mediated phosphorylation (Fig. 42.5).

While the signaling pathways described above are linear, a significant amount of cross-talk occurs amongst these cascade elements

For example the ERK-MAPK signaling cascade is capable of regulating mTORC-1, by activating the kinase RSK1, which in turn phosphorylates and inhibits TSC1/2, thus resulting in activation of mTORC-1. Examples such as this illustrate the complexity that exists in intracellular signaling pathways downstream of receptors. Moreover, cross-talk goes some way to highlighting how a mutation in a single signaling component can impact on a wide array of biological responses within a single cell.

Cytokines are growth factors that mainly coordinate the development of hematopoietic cells and the immune response, although they also have multiple effects on nonhematopoietic cell types

As described above for growth factors, cytokines also exert their effects on cells by binding to cell surface receptors. There are many classes of cytokine receptors, many of which belong to a superfamily called the hematopoietic receptors. These are transmembrane glycoprotein receptors, characterized by the conserved extracellular ligand-binding domains, which contain characteristic cysteine pairs and a pentapeptide motif WSXWS (tryptophan-serine-X-tryptophan-serine, where X is any amino acid). Many consist of multisubunit receptors that contain a unique ligand-binding subunit that gives specificity, and a common signal transducer subunit that is often shared by several related cytokines. The sharing of the signal transducing subunit gives the basis for classification of cytokines into distinct subfamilies and helps to explain the severe immunodeficiencies that result from naturally occurring defects in these receptors.

Janus kinases (JAKs) link the hematopoietic receptors with the downstream signaling and gene transcription

In contrast to growth factor receptors, cytokine receptors do not possess an intrinsic catalytic activity. However, a family of cytosolic PTKs, called Janus kinases (JAKs), are essential for linking the hematopoietic receptors with the downstream signaling and gene transcription. Thus, after ligand engagement with the receptor, tyrosine kinase activity is induced, and most cytokines, in common with classic growth factors, can signal through PLC, PI3K and Ras-MAPK signaling cascades, JAKs associate with the receptors by binding to the conserved regions near the transmembrane domain. Upon cytokine binding, which causes receptor oligomerization, JAKs become phosphorylated and activated to mediate phosphorylation of their downstream targets, which are transcription factors called signal transducer and activators (STATs) (Fig. 42.6). In unstimulated cells, STATs are found in the cytoplasm in monomeric forms. Cytokine stimulation leads to the JAK-mediated STAT phosphorylation and dimerization. STAT dimers then translocate to the nucleus, where they mediate transcription of the target genes by binding to their specific DNA sequences. Recently, growth factors such as EGF and PDGF have also been shown to induce activation of JAK/STAT pathways; therefore, JAK/STAT signaling may be a universal mechanism utilized by growth factors to regulate gene induction and cellular responses.

Cyclin-dependent kinase (CDK) family of serine/threonine kinases, and cyclins, regulate cell cycle transition points

As cells progress through the cell cycle in response to growth factor stimulation, they must pass through three switch-like transition/restriction points, positioned at the G1/S phase boundary and at the entry and exit of M phase. Master regulators of these transition points include members of the cyclin-dependent kinase (CDK) family of serine/threonine kinases and a family of proteins known as cyclins.

CDKs are expressed as heterodimers comprising a protein kinase subunit and a regulatory cyclin subunit. Their activity is tightly regulated by different mechanisms, including the phosphorylation status of the kinase subunit, levels of cyclins and/or interaction with inhibitory proteins (CDKIs) that block their catalytic activity. While CDK expression levels are relatively constant throughout the cell cycle, the expression levels of cyclins are strictly controlled at both the mRNA and the protein level (transcriptional and translational control). Indeed, cyclins were originally defined as proteins that were specifically degraded during every mitosis.

The traditional model of cell cycle states that a specific cyclin-CDK partner drives distinct parts of the cell cycle: D-type cyclins and CDK4/6 regulate the events in early G1 phase, cyclin E-CDK2 triggers S phase, cyclin A-CDK2 and cyclin A-CDK1 regulate the completion of the S phase, while cyclin B-CDK1 controls mitosis (Fig. 42.7).

Mitogenic signals activated by growth factors exert their effects between the onset of G1 phase and a point late in the G1 phase, called the restriction point

The key event for the initiation of the cell cycle in G1 phase is the phosphorylation of retinoblastoma protein, Rb, at various residues. Rb controls the expression of genes that commit cells that have reached restriction point late in G1 phase to enter the S phase (DNA synthesis) of the cell cycle. In the early G1 phase, Rb molecules are in the hypophosphorylated state. This allows them to bind and repress the DNA-binding activity of the main regulators of the G1/S phase transition, members of E2F family of transcription factors, thus inhibiting cell cycle progression (Fig. 42.7). Additional molecules that play an important role at this stage are histone deacetylases and chromatin remodeling complexes that epigenetically regulate gene transcription. Stimulation with growth factors and/or mitogens affects the entry into the cell cycle by triggering the expression and/or activation of proto-oncogenes, such as Ras and Myc, which results in the induction of expression of cyclins from the D-type family (D1, D2 and/or D3), followed by the cyclins from E-type family (E1 and E2). The D-type cyclins partner with CDK4/6 and stimulate their activity, whereas E-type cyclins increase the kinase activity of CDK2. These cyclin-CDK partners modulate the phosphorylation state of Rb by converting it from hypophosphorylated into hyperphosphorylated state, thus inactivating Rb, and promoting the release of E2F from Rb inhibitory complex. Free E2F proteins mediate gene transcription, the products of which are important for entry into S phase, and beyond, such as A- and B-type cyclins (Fig. 42.7). From this point onward, cell cycle progression is independent of growth factors.

When E-type cyclins are degraded, CDK2 binds to the A-type cyclins and these complexes phosphorylate many protein targets necessary for the proper completion and exit from the S phase. At the end of S phase, A-type cyclins also associate with CDK1, and these complexes share substrates with cyclin A-CDK2. The importance of the existence of both cyclin A-CDK2 and cyclin A-CDK1 complexes is still not clear.

Nevertheless, during G2 phase A-type cyclins are degraded by ubiquitin-mediated proteolysis and B-type cyclins are synthesized and interact with CDK1. It is estimated that cyclin B-CDK1 complexes phosphorylate more than 70 target proteins, which are important mediators of both regulatory and structural processes (chromosomal condensation, fragmentation of the Golgi network and breakdown of the nuclear envelope) during G2/M transition. Finally, the inactivation of cyclin B-CDK1 complexes is necessary for the exit from mitosis. This is achieved by the ubiquitin-labeling and subsequent proteasomal degradation of B-type cyclins regulated by the anaphase-promoting complex (APC). The main phosphatase that mediates return to interphase after mitosis is a form of protein phosphatase-2A (PP2A), whose activity is increased after degradation of the mitotic cyclins.

Molecular checkpoints that mediate appropriate progression through the cell cycle sense problems that may occur during DNA synthesis and chromosome segregation

The ultimate role of these checkpoints is to inhibit cyclin-CDK activity and thus delay or arrest cell division. During replication, DNA is less condensed and therefore protected to a lesser degree from the attack of exogenous and endogenous genotoxic agents, which may cause DNA damage.

In the case of damage occurring, the DNA damage checkpoints sense alterations and activate signaling pathways that mediate DNA repair

If the damage is beyond repair, they induce apoptosis. The core molecules of the DNA damage checkpoint are sensor kinases, ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR), which detect double-strand breaks and replication stress, respectively, and checkpoint kinases, CHK1 and CHK2, which relay the signals from the sensor kinases. These molecules prevent G1/S and G2/M phase transitions by inhibiting the CDK activity through increasing the expression of CDK inhibitor (CDKI) protein, p21 via p53 stabilization and/or inhibiting the CDK activator Cdc25 phosphatases.

The tumor suppressor protein p53 is predominantly a DNA damage-sensing protein which monitors DNA damage throughout the cell cycle

If DNA damage is detected, ATM and subsequently CHK2 kinases are activated, action that contributes to the stabilization of p53 and thus enables the induction of DNA repair mechanisms. One of the roles of p53 is to act as a transcription factor, increasing the expression of the CDKI p21 (WAF1). In turn, p21 inhibits cyclinD-CDK4 complexes, preventing Rb phosphorylation and thus promoting Rb/E2F binding and suppression of E2F-mediated gene transcription. Moreover, type-E cyclin-CDK2 complexes are inhibited, enabling cell cycle arrest at the G1/S transition. This allows the cell time to repair the DNA damage, and hence prevents the incorporation of mutated genetic material into daughter cells. However, if the DNA damage is beyond repair, p53-dependent programmed cell death by apoptosis is triggered.

A p53-independent pathway involving the INK4 family of proteins can also induce cell cycle arrest in the G1 phase in response to DNA damage

These proteins, including p16 (INK4A), p15 (INK4B), p18 (INK4C) and p19 (INK4D), mediate cell cycle arrest by binding to CDK4/6 or their binding partner, cyclin D, thereby causing the inactivation of the cyclin cyclin D-CDK4/6 complexes. Another important checkpoint is the spindle assembly checkpoint (SAC), which ensures proper alignment and segregation of chromosomes in the metaphase of mitosis. The SAC signal is generated by the presence of unattached or improperly attached kinetochores (protein complexes on chromatids which mediate attachment to the mitotic spindle), ultimately leading to the inhibition of the anaphase-promoting complex (APC) and thus preventing the onset of anaphase.

Defects in the DNA damage checkpoints allow accumulation of the DNA alterations, thus contributing to the genomic instability, whereas defective SAC may lead to the unequal segregation of the genetic material among the daughter cells, thus creating chromosomal aberrations. Both genomic instability and chromosomal aberrations are major culprits in cell transformation and oncogenesis.

Cell death is a fundamentally important part of a cell's life cycle, and appropriate regulation of this process is critical to maintain the homeostatic regulation of a multicellular organism

Cell death may be accidental or programmed, initiated and executed through distinct biochemical pathways. Programmed cell death (PCD) is genetically regulated and its role is to remove superfluous, damaged or mutated cells. For many years, apoptosis was a synonym for PCD; however, this concept is changing due to the recent findings identifying different modes of controlled cell death.

Both initiation and execution of cell death are complex processes, with scientists classifying the various forms of cell death on the basis of morphological and/or biochemical features as the most common criteria. According to morphological criteria, cell death can be classified as:

Apoptosis is initiated and executed through either perturbation of intracellular homeostasis by the intrinsic (mitochondrial) or extrinsic pathways, the latter of which involves ligation of death receptors such as FAS, TNFR, TRAIL and TWEAK

For both of these pathways, two families of proteins are considered quintessential regulators: cysteine proteases called caspases, and the B cell lymphoma protein 2 (Bcl-2)-related family members, which interact to serve as modulators of life versus death decisions (Fig. 42.8). However, there is a growing body of evidence for other PCD pathways that sense stress and damage in other cellular organelles (such as endoplasmic reticulum and lysosomes), and result in the initiation of death programs. These programs can occur in association with, or independently of, the intrinsic mitochondrial pathway.

Caspases are cysteine proteases with aspartate substrate specificity

They are synthesized as inactive proenzymes (zymogens) referred to as procaspases. According to their role in the death pathways, caspases can be categorized as initiator or effector caspases.

Initiator caspases (caspase-2, -8, -9, -10) are synthesized as monomers and upon cell receiving a death signal they undergo activation, resulting from proximity-induced conformational changes and dimerization within multimeric complexes, as well as auto-proteolytic cleavage, which induces full enzymatic activity. Once cleaved/activated, caspases mediate proteolytic cleavage of other caspases in the death pathway cascade.

Effector caspase proenzymes are expressed as pre-formed dimers, which are activated by the direct proteolytic attack of initiator caspases. These effectors then execute the cell demise program by cleaving many vital cellular proteins (such as lamins, gelsolin), inducing cell cycle arrest, and disabling the initiation of homeostatic and repair mechanisms. All these events lead to the detachment of the cell from its surrounding tissue, dismantling structural components and ultimately flagging the phosphatidylserine (PS) ‘eat-me’ signal for phagocytosis. Overexpression of active caspases is sufficient to induce cellular apoptosis.

IAP gene family: its main function is to inhibit apoptosis

The inhibitor of apoptosis (IAP) gene family, composed of nine family members (X-linked IAP, cIAP1, cIAP2, melanoma IAP, IAP-like protein, neuronal apoptosis inhibitory protein, survivin, livin and apollon), is evolutionarily conserved from Drosophila through to humans. The main function of this gene family is, as their name indicates, to inhibit apoptosis by either directly blocking caspases and/or activating survival pathways via NFκB function. For example, X-linked IAP inhibits caspase-3, -7 and -9 by direct binding to the active pocket of caspase-3 and -7, while in the case of caspase-9 it prevents the dimerization necessary for full activation. On the other hand, cIAP1 and cIAP2 are positive regulators of both canonical and non-canonical pathways of NFκB activation. Survivin, in addition to inhibiting apoptosis, plays a role in cell cycle progression by blocking the activity of caspase-3, thus preserving the integrity of p21 within the survivin–caspase-3–p21 complex, and by mediating proper chromosome segregation as a part of the complex that binds chromosomes to kinetochores. It is not surprising, therefore, that IAP family members are found to contribute to tumor cell survival, cell invasion and metastasis in many human cancers. Interestingly, loss of cIAP1/cIAP2 is implicated in the development of multiple myeloma.

Bcl-2 gene family is composed of structurally related proteins that form homo- or heterodimers and act as positive or negative regulators of apoptosis

The Bcl-2 gene was initially discovered in a follicular B cell lymphoma as a protein that is constitutively expressed due to a t(14;18) chromosome translocation, which placed the Bcl-2 gene under the control of the immunoglobulin heavy chain promoter. Bcl-2 family members have traditionally been classified into three groups: pro-survival family members (Bcl-2, Bcl-xL, Bcl-W, Mcl-1); the pro-apoptotic BAX/BAK family; and the pro-apoptotic BH3-only proteins (BIM, BID, PUMA, NOXA, BAD, BIK). While the function of Bcl-2 appears to be to preserve the integrity of the outer mitochondrial membrane, pro-apoptotic family members BAX and BAK are responsible for inducing mitochondrial outer membrane permeabilization and subsequent release of apoptotic mediators (such as cytochrome c), leading to the caspase activation. Bcl-2 and Bcl-xL prevent apoptosis induction by inhibiting BAX and BAK, while BH-3-only family members prevent this inhibition by a direct binding to the Bcl-2 and other anti-apoptotic family members (Fig. 42.8).

There are alternative routes to apoptosis

The rupture of lysosomal membranes causes release and activation of lysosomal proteases (i.e. cathepsins), which either mediate direct proteolytic cleavage of cellular components or activation of the intrinsic pathway. The endoplasmic reticulum (ER) stress is generally caused by the accumulation of unfolded proteins in the lumen, giving rise to the unfolded protein response or irregular intracellular Ca²⁺ flux. These perturbations usually induce apoptosis through the intrinsic pathway. Accumulation of unfolded or misfolded protein aggregates is associated with many neurodegenerative disorders, including Alzheimer's, Huntington's and Parkinson's disease. For example, amyloid-β-protein aggregates and mutations in the ER-associated presenilin 1 have been associated with the development of the familial Alzheimer's disease. Interestingly, all these pathways may cross-talk and perform rather complex death programs in both the morphologic and biochemical sense.

Autophagy is a degradation process of cellular components in which a part of cytoplasm is engulfed by a specific membrane, and the contents are subsequently degraded by lysosomal enzymes

Autophagy is a highly regulated homeostatic process, which plays a role in the turnover of long-lived proteins or in the elimination of damaged organelles such as mitochondria (mitophagy) or ER (reticulophagy). In addition to being associated with cell death, autophagy can also enable cells to survive starvation conditions in the circumstances of decreased availability of extra- and intracellular nutrients. In this case, autophagy induces catabolic processes, which generate metabolic substrates from ‘self’-components, thus allowing cells to meet their bioenergetic needs and initiate so-called adaptive protein synthesis in the time of scarcity.

Autophagy is induced by a variety of stress stimuli, including nutrient and energy stress as well as hypoxia, redox stress, infections, ER stress, and mitochondrial damage

All of these stresses induce distinct signaling pathways that regulate autophagy. A characteristic signaling event that induces autophagy as a result of nutrient deprivation is the inhibition of mTORC-1 signaling and/or AMPK activation. The process of autophagy involves the formation of a membrane structure, termed a phagophore (most likely by the de novo synthesis), which encases part of the cytoplasm or a whole organelle, forming a double-membrane structure called the autophagosome. The autophagosome then may fuse with an endosome, creating an amphisome. Subsequently, these structures fuse with a lysosome, generating an autolysosome where acid hydrolases break down the inner membrane and the cargo. Digested macromolecule building blocks are then recycled back into the cytoplasm through protein channels referred to as permeases (Figs 42.9 and 42.10).

Apart from its roles in maintenance of cell homeostasis, autophagy plays a role in both innate and adaptive immunity. For example, autophagy is used for the elimination of intracellular bacteria such as Streptococcus pyogenes and Mycobacterium tuberculosis. Moreover, the Epstein–Barr virus nuclear antigen 1 (EBNA1) is processed through an autophagic pathway, and loaded on MHC class II molecules for presentation to the CD4⁺ T lymphocytes.

Cells that develop mutations affecting normal regulation of the cell cycle are able to undergo unchecked proliferation, resulting in a loss of homeostatic regulation and the development of a tumor or neoplasm

As long as the neoplastic cells remain as an intact tumor, the tumor is considered benign and can be removed surgically. However, if further mutations allow such tumor cells to invade and colonize other tissues, creating widespread secondary tumors or metastases, the tumor is described as malignant and classified as a cancer. Each cancer is derived from a single cell that has undergone some germline mutation that allows it to outgrow its surrounding cells; by the time they are first detected, tumors typically contain a billion cells. Cancers are classified according to the tissue and cell type that they are derived from: those from epithelial cells are carcinomas, those from connective tissue or muscle cells are termed sarcomas, and those from the hematopoietic system are called leukemias. About 90% of human cancers are carcinomas, the five most common being those of the lung, stomach, breast, colon/rectum, and uterine cervix.

In the majority of cases, a single mutation is not sufficient to convert a healthy cell to a cancer cell; several rare mutations have to occur together

Mutations in DNA occur spontaneously at a rate of 10⁻⁶ mutations per gene, per cell division (even more in the presence of mutagens). Thus, since approximately 10¹⁶ cell divisions occur in the human body over an average lifetime, every human gene is likely to undergo mutation on about 10¹⁰ occasions. Clearly then, a single mutation is not normally sufficient to convert a healthy cell to a cancer cell; several rare mutations have to occur together, as demonstrated by epidemiologic studies showing that, for any given cancer, the incidence increases exponentially with age. It has been estimated that 3–7 independent mutations are usually required, leukemias apparently needing the fewest mutations and carcinomas the most.

Mutations need to occur in the appropriate cells in order to enable the neoplasm to develop, indicating that cell context has an important bearing on the type of cancer that subsequently develops

In addition to developing cancer-promoting mutations, the cell in which a mutation occurs must then be permissive to being a cancer-initiating cell. This relates to the cellular context in which an oncogene is expressed, and the properties bestowed upon a cancer cell by expression of a particular oncogene. In order to become oncogenic and promote cancer cell growth, the cell must possess self-renewal capacity. If oncogene expression occurs in a stem cell, then oncogene expression could inhibit the negative regulatory networks in place that would ordinarily stop cell growth and proliferation, thus generating a cancer stem cell. Cancers such as chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) can arise from mutations in stem cells. However, it is not essential for the cell of origin of individual cancers to arise from stem cells. Indeed, cancers can arise from mutations arising in committed progenitor cells that enable those cells to acquire the potential to self-renew, therefore providing a cellular source for cancer.

Mutations that lead to the expression of established oncogenes do not necessarily lead to the development of cancer if it occurs in nonsusceptible cells

For example, the Philadelphia chromosome (t(9;22)) that generates a fusion protein BCR-Abl, encoding a constitutively active from of the protein tyrosine kinase (PTK) c-Abl (which is the causative mutation of the development of >95% CML cases), is detected at a very low level in circulating peripheral blood cells isolated from around 30% of healthy individuals. Studies have demonstrated that expression of BCR-Abl alone does not confer self-renewal properties on committed progenitor cells, indicating that secondary mutations are required to make committed progenitor cells cancerous. This finding suggests that the expression of oncogenes such as BCR-Abl within a stem cell environment can enable it to hardwire into the initiation of the neoplastic program.

Oncogenes were first identified as viral genes that infect normal cells and transform them into tumor cells

The Rous sarcoma virus, which is a retrovirus that causes connective tissue tumors in chickens, will infect and transform fibroblast cells grown in cell culture. The transformed cells outgrow the normal cells and exhibit a number of growth abnormalities, such as a loss of cell contact-mediated inhibition of growth and loss of anchorage dependence of growth. In addition, the cells have a rounded appearance and can proliferate in the absence of growth factors. Moreover, the cells are immortal, do not senesce, and can induce tumor formation when injected into a suitable animal host, confirming their ability to self-renew.

The key to understanding cell transformation lies in the mutation of a normal cellular gene that controls cell growth

The use of mutant Rous sarcoma viruses that, despite multiplying normally, have lost the ability to transform host cells, showed that it was the Src gene that was responsible for such cell transformation. The breakthrough in our understanding of how this single gene could transform cells in culture came when it became apparent that the viral oncogene was a mutated homologue of a normal cellular gene. This gene is now called the c-Src proto-oncogene, and has been identified as a PTK signal transducer involved in the normal control of cell growth. As expression of this gene is not essential to the survival of the retrovirus, it is likely that Src was accidentally incorporated by the virus from a previous host genome and was somehow mutated in the process. In the case of the Rous sarcoma virus, the introns normally present in c-Src are spliced out and, in addition, there are a number of mutations causing amino acid substitutions, resulting in a constitutively active PTK.

Cell transformation can, however, also result from oncogenes that are not constitutively activated but, rather, are overexpressed in an abnormally high number of copies, as a consequence of the gene being under the control of powerful promoters or enhancers in the viral genome. Alternatively, for retroviruses, DNA copies of the viral RNA can be inserted into the host genome at or near sites of proto-oncogenes (insertional mutation), causing abnormal activation of these proto-oncogenes. In this situation, the altered genome is inherited by all progeny of the original host cell.

Most human tumors are nonviral in origin and arise from spontaneous or induced mutations

Approximately 85% of human tumors arise as a result of point or deletion mutations. These mutations may be spontaneous or induced by carcinogens or radiation, resulting in overexpression or hyperactivity of the proto-oncogenes. Indeed, Ras, which has been found to be mutated to a constitutively activated form in approximately 25% of all tumors, appears to exert many, if not all, of its effects ultimately by upregulating concentrations of cyclin D and, hence, by stimulating cell cycle progression. This upregulation of cyclin D expression results from stimulation of the MAPK cascade by Ras, and the induction of the transcription factor AP-1, which regulates induction of cyclin D expression.

Whole-exome/genome sequencing of individual patients, utilized to determine the specific mutational landscape within cancer subtypes, has enabled links to be established between seemingly diverse cancers that result from similar genetic mutations

Of note, B-Raf^V600E mutation was recently found to be present in all in hairy cell leukemia (HCL) patients assessed. B-Raf^V600E is oncogenic in a number of tumors, including melanoma, encoding active B-Raf kinase leading to the constitutive activation of the MEK/ERK signaling pathway. This mutation has a major impact on cell cycle. Interestingly, now that the B-Raf^V600E mutation has been linked as a causal/driver event of HCL, it presents the opportunity for targeted therapies, such as PLX-4720, a specific inhibitor of active B-Raf, which would otherwise have not been considered for the treatment of HCL. Identification of specific driver mutations in a particular cancer cell opens up the possibility of treating patients with a targeted therapy towards that mutation.

Karyotyping of tumor cells has also shown that chromosomal translocation can bring the oncogene under the control of an inappropriate promoter. For example, in Burkitt's lymphoma, overexpression of the Myc gene occurs through its translocation into the vicinity of one of the Ig loci. Because Myc normally acts as a nuclear proliferative signal, overexpression of Myc induces the cell to divide, even under conditions that would normally dictate growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated proteins in sporadic cancer

As described above, PI3K-mediated signaling pathways are activated in response to a multitude of growth factor stimuli, resulting in the promotion of cell growth, survival and proliferation. The main protein responsible for attenuating PI3K activity and downstream pathways is the dual-specificity protein and lipid phosphatase PTEN. It reverses the activity of PI3K by dephosphorylating PIP₃. The tumor suppressor PTEN is one of the most commonly inactivated proteins in sporadic cancer, resulting in sustained PI3K/Akt signaling and uncontrolled cell survival and proliferation. Mutations in PTEN have been identified in a wide range of cancers, including breast, thyroid, prostate and brain cancers. Interestingly, individuals with inherited germline mutations in PTEN, known as PTEN hamartoma tumor syndrome, develop benign tumors associated with breast, thyroid, colorectal, endometrial and kidney. However, these patients also carry an elevated lifetime risk of developing malignant cancers in these tissues. The increased susceptibility to cancer in cells carrying germline PTEN mutations underlines the importance of this protein as a tumor suppressor.