hall 17

Tissue homeostasis depends on the regulated cell division and self-elimination (programmed cell death) of each of its constituent members except its stem cells. In fact, a tumor arises as a result of uncontrolled cell division and failure for self-elimination. One can consider cancer as a Darwinian-like process whereby the fittest cells reproduce to become the dominant population of a tumor. Alterations in three groups of genes are responsible for the deregulated control mechanisms that are the hallmarks of cancer cells:

1. Proto-oncogenes are components of signaling networks that act as positive growth regulators in response to mitogens, cytokines, and cell-to- cell contact. A gain-of-function mutation in only one copy of a proto-oncogene results in a dominantly acting oncogene that often fails to respond to extracellular signals.

2. Tumor-suppressor genes are also components of the same signaling networks as proto-oncogenes, except that they act as negative growth regulators. They modulate proliferation and survival by antagonizing the biochemical functions of protooncogenes or responding to unchecked growth signals. In contrast to oncogenes, inactivation of both copies of tumor-suppressor genes is required for loss of function in most cases.

3. DNA stability genes form a class of genes involved in both monitoring and maintaining the integrity of DNA. Loss of these genes results in defective sensing of DNA lesions as well as improper repair of the damaged template.

The malignant progression from normal tissue to tumor to metastasis occurs in a number of discrete “steps” over a period of time. These steps, which are the result of mutations, deletions, or gene changes in the three groups of genes described here, may occur spontaneously as a consequence of random errors or result from exposure to agents as diverse as chemical mutagens, ionizing radiations, ultraviolet light, and viruses and provide a growth or survival advantage that allows the cells to become the clonal origin of the tumor. To summarize, tumor evolution results from the accumulation of gene mutations that arise in a single cell that has suffered a disruption in its regulatory mechanisms for proliferation, self-elimination, immortalization, and genetic stability. This is illustrated in Figure 17.1.

MECHANISMS OF CARCINOGENESIS

A single genetic alteration that leads to the activation of an oncogene or loss of a tumor-suppressor gene does not by itself lead to the formation of a solid tumor. Instead, carcinogenesis appears to be a multistep process with multiple genetic alterations occurring over an extended period of time; at least, that is how it appears. Sometimes these genetic alterations are carried in the germ line, as, for example, in the cancer-predisposing syndrome retinoblastoma; however, heritable mutations are rare. Most alterations that lead to cancer are acquired in the form of somatic mutations: chromosomal translocations, deletions, inversions, amplifications, or simple point mutations.

Initially, it was thought that cancer was the result of deregulated growth signals by oncogenes, a concept supported by increased proliferation in many types of cancer. In the last decade, the finding that many cancers possess diminished apoptotic (programmed cell death) programs or loss of cell- cycle control has led to the concept that mutations in proto-oncogenes and tumor-suppressor genes that inhibit apoptosis provide a selective growth advantage to a premalignant cell that allows it to clonally expand. Mutations in DNA stability genes increase the rate of acquiring genetic mutations that will result in a malignant tumor. Thus, while tumor cells are considered clonal in origin, most tumors contain heterogeneous populations of cells that differ in their ability to repopulate the tumor or form metastasis. In fact, only a small percentage of tumor cells possess the ability to form a tumor, leading to the concept that tumors possess “stem cells” and that elimination of these stem cells is essential for controlling tumor growth.

ONCOGENES

The first demonstration that a tumor was initiated by a cellular component found in tumor cells but not normal cells was shown by Rous in the early 1900s. His landmark studies demonstrated that cell-free extracts derived from chicken sarcomas could cause a new sarcoma if injected into healthy chickens. In the l970s, with the advent of molecular biology, several groups identified the etiological agent for sarcoma formation in chickens as an RNA virus, designated the Rous sarcoma virus (RSV), which belongs to a group of viruses designated retroviruses - viruses whose genomes are of RNA. Thus, oncogenes were first discovered from a study of retroviruses that cause cancers in animals. Although the virus had been identified, it still remained to be elucidated how this retrovirus causes a sarcoma, since another virus belonging to this same group of RNA viruses, avian leukosis virus (ALV), docs not transform cells in culture or induce sarcomas. Analysis of the genomes of ALV and RSV revealed that RSV contains approximately 1,500 more base pairs of DNA than ALV. It was hypothesized, therefore, that these extra base pairs of DNA in the RSV genome are responsible for the tumorigenic activity. This was supported by the observation that deletion mutants of RSV that are missing this 1,500-hp region lose their transformation potential but can still replicate and produce viral progeny normally. This led to the conclusion that the transforming activity and replicative activity of RSV are encoded by genetically distinct regions of the virus and that only a small portion of the RSV genome is needed for transformation.

From these early studies, several important conclusions could be derived: (1) Cancer can be caused by a genetically transmissible agent - in the case of chicken sarcoma, by a retrovirus containing a unique piece of genetic information that was latter designated the src gene; (2) only a certain region of a retrovirus is needed for transformation; and (3) the region of the viral genome necessary for transformation is not involved in the normal replicative life cycle.

Huebner and Todaro later proposed that cancer- causing viral genes such as src are normally inactive but can be activated when they recombine with a retroviral genome. Once they do so, they pass from being a benign proto-oncogene (i.e., c-crc) to a malignant form (v-src) capable of causing cancer when introduced into the appropriate host cell. Although we now know that viruses represent only one of several mechanisms that cause the deregulated expression of a proto-oncogene, these studies helped to define oncogenes as mutant forms of normal cellular genes that are altered in expression and/or function by various agents, including radiation, chemicals, and viruses. Consequently, very different agents produce tumors that are indistinguishable one from another. This is illustrated in Figure 17.2.

MECHANISMS OF ONCOGENE ACTIVATION

Although many mechanisms are involved in oncogene activation, transcriptional deregulation by overexpression or abnormal expression of the mRNA of a proto-oncogene is a common theme. At least four mechanisms exist for oncogene activation in human neoplasms (Fig.
17.3).

Retroviral Integration through Recombination

Retroviral integration of proto-oncogene sequences in retroviral genomes occurs through two possible recombinational mechanisms. In the first, mRNAs from a proto-oncogene recombine with viral genomic RNAs. During the recombination process, the proto-oncogene mRNA becomes deregulated as it comes under the control of the viral promoter, termed LTR. However, the probability of RNA recombination events between proto-oncogene mRNA and viral mRNA generating an oncogenic retrovirus is quite low and undermines the importance of this mechanism.

A second more probable mechanism is as follows: First a retroviral genome integrates in dose proximity to a proto-oncogene, where the proto-oncogene is under the transcriptional control of the retrovirus LTR promoter. Then the viral and proto-oncogene sequences become closely associated through a DNA recombination event that permits the production of mRNAs that contain both viral and proto-oncogene sequences. In this scenario, the proto-oncogene becomes transcriptionally deregulated as it is under the control of the viral promoter LTR. In addition. it can acquire mutations in its coding sequence. Although the proto-oncogene can become mutated during the recombination process, the key point is that its deregulated expression by the viral LTR increases its expression and promotes cell growth.

DNA Mutation of Regulatory Sites

The union of the technique for gene transfer with mouse transformation assays facilitated the isolation of human oncogenes that were activated by DNA mutation. Transfection of human DNA into immortalized but untransformed mouse cells was first used to isolate the H-ras oncogene from bladder carcinoma cells. The key to this approach is that only transformed cells possess the ability to grow in soft agar (Fig. 17.4). The implicit assumption is that a specifìc gene (or more than one) is responsible for causing the bladder carcinoma and that it will act in a dominant fashion to induce a tumor. Indeed, multiple groups were successful in isolating the H-ras oncogene by this approach.

Several steps are needed for the molecular cloning of an oncogene from transformed rodent cells using the rodent fibroblast transformation assay. First, human DNA containing the transforming oncogene is transfected into mouse cells, and then DNA from the transformed mouse cells is serially transfected to reduce the amount of human DNA that is not associated with the transforming oncogene. After several rounds of transfection, the DNA is isolated from a soft agar colony and digested with restriction enzymes to make a genomic DNA library. The library is then screened with a human-specific repetitive probe that does not cross-react with mouse DNA, thereby identifying human sequences in a mouse background. Clones that possess human repetitive sequences are then isolated and digested with restriction enzymes to identify a similar-length fragment that is common to all transformants. Finally, DNA from the clones is transfected into mouse cells to confirm its oncogenic potential. If the oncogene is present in this genomic clone, then a significant percentage of the transfected mouse cells should be transformed when compared with transfecting genomic DNA from untransformed cells.

Perhaps the prototypical example of oncogene activation by DNA mutation is the H-ras oncogene. The H-ras oncogene was isolated by the approach just described, and its DNA sequence was compared with its normal cellular counterpart. At first comparison, there did not seem to be any difference between oncogenic and proto-oncogenic forms. However, because H-ras is a relatively small oncogene. It was possible to sequence the entire gene to rigorously search for small mutations. It did not take long to find the difference between the two forms of the gene. The transforming, oncogenic H-ras gene possesses a single base-pair mutation that changes the 12th amino acid from glycine to valine. This single DNA mutation is responsible for changing H-ras from a benign proto-oncogene into a malìgnant oncogene. We now know that mutations in codons 13 and 61 will also produce oncogenic H-ras genes that are constitutively locked in an active state.

Gene Amplification

Oncogene amplification occurs through bridge breakage fusion cycles in anaphase during mitosis. In contrast to the other mechanisms discussed that involve transcriptional deregulation as a key mechanism of oncogene activation, gene amplification represents an alternative means of increasing proto-oncogene expression by increasing the number of DNA copies of the proto-oncogene. Gene amplification can result in an increased number of copies of extrachromosomal molecules called double minutes or can result in intrachromosomal amplified regions called homogeneously staining regions (HSRs), both of which are detectable by fluorescence in situ hybridization or Giemsa banding of chromosomes. The N-myc oncogene is a classic example of an oncogene amplified in leukemia, neuroblastoma, and breast cancer.

Chromosome Translocation

It had long been known that tumors possessed abnormal karyotypes. However, the chromosome content of many solid tumors is unstable, making it difficult to determine which cytogenetic alterations are causative for tumorigenesis and which are the consequence of the neoplastic process. The first real breakthrough in identifying tumor-specific chromosome alterations occurred in the late 1950s when Dr. Peter Nowell found a consistent shortened version of chromosome 22 in individuals afflicted with chronic myelogenous leukemia (CML). Because many patients with CML possess an abnormal chromosome 22 in their leukemic cells, this was a strong indication that a specific chromosome alteration is involved in the pathogenesis of this malignancy. With the advent of more sophisticated cytogenetic and molecular techniques, it was discovered that this shortened version of chromosome 22 is due to a symmetric translocation with chromosome 9. It was hypothesized. therefore, that the translocation between chromosomes 9 and 22 gives rise to CML. Further molecular analysis revealed that the bcr gene on chromosome 9 translocates in front of the abl gene on chromosome 22, producing a fusion transcript with abnormal expression (Fig. 17.5).

With the recent advent of molecular cytogenetics - that is, fluorescent in situ hybridization (FTSH) - many translocation partners have been identified. In fact, a common strategy has been to use proto-oncogenes, which chromosomally map near translocation breakpoints, as markers to identify potential translocation partners. Although numerous translocation breakpoints have been identified in hematopoietic neoplasms, few consistent translocations have been found in solid-tissue tumors. The reason for this is still unclear, but may be attributed to the fact that hematopoietic cancers require fewer alterations for neoplasia than solid tumors. Table 17.1 provides examples of the chromosomal changes that result in oncogene activation and the associated human malignancies. Interestingly, there are no known examples of oncogenes activated by retroviruses in human malignancies.

MUTATION AND INACTIVATION OF TUMOR-SUPPRESSOR GENES

The Retinoblastoma Paradigm

Oncogenes result from a mutation, deletion, or alteration in the expression of one copy of a gene. Thus, oncogenes are dominant genes, because a mutation in only one copy will cause their activation, even though the other copy of the gene is unchanged. This concept led to speculation that another class of genes, termed “anti-oncogenes,” suppress the effect of oncogenes on transformation and tumor formation. The existence of tumor suppressor genes was supported by cell fusion studies between tumor ceils and normal cells and by the family history studies of people afflicted with inherited cancer-prone disorders such as retinoblastoma or Li - Fraumeni syndrome. Although one mutated version of an oncogene is sufficient to drive malignant progression, one functional copy of a tumor suppressor gene is sufficient to suppress transformation, suggesting that both copies of a tumor suppressor gene must be inactivated to inhibit tumor growth (Fig. 17.6). Insight into the mechanism of tumor-suppressor gene inactivation came from Knudson’s epidemiological studies of families in which retinoblastoma appeared to be inherited in an autosomal dominant manner. Patients with familial retinoblastoma develop bilateral or multifocal disease at an earlier age than patients with sporadic retinoblastoma. Based on these observations, Knudson proposed that in the inherited form of retinoblastoma, individuals possess a gem-line mutation of the retinoblastoma gene (Rb) in all the cells of their body, but inactivation of one Rb allele does not give rise to retinoblastoma. Thus, the disease appeared to be autosomal dominant because individuals are born with one mutated allele. However, a second mutation in a retinal cell is required to develop retinoblastoma. In the sporadic form of the disease, an individual has to acquire two Rb mutations in the same retinal cell to develop retinoblastoma. The “two-hit hypothesis” by Knudson provided a genetic basis to understand the differences in inherited and sporadic mutations in the onset of tumors and to advance the concept that both alleles of a tumor-suppressor gene need to he inactivated to promote tumor development. Thus, tumor-suppressor genes are recessive genes that require the inactivation of both functional gene copies before malignancies develop, whereas loss of one functional copy results only in increased cancer susceptibility. In addition, it is often the case that the same tumor-suppressor gene involved in hereditary cancer syndromes, such as retinoblastoma, is also inactivated in other forms of cancer. The Rb gene itself has now been implicated in several other human cancers, which indicates that it may play a
generalized role in tumor growth suppression in a variety of tissues. For example, patients who are cured of familial retinoblastoma are at increased risk of osteosarcoma, small-cell lung cancer, and breast cancer; although the loss of the Rh gene alone is sufficient for retinoblastoma, further changes are required for the development of these other tumors.

The Li - Fraumeni Paradigm

The Li - Fraumeni syndrome (LPS) is a rare autosomal dominantly inherited disease that predisposes individuals to develop osteosarcomas, soft tissue sarcomas, rhabdomyosarcomas, leukemias, brain tumors, and carcinomas of the lung and breast (Fig. 17.7). Initial attempts to identify the genetic mutations that underlie LPS were unsuccessful because of the rarity of the syndrome and the high mortality of the patients. The major insight into the underlying cause of Li - Fraumeni came when it was found that mice that overexpress a mutant version of the p53 tumor-suppressor gene in the presence of the wild-type pS3 gene develop a spectrum of tumors similar to that seen in Li - Fraumeni patients. Sequencing of p53 in affected family members revealed germ-line missense mutations in the p53 tumor-suppressor gene located on chromosome 17p13 that resulted in its inactivation, and tumors derived from affected individuals had lost the remaining wild-type allele of p53. Similar to retinoblastoma, loss or inactivation of both wild type copies of p53 is needed for tumor formation. However, functional loss of one germ-line inherited copy of mutant p53 accelerates the onset of spontaneous tumor formation. Therefore, Li - Fraumeni patients follow a similar paradigm as retinoblastoma patients in developing spontaneous tumors, but unlike retinoblastoma, in which germ-line mutations mainly give rise to retinal tumors, loss of p53 results in a wide spectrum of tumors. Table 17.2 provides examples of other cancer predisposition genes and their associated syndromes.

SOMATIC HOMOZYGOSITY

How are recessive tumor-suppressor genes lost? Cytogenetic studies are used to identify chromosomal changes in peripheral blood lymphocytes or fibroblasts from cancer patients, especially those with a family history of cancer, to identify chromosomal rearrangements or deletions. At the subchromosomal level, genome-wide linkage analysis is used to determine that a certain chromosome region is tightly linked with cancer predisposition. Both copies of a suppressor gene in the sporadic form of retinoblastoma and other solid tumors may result from two independent allelic mutations, but in practice, it occurs more often by the process of somatic homozygosity. The steps appear to be as follows: One chromosome of a pair is lost, a deletion occurs in the remaining chromosome, and the chromosome with the deletion replicates. Instead of having each of the two alleles contributed by different parents, the cell has both alleles from the same parent, with loss of a vital piece containing the tumor-suppressor gene (Fig. 17.8). This process has been documented for chromosome 13 in the case of retinoblastoma, chromosome 11 in Wilms’ tumor, chromosome 3 for small-cell lung cancer, and chromosome 5 for colon cancer. Most interesting of all, perhaps, is the case of astrocytomas, in which somatic homozygosity is observed for chromosome 10 in grade 11 and III astrocytoma and for both chromosomes 10 and 17 for grade IV glioblastoma.

THE MULTISTEP NATURE OF CANCER

Perhaps the most pervasive dogma in cancer research is that carcinogenesis is a multistage process. The implication is that there are a number of distinct events that may be separated in time. This idea is almost 70 years old and is exemplified by the skin cancer experiments in mice that introduced the concepts of initiation, promotion, and progression as stages in tumor development.

Genetic analysis of cells from solid tumors, too, suggests alterations, mutations, or deletions in multiple signaling genes, either oncogenes or suppressor genes; 6 to 12 mutations have been suggested for the formation of a carcinoma. In the case of colorectal cancer, a model has been proposed that correlates a series of chromosomal and molecular events with the changes in the histopathology of normal epithelium during the multistage formation of colorectal cancer and metastatic carcinoma. This concept is illustrated in Figure 17.9.

A more general model of the series of events in carcinogenesis is shown in Figure 17.10. The first event, from whatever cause (including ionizing radiation), causes a mutation in a gene in one of the families responsible for the stability of the genome. This leads to a mutator phenotype, so that with many cells dividing, multiple mutations are likely in cancer-associated genes, both oncogenes and tumor-suppressor genes. This in turn leads to progression of the cancer and ultimately its invasive and metastatic properties.

Therefore, it is not surprising that restoration of one copy of a tumor-suppressor gene is sometimes not sufficient to restore tumor-suppressor activity, as solid tumors accumulate mutations that can make them refractory to the restoration of a single tumor suppressor gene. Although tumor-suppressor genes are functionally quite different from oncogenes, they modulate similar cellular targets as oncogenes.

FUNCTION OF ONCOGENES AND TUMOR-SUPPRESSOR GENES

The myriad of genetic and epigenetic changes that drive tumor evolution is a systems biology problem in which cells can be thought of as circuits, where an alteration of the circuit can lead to increased output, decreased output, complete loss of output, or no change. Therefore, cancer biologists attempt to determine how a specific gene, when mutated, alters normal tissue function. To understand how oncogenes and tumor-suppressor genes lead to neoplasia, we need to understand how each of these circuits impacts normal cellular physiology. What cellular functions are disrupted by oncogene activation and tumor-suppressor gene inactivation, and how do these disrupted functions affect the differentiation, growth, and death of cells? What follows is a description of the general categories of cell functions that are perturbed by deregulation of these two classes of genes during malignant progression.

Deregulated Proliferation

The loss of proliferative control of cancer ceils is evident to all who study cancer. In fact, the earliest concept suggested by tumor biologists was that cancer was a disease of uncontrolled proliferation. Untransformed cells respond to extracellular growth signals known as mitogens through a transmembrane receptor that signals to intracellular circuits that increase growth. Thus, the growth factor, the receptor, and the intracellular circuits can all lead to self-sufficiency when deregulated. Typically, one cell secretes a mitogenic signal to stimulate the proliferation of another celi type. For example, an epithelial cell can secrete a signal to stimulate fibroblasts to proliferate. In contrast to untransformed cells, transformed cells have become autonomous in regulating their growth by responding to the mitogenic signals they themselves produce. In this manner, they use an autocrine circuit to escape the need for other cell types. For example, mesenchymal cells are responsive to transformation by v-sis, as they possess receptors for PDGF, and breast epithelial ceils are responsive to int-2, as they possess receptors for FGF.

If overexpression of growth factors can lead to uncontrolled proliferation, then continuous activation or overexpression of growth-factor receptors will do the same. Several well-known oncogenes, such as v-erb-2 (HER-2/neu) and v-fins, encode growth-factor receptors. These receptors are mutated at their amino terminal residues so that they no longer require their respective growth factor (ligand) to signal induction of their kinase activity. Growth-factor receptors can structurably be divided into extracellular ligand-binding domains, transmembrane-spanning domains, and intracellular kinase domains. Although mutations have been found in all three domains, mutations in the ligand binding domains are a common alteration that results in constitutive kinase activity that transduces the signal for the cell to proliferate. In addition to structural alterations in the receptor, some tumors overexpress growth-factor receptors that make them hyperresponsive to physiologic levels of growth-factor stimulation. In contrast to mitogenicresponsive growth-factor receptors, a second class of receptors that transmit signals from the extracellular matrix can also regulate proliferation. Integrin receptors are the prototypical example of this class of regulators that transmit signals from different components of the ECM to signal proliferation or quiescence.

There are numerous intracellular circuits that transduce the signal from the cell surface to the nucleus of the cell. The Src, Ras and Abl proteins are all members of this group. By and large, most members of this group are tyrosine - kinases or serine/threonine - kinases. Src and Abl are tyrosine - kinases located on the cytoplasmic side of the cell membrane. H-ras, K-ras and N-ras are a family of GTP-binding proteins also located on the cytoplasmic side of the cell membrane and are the most frequently mutated oncogene family in human cancers. The Ras-Raf-ERK, Ras-RalGDS-Ral, and Ras-Pi(3)K-Akt-TOR pathways play critical roles in transducing signals from growth-factor receptors at the cell surface to the nucleus in untransformed cells. Oncogenic forms of ras bypass the normal growth regulatory signals of a cell by being locked in an “on” state, obviating the need for external signals from growth factors to activate them (Fig. 17.11).

Failure to Respond to Growth-Restrictive Signals

If signal transduction cascades initiate at the cell membrane, then they end in the nucleus. Normally, the majority of cells in the body are at rest in a nonproliferntive state (G0). The oncogenic activation of these nuclear oncogenes stimuintes the cell into the synthetic phase (S phase), where it duplicates its genetic material before cell division. Nuclear control proteins that regulate entry into S phase include transcription factors, such as c-myc, c-rel, c-jun and c-fos, and cell-cycle regulatory proteins, such as E2F and cyclin D1 (PRAD1). These nuclear proto-oncogenes can work as transcription factors by binding to DNA in a sequence-specific manner and forming complexes with themselves or other proteins that will increase mRNA transcription of genes such as cyclin D that promotes cell division.

To understand how tumor cells evade antiproliferative signals, one must appreciate how the cell cycle is regulated - in particular, the G1 phase. It is during a cell’s transit through the G phase that it makes the decision to continue to the S phase and duplicate its genetic material or enter into a reversible state of quiescence (reversible growth arrest) or enter a permanent state of senescence (irreversible growth arrest). The Rb family of proteins arc the most critical determinants of the fate of a G phase cell. When Rb protein is in a hypophosphorylated state, Rb protein blocks progression into 8 phase by sequestering E2F transcription factors that regulate the expression of genes that are essential for the transition from C to 8 phase. As previously discussed, the ECM can signal cell proliferation through integrin receptors. In addition, it can also produce antiproliferative signals through the secreted protein TGF-β. At the molecular level, TGF-β inhibits Rb protein phosphorylation and prevents the release and activation of the E2F family of transcription factors. it does this through the induction of inhibitors such as p21 and p15 that inhibit the activity of the kinases that are essential to phosphorylate Rb protein. Thus, loss of Rb or faliure to induce p21 and p15 wiii aid celis in escaping antiproliferative signais.

In addition to extracellular antiproliferation signals, endogenous proteins that down-regulate signal-amplifying kinases are also important in keeping signal transduction cascades in check. For example, the NFl protein is a GTPase-activating protein that facilitates the hydrolysis of GTP by Ras. When Ras protein is complexed with GDP, it is inactive; when it is bound to GTP, it is active. Individuals afflicted with neurofibromatosis lack NFl protein and have lower levels of GTPase activity and more Ras protein complexed with GTP, resulting in a more active Ras protein. Therefore, lack of NFl protein activity will result in enhanced activity of Ras protein similar to what is found in Rastransformed cells as already described. The NFl protein and the Ras protein are excellent examples of how a tumor-suppressor gene and an oncogene work in unison to control a signal transduction pathway.

Failure to Commit Suicide (Apoptosis)

Two major pathways that mediate cell death emanate either from the cell membrane or from the mitochondrion (Fig. 17.12). The signals transmitted by each pathway results in the activation of intracellular cysteine proteases, termed caspases, that cleave a diverse and ever-increasing number of substrates, including themselves, at aspartic acid residues. Caspases can broadly be divided into initiator caspases and effector caspases. The binding of ligands such as Fas to specific death receptors on the cell surface induces receptor activity and the recruitment of the initiator procaspase 8. The recruitment of procaspase 8 proteins in close proximity to each other results in active caspase 8 and effecto caspases, such as caspases 3, 6, and 7, that are responsible for the ultrastructural changes in the cell. In response to mitochondrial-dependent cell death, activation of initiator caspases (e.g., caspase 9) is achieved by proteolytic cleavage of their inactive pro-forms through their recruitment and interaction with specific adapter proteins. The adapter proteins are in turn regulated by the mitochondria through the release of cytochrome c. The release of cytochrome c from the mitochondria is controlled by the Bcl-2 protein family, which is composed of proapoptotic regulators such as Bax, Bak, Bid, and Bim and antiapoptotic family members Bcl-2. Bcl-xl, and Mel-i. The bcl-2 oncogene is the prototypical example of a membrane-associated oncogene whose overexpression protects the cell from death-inducing stimuli by a wide variety of agents.

How does Bcl-2 protect cells from undergoing apoptosis? Both proapoptotic and antiapoptotic Bcl-2 family members can form dimers with themselves (homodimers) or with other family members (heterodimers). This ability to form dimers with a pro-death-promoting family member has been proposed as one mechanism of how Bcl-2 prevents apoptotic cell death that is signaled by the release of cytochrome c from the mitochondria. In the cytoplasm, cytoehrome c forms a complex with the adapter protein Apaf- 1, and together they recruit the inactive form of the initiator caspase, Procaspase 9. In this complex. Caspase 9 becomes activated and in turn activates effeetor caspases such as Caspases 3, 6, and 7 through proteolytic cleavage. Cell lines deficient in Caspases 3 and 9 exhibit substantially reduced levels of apoptosis during development and in response to exogenous stress-inducing stimuli (Fig. 17.13).

The p53 tumor-suppressor gene is an important modulator of oncogene-induced apoptosis. Levels of p53 are kept low in unstressed cells through the binding of a specific E3-like ubiquitin ligase, Mdm2. Binding of Mdm2 to the N-terminus of p13 results in the complex being shuttled to the cytoplasm, where it is quickly degraded by the proteosome. However, in response to a variety of stresses, including ionizing radiation, serum starvation, and hypoxia, p53 protein levels increase both through protein stabilization and increased protein synthesis. Stabilization of p53 in response to stress is thought to occur through a number of mechanisms, including prevention of Mdm2 binding and phosphorylation of p53. Once stabilized, p53 is a powerful proapoptotic molecule capable of transcriptionally activating gene expression by sequence-specific DNA binding to regulatory sequences. Transcriptional targets of p53 that induce apoptosis include bax, puma, noxa, and perp and provide a link between the tumor-suppressor activity of p53 and apoptosis. This list of p53-regulated apoptotic genes is always growing and very dependent on the cell type and stress. Many of the mutations in the p53 gene found in human tumors are found within the DNA-binding domain, highlighting the importance of this region to the role of p53 as a tumor suppressor and its ability to induce apoptosis. However, recent reports in the literature indicate a new role for p53 in the cytoplasm and specifically at the mitochondna, where it may function directly to release cytochrome c to initiate the caspase cascade tmd apoptosis, bypassing the need for its transcriptional activity.

Seemingly paradoxical to its role in proliferation and oncogenic transformation is the fact that overexpression of the myc oncogene primes cells for apoptotic cell death under growth-restrictive conditions generated by nutrient deprivation or low-oxygen conditions (Fig. 17.14). It is this paradox that has set forth the hypothesis that myc deregulation results in a cellular state where increased proliferation or apoptotic death are both cqually possible, depending on the cellular microenvironment and the activity of certain crucial genetic determinants, such as the p53 tumor- suppressor gene. Evidence has accumulated that oncogenes such as myc and the adenovirus EM gene increase p53 protein stabilization and sensitize cells to killing by growth-restrictive conditions. Loss of p53 through mutation or functional inactivation severely attenuates the sensitivity of these same oncogene-expressing cells to stress-induced apoptosis. Analysis of cells deficient in 19ARF (a cell-cycle inhibitor protein) indicates that ‘nyc signals to p53 through pl9. Loss of i9Am attenuates the sensitivity of myc-expressing cells to apoptosis even in the presence of wild-type p53, suggesting that myc needs to signal pl9AM to activate p53-dependent apoptosis. Furthermore, genetic analysis of tumor cells indicates that they possess either p53 mutations or p1 9F mutations, but rarely both. Implicit in this observation is that myc deregulation favors proliferation and that a growth- restrictive state induced by DNA damage, lack of nutrients, or oxygen starvation is needed to substantially tip the cellular balance to favor apoptotic cell death. Therefore, increased sensitivity to growth-restrictive conditions that induce apoptotic cell death will result in a selective pressure for the loss or inactivation 0fJ9M, p53, or other components of this stress-induced pathway. In addition to inactivating p5.3, overexpression of antiapoptotic proteins such as Bcl-2 can accelerate tumor expansion. Myc and bcl-2 cooperate in lymphomagenesis in viva, suggesting that overcoming apoptosis is an important step in stimulating tumor growth by the inyc oncogene.

Escaping Senescence

Just like Janus, the mythological Roman god who had two faces, oncogenes that stimulate transformation, such as ras, can also drive cells into senescence. The observations that led to this discovery were based on the ability of the ras oncogene alone to transform and immortalize primary rodent fibroblasts only if they lacked the p53 tumor- suppressor gene or the cell-cycle inhibitor p16 that regulates the Rb pathway. The loss of p53 and p16 is also important for human cell immortalization. In addition, senescent cells have elevated levels of p53 or pl6, suggesting that senescence is ultimately an irreversible cell-cycle arrest. In primary cells, such as fibroblasts, the activation of the constitutive growth signal by the ms oncogene will induce the activity of p16 and p53 that will counter this signal and result in the induction of cellular senescence. Therefore, for a cell to develop independence of extracellular mitogenic growth signals, it must develop mutations in pathways that send a continuous proliferation signal as well as in pathways that attempt to restrict this signal. Cell immortalization can be viewed as a competing process that requires both the activation of dominant activating oncogenes to induce proliferation and the loss of recessive tumor-suppressor genes that induce a cell- cycle arrest in response to this constitutive activating signal.

Although cellular senescence can be delayed by mutations in the p53 and Rb pathways, cells will ultimately encounter another junction in their road to transformation, namely, crisis (Fig. 17.15). This term crisis is highly appropriate, as this roadblock to cell immortalization results in chromosomal rearrangements and cell death. Less than I in 10 million cells that enter crisis survives and gains the ability to replicate indefinitely. One clue to what drives cells to the crisis stage comes from the end- to-end fusions of chromosomes. From this it is apparent that crisis results from the progressive shortening of the protective caps (telomeres) on the ends of chromosomes.

Mammalian telomeres consist of long arrays of the repeat sequence TTAGGG that range in length anywhere from 1.5 to 150 kb. Each time a normal somatic cell divides, the terminal end of the telomere is lost; successive divisions lead to progressive shortening, and after 40 to 60 divisions, vital DNA sequences are lost. At this point, the cell cannot divide further and undergoes senescence. Telomere length has been described as the “molecular clock.” because it shortens with age in somatic tissue cells during adult life. Stem cells in self-renewing tissues, and cancer cells in particular, avoid this process of aging by activating the enzyme telomerase. Telomerase is a reverse transcriptase that polymerizes TTAGGG repeats to offset the degradation of chromosome ends that occurs with successive cell divisions; in this way, the cell becomes immortal. Telomere shortening inhibits tumor expansion when the p53 tumor-suppressor gene is intact. Although it has not been proven, telomeres seem to engage the p53 pathway by inducing a damage response signaled through the ATM pathway (Chapter 5). Studies have shown that telomere shortening leads to senescence and tumor suppression in cells with an intact p53 pathway. Surprisingly, telomere shortening accelerates tumorigenesis in cells that were deficient in p53 aetivity The ability of shortened telomeres to accelerate tumorigenesis in p53-deficient cells results from increased chromosomal instability and rearrangements and gene amplification.

Angiogenesis

Angiogenesis, the recruitment of new blood vessels to regions of chronically low blood supply, is essential for the progression of solid tumors to malignancy. Increasing evidence supports the hypothesis that tumor angiogenesis is controlled by an angiogenic switch, a physiologic mechanism involving a dynamic balance of angiogenic factors that include both inhibitors and inducers. Numerous angiogenic factors have been identified, including specific endothelial cell growth factors (e.g., vascular endothelial growth factor, or VEOF), cytokines and inflammatory agents (e.g., tumor necrosis factor a, or TNF-a, and interleukin-8, or IL-8), fragments of circulatory system proteins (e.g., angiostatin and endostatin), and extracellular matrix components (e.g., thrombospondins, or TSPs). Presumably, this diversity of angiogenic factors reflects a strict requirement for controlling angiogenesis under normal physiologic conditions and in response to oncogenic events by modulating the expression of both angiogenic inducers and inhibitors.

Although the list of proangiogenic growth factors is expanding, VEGF was the first growth factor isolated that could stimulate endothelial proliferation and migration. in tumors, VEGF can be regulated by oncogenic stimuli such as ras and raf, hypoxia, and deregulated growth-factor receptor signaling. Both tumor cells and host stromal cells produce VEGF. However, the target for VEGF lies on the cell surface of endothelial cells. These cells possess several specific transmembrane receptor tyrosine - kinases that bind VEGF, which in turn initiate endothelial migration and proliferation. In regard to neoangiogenesis, VEGF receptor II is the most important for stimulating new blood vessel formation. Studies have shown that blocking the binding of VEGF to its receptor inhibits tumor angiogenesis and tumor growth. These findings have led to the development of new antibody approaches for antiangiogenesis therapy for clinical use.

Tumors such as renal cell carcinomas that possess mutations in the von Hippel-Lindau gene (VHL) exhibit high aerobic expression of proangiogenic genes such as VEGF, whereas reintroduction of wild-type VHL substantially reduces VEOF levels to those found in untransformed cells. The mechanism underlying this observation is that VHL inhibits HIP levels under aerobic conditions by targeting HIP for ubiquitin-mediated degradatinn. Cells that have lost VHL are impaired in their ability to degrade HIP and have constitutive elevated levels of HIF and HIP target genes, of which VEGE is one under aerobic conditions (see Chapter 6 for further explanation).

TSP- 1 is a secreted adhesive glycoprotein that has been shown to have antiangiogenic activity by preventing angiogenic factors such as VEGF from binding to their target receptors. Compelling evidence of an antiangiogenic function for TSP-i is provided by studies showing that activation of the angiogenic switch in Li - Fraumeni fibroblasts lacking functional p53 is associated with diminished TSP-I expression. Furthermore, introduction of functional p53 into carcinoma cells generates an antiangiogenic activity involving induction of TSP-1 expression. Reports that endogenous TSP-1 expression can be induced by p53, repressed by oncogenic signals such as c-jun overexpression, and silenced by DNA methylation, indicate that down- regulation of TSP-l contributes to oncogenesis. As both p53 and c-jun are inducible by hypoxia, the regulation of TSP-i expression by these proteins suggests that it is also a hypoxia-responsive angiogenic inhibitor.

In summary, TSP-1 and VEGF are genetically controlled by both tumor-suppressor gene and proto-oncogene activity, providing molecular mechanisms that could contribute to the switch to the angiogenic phenotype when these controls are deregulated during oncogenesis.

Invasion and Metastasis

Many years ago, Paget realized that cancer spreads in defined patterns and is influenced by both lymph and blood flow patterns as well as the tissue being invaded. He proposed that a metastatic cell is analogous to a vegetable seed, in that without the right soil conditions, it would never grow. Malignant tumor cells become locally invasive and escape their tissue confines by invading the substratum beneath them, before they can colonize to distant tissues (Fig. 17.16). Local invasion necessitates the breakdown of epithelial integrity that is influenced by cell - cell and cell - matrix interactions through the loss of adhesion molecules such as if-cadherin. Decreased expression or impaired function of E-cadherin (E-CAD) leads to deregulated intercellular adhesion and increases the invasive growth and spread of the primary tumor, whereas overexpression of reduces the invasive and metastatic growth of transformed cells. In gastric carcinomas and lobular breast carcinomas, F-cAD has been found to be mutated and functionally inactivated. Thus, loss of F-CAD can permit local invasion of tumor cells and may be a common step in invasion. In addition to E-cadherin, cell adhesion molecules such as N-CAM also appear to play an important role in invasion. Their expression is decreased in invasive cells and when overexpressed can decrease invasion and metastasis. One final group of proteins that contribute to the invasive capability of tumor cells are integrins, which relay signals from the ECM to epithelial cells. In this case, the cell-surface repertoire of integrins changes when tumor cells become invasive.

The genetic circuits that regulate metastasis remain mainly undiscovered and elusive. Some broad concepts about metastasis have been proposed, such as the need for proteases to degrade the ECM. However, which proteases and how they are regulated differently in tumor cells compared with untransformed cells are unknown. An important question is whether or not tumor cells must acquire additional mutations to invade and metastasize. In returning to Paget’s concept of the seed and the soil, only a small percentage of tumor cells are able to metastasize. Some die rapidly by apoptosis, some remain in the circulation, and a small number invade and colonize other tissues. One noteworthy point is that loss of apoptosis in response to detachment from neighboring cells and the ECM (anoikis) is essential for metastatic spread. Metastasis represents a major challenge in the treatment of cancer, especially as the ability to control local tumor growth is increasing though the combination of surgery and radiotherapy. Metastasis research represents the most critical frontier in cancer research.

THE CONCEPT OF GATEKEEPERS AND CARETAKERS

It appears that most tumor-suppressor genes can be broadly divided into two classes that have been called “gatekeepers” and “caretakers.” Gatekeepers are genes that directly regulate the growth of tumors by inhibiting cell division or promoting cell death. The function of these genes, therefore, is rate limiting for tumor growth; both alleles (maternal and paternal) must be lost or inactivated for a tumor to develop. Predisposed individuals inherit one damaged copy of such a gene and so require only one additional mutation for tumor initiation. The identity of gatekeepers varies with each tissue, such that inactivation of a given gene predisposes to specific forms of cancer; inherited mutations in APC predispose to colon cancer, for example; mutations in VHL predispose to kidney cancers; and so on. Because these gatekeeper genes are rate limiting for tumor initiation, they tend to be mutated in many cancers. They can arise both through somatic or germ-line mutations.

By contrast, inactivation of caretaker genes does not directly promote the growth of tumors, but leads instead to genomic instability that only indirectly promotes growth by causing an increase in mutation rate. This increase in genetic instability can greatly accelerate the development of cancers, especially those that require numerous mutations for their foIl development. Colon cancer is a good example. The targets of the accelerated mutation rate that occurs in cells with defective caretakers are the gatekeeper tumor-suppressor genes, oncogenes, or both. Table 17.3 provides examples of cancer predisposition syndromes caused by mutations in DNA repair and stability genes. The evidence is highly suggestive that mutations in these predisposition genes increases the rate of acquiring cancer after exposure to DNA-damaging agents such as ionizing radiation.

Evidence for this comes from a review by Swift of 161 families affected by AT. In this prospective study, new cases of cancers were observed in blood relatives of persons with AT (of whom about half may he heterozygotic), in those who are definite heterozygotes (obligates), and in spouses who were assumed to be normal but who lived in the same environment. This extensive study also divided blood relatives of AT homozygotes into those with and those without a “radiation history:’ A radiation history was interpreted loosely as fluoroscopy of the chest, back, or abdomen, therapeutic irradiation, or occupational exposure. Table 17.4 shows the results of the survey: 53% of blood relatives with cancer had a radiation history, compared with 19% of those without cancer.

From these data, the study purported to show that AT heterozygotes are very sensitive to radiation- induced cancer; a control study of this kind does not provide proof of this, but the possibility certainly exists. It is a challenging and sobering thought to diagnostic radiologists that a proportion of the women routinely screened by mammography may be exquisitely sensitive to radiation-induced carcinogenesis because of repair deficiencies associated with being heterozygotic for AT.

MISMATCH REPAIR

Interest in mismatch repair genes heightened with the discovery that they were responsible for the mutator phenotype associated with a predisposition for hereditary nonpolyposis colon cancer (HNPCC) and possibly other familial cancers. The initial clue to this novel molecular mechanism was the discovery of deletions of long monotonic (dA-dT) runs in a subset of human colon cancers. Soon after, insertions or deletions at mono-, di-, and trinucleotide repeat sequences were discovered in subsets of colon tumors as well as in a majority of colon cancers from individuals with HNPCC. This phenntype also has been detected in several other types of human malignancies, especially those associated with type 2 Lynch syndrome. These various investigations culminated in the identification and cloning of the human hMSH2 gene, which maps to a locus linked to HNPCC on chromosome 2p2l—22 and whose homologues in Saccharomyces cerevisiae and Escheri chin coil are involved in the process of DNA mismatch repair.

The primary function of mismatch repair genes in E. coil appears to be to scan the genome as it replicates and to spot errors of mismatch as the DNA is replicated, that is, as the new strand is laid down using the stable methylated strand as a template. A growing number of human genes have been associated with HNPCC by means of linkage analysis and studies of mutational mapping. Table 17.5 lists human mismatch repair genes associated with HNPCC. The mismatch repair process in yeast and hacteria involves a large number of proteins, and so it is likely that additional causes of HNPCC remain to be uncovered.

Cells with defective or nonfunctioning mis- match repair genes can be identificd by two quite different techniques:
1. By using a selectable reporter system that inserts an exogenous long repeat sequence into the cells in question and measores the mutation rate in it.
2. By measuring the mutation rate in one or more of the many endogenous repeat sequences that already exist in every human cell—the so-called microsatellite instability assay. lVlicrosatellite instability appears to be a factor of some importance in a wide variety of human tumors.

Both techniques have strengths and weaknesses and are far from perfect.

RADIATION-INDUCED SIGNAL TRANSDUCTION

Ionizing radiation can regulate the expression of early-response genes such as c-fos, c-jan, and c-myc, genes that control lipid signaling such as acid sphingomyelinase, and the PIKK family that includes ATM and DNA-PK.

Early-Response Genes

The activation of early-response genes by ionizing radiation suggests that radiation in some way mimics the mitogenic activation of quiescent cells. Evidence for the importance of such effects is that the overexpression of proto-oncogenes such as c-ras and c-raf, whose products are intermediates in the pathways of mitogenic signal transduction, is associated with radiation resistance. The induction of these pathways results in gene activation through elements such as AP-1, serum response element, and CREB.
The role of these early-response genes is not clear at the present time, but the stimulation of signal transduction pathways and activation of transcription factors may enhance secondarily the response of the cell to radiation in terms of repair and cell-cycle arrest. In addition, the activation of these early-response genes in turn could provide a mechanism for secondary stimulation of various late-response genes such as TNF-a, PDGF, FOE, and IL-1. Activation of these genes may allow the cell to adapt to acute changes in the microenvironment and may be responsible for some of the chronic responses of the cell to ionizing radiation, including apoptosis.

A cell’s response to DNA damage depends on the type of damage. The PIKK family is comprised of proteins such as ATM and DNA-PK that sense and respond to DNA strand breaks. In unstressed cells, ATM exists as a homodimer in which the kinase domain is sterically blocked by its tight binding to a region that includes serine 1981. Recruitment of ATM to sites of DNA damage is still under investigation and may involve the MRE11/RAD50/NBS (MRN) complex. In response to DNA strand breaks, ATM changes conformation and autophosphorylates at set-inc 1981, resulting in dissociation of the inactive dimer into a monomer. The resulting ATM monomer is the active species that in turn phosphorylates target proteins involved in cell-cycle control and DNA repair such as Nijmegen breakage syndrome (NBS), breast cancer 1 (BRCA1), structural maintenance of chromosome 1 (SMC1), H2AX, and p53BP1 (Fig. 17.l7). The p53 protein is also an ATM target that plays a major role in the regulation of the mammalian cellular stress response, in part through the transcriptional activation of genes involved in cell-cycle control, DNA repair, and apoptosis. Past studies indicate that the p53 protein can also be phosphorylated by other PIKK family members, such as DNA-PK and ATR, various cyclin-dependent kinases, and the c-abl protooncogene product under different cell-cycle phases in different cell types. It has been proposed that changes in p13 phosphorylation status and protein - protein interactions are important for p53 protein stability and function.

The Ceramide Pathway

Exposure of cells to DNA damage induces the production of ceramide through two major pathways. In one pathway that is activated by ionizing radiation and DNA - damaging agents, ceramide is generated from sphingomyelin hydrolysis by the enzyme acid sphongomyelinase (Fig. 17.18). This results in the catalysis of up to one half of total cellular sphingomyelin, the majority of which would he presumably associated with the plasma membrane. Ceramide can also be generated by ceramide synthase, which is inhibited by the ATM kinase. Therefore, in response to ionizing radiation, individuals who have lost ATM have elevated levels of ceramide synthase and increased apoptosis of certain cell types (e.g., crypt cells). It is important to note that ionizing radiation can activate both nuclear and membrane - cytoplasmic signal transduction pathways, which can lead to either cell-cycle arrest or, alternatively, apoptosis.

In summary, although there is little doubt that the lethal effects of ionizing radiation result from extensive DNA damage leading to chromosomal aberrations, radiation can also stimulate signal transduction pathways that can lead to cell-cycle arrest or cell death by apoptosis, depending on the dose of radiation and genetic background of the cell.

THERAPEUTIC EXPLOITATION OF RADIATION-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS

The molecular exploitation of oncogenic protein products should soon be possible. For example. it is still controversial whether the Erb-2 receptor is predictive of a poor prognosis for breast cancer. However, as already stated, many growth-factor receptors possess truncated extracellular ligand - binding domains. Therefore, it is possible to specifically target monoclonal antibodies coupled to radionuclides or chemotherapeutic agents to these truncated receptors. It also seems possible to reverse or decrease the oncogenic effects of certain oncogenes by altering essential interactions with subcellular compartments. For example, the ras oncogene protein product requires the addition of an isoprenoid lipid (a process termed prenylation) so that it can attach to the cell membrane where it is active. Prenylation of ras is performed by farensyltransferase or geranylgeranyltransferase. Recent studies have shown that the first generation of farensyltransferasc inhibitors is able to alter ras activity and decrease growth of transformed cells. Since ras is found to be mutated in a wide variety of human tumors, and ras requires this modification for activity, the farensyltransferase would therefore be an excellent candidate enzyme to inhibit.

A second example of an oncogenic target is the NF-KB transcription factor (a member of the c-rel family of proto-oncogenes), which is found to be constitutively active in a wide variety of tumor cells by multiple mechanisms (Fig. 17.19). If NP-KB activity is inhibited in these tumor cells, they fail to proliferate in soft agar, become more sensitive to apoptotic cell death induced by ionizing radiation or chemotherapy, and exhibit decreased migration and apoptosis. What makes this transcription factor a good target for therapy is that it is normally held in the cytoplasm by an inhibitory molecule called 1KB (inhibitor of KB). If 1KB is overexpressed or prevented from degradation, then NF-KB will be held in check in the cytoplasm, where it is inactive and kept out of the nucleus. At present, several drugs exist that could therapeutically inhibit 1KB degradation. These are but several examples of molecular strategies that target key components of oncogene regulation for the treatment of cancer.

THE CELL CYCLE

The ability of cells to produce exact, accurate copies of themselves is essential to the continuance of life; it is accomplished through highly organized processes, well conserved through evolution. Lack of fidelity in cellular reproduction as manifested by DNA and chromosome alterations is a hallmark of cancer.

The only event in the cell cycle that can be identified with a simple light microscope is the condensation of the chromosomes during mitosis (M); this was observed in the late 19th century. Using autoradiography, Howard and Pelc in the early 1950s divided up the cell cycle by showing that DNA was synthesized only during a discrete time interval, which they called S phase. Between mitosis and the S phase was the “first gap in activity” (G), and between S phase and the next mitosis was the “second gap in activity” (02). If the cells stop progressing through the cycle—that is, if they are arrested— they are said to be in 0o The cell cycle was discussed in detail in Chapter 4.

Howard and Pelc also showed that it was in these gaps that radiation affects cell-cycle progression, because in their early studies it was obvious that cells arrest cell-cycle progression after low-dose radiation damage not in S or M but in either G or 02. It was subsequently recognized that these arrests also were related to the process of malignancy, because primacy cells would arrest in both 0, and 02, but tumor cells often would show only the 02 arrest point. Breakthroughs in understanding these events and the nature of the cell cycle itself came with the discovery of the cyclins, the cyclin - dependent kinases, and the cyclin - kinase inhibitors and with the elaboration by Weinert and Hartwell of the concept of cell-cycle checkpoints. The current concept of the cell cycle and its regulation is illustrated in Figure 17.20.

CYCLINS AND KINASES

Regulation of the complex processes that occur as a cell passes through the cycle is a result of a series of changes in the activity of intracellular enzymes known as cyclin-dependent kinases (Cdks). The active forms of these enzymes exist in protein complexes with a cell-cycle phase-specific protein known as a cyclin. Transitions from one phase to the next in the cycle occur only if the enzymatic activity of a given kinase activates the proteins required for progression.

In mammals, cyclins A through H have heed described. Each cyclin protein is synthesized at a discrete phase of the cycle: cyclin D and B in G1, cyclin A in S and G2, and cyclin B in G2 and M. Cyclin levels oscillate with phase of the cycle.

Seven Cdks have been described. Cdk levels are constant throughout the cell cycle, but their activity is regulated by cyclin-dependent activating kinases, the protein level of cyclin regulatory subunits, and association with Cdk inhibitors.

Molecular events in G prepare the cell for DNA synthesis. There is a stage in G1, known as the G1 restriction point, after which cells are committed to enter the S phase and no longer respond to growth conditions. Prior to this point, cells may take several routes: They may progress, differentiate, senesce, or die, depending on external signals. Key players in the G1 restriction point include the protein of the Rb gene, D-type cyclins, and Cdk4 and Cdk6, as well as Cdk inhibitors (Fig. 17.21). If extracellular signals stimulate a cell to enter the cycle from quiescence, D-type cyclins are stimulated and continue through G and form a complex with Cdk4 or Cdk6. The activated cyclin—Cdk4 or cyclin—Cdkfl complex then phosphorylates the Rb protein, which releases it from E2F and its growth-suppressive function. E2F that is released from the Rb protein binds to the promoter of the cyclin E gene, resulting in increased cyclin E mRNA and protein. There is more cyclin B available to bind Cdk2 and phosphorylate Rb, resulting in a positive feedback loop that is now refractory to mitogenic signals. Although numerous studies have documented the importance of all three D-type cyclins, two B-type cyclins, and Cdk2, Cdk4, and Cdkti, gene knockout studies in mice have indicated that all of these cyclins and their dependent kinases are not essential for normal cell-cycle progression. However, the ability of cells to be transformed by oncogenes is dependent on G1 cyclins and their dependent kinases. The current thinking is that untransformed cells require a lower level of G1 cyclins to proliferate and differentiate, whereas transformatioa requires a quantitatively different level of G1 cyclins. Thus, if increased G1 cyclin activity is needed for transformation, then loss or diminished G1 cyclin activity could act to suppress tumor formation.

Once a cell has committed to entering 5, it must begin the incredibly difficult task of accurately copying over 3 billion bases of the genome; this feat is completed in a matter of a few hours. DNA polymerases are the enzymes involved in this copying process, which must be completed with high fidelity, aided by repair and misrepair genes that remove and replace mismatched DNA bases. Cyclin A is maximally expressed in S phase and enhances transition of the cell through this phase of the cycle.

After the cell has copied its entire genome, the next important task is to segregate the two copies of the DNA equally into the progeny cells. There is a gap (G2), however, between the end of all detectable DNA synthesis and the beginning of cell division, at which the process of condensing and segregating the chromosomes begins. Events during this period are controlled by Cdk activity analogous to that occurring at the G1/S transition, but this time it is a complex of cyclins B and A with Cdk 1.

Although the cell is progressing through this complicated process of DNA replication and division, it must respond constantly to extracellular signals concerning nutrient status, cell-to-cell contact, and so forth, that arrive at the nucleus through one or another signal transduction pathway.

CHECKPOINT PATHWAYS

Events in the cell cycle must take place in a specific order, and it is the function of a number of checkpoint genes to ensure that the initiation of late events is delayed until earlier events are complete.

There are three principal places in the cell cycle at which checkpoints function:
1. G1/S checkpoint
2. S phase checkpoint
3. G2/M checkpoint

If DNA is damaged, normal cells stop progressing through the cycle and are arrested at one of these checkpoints, depending on their position in the cell cycle at the time at which the damage occurs.

Cells with damaged DNA in G avoid replicating that damage by arresting at the G1IS interface, or if they have already passed the restriction point governed by phosphorylation of Rb, they will transiently arrest in the S phase. Avoiding the replication of damaged DNA and allowing time for repair prevents cell death and the accumulation of heritable mutations. The tumor-suppressor gene p53 is critical in the pathway that leads to G, arrest. DNA damage initiates a chain of events: First ATM autophosphorylates and releases an active monomer that can directly phosphorylate p53 and Mdm2, the ubiquitin ligase that targets p53 for degradation. In addition, the checkpoint kinases (Chk), also targets of ATM, can also phosphorylate p53 and Mdm2. Phosphorylation of both p53 and Mdm2 results in increased levels of p53 protein. Activated p53 enhances 21wAFI!cIPI gene expression, which results in a sustained inhibition of Gi cyelin/Cdks. G1 cyclin inhibition prevents phosphorylation of Rb and progression from G into S. Mutations in p53 (which are present in so many human tumors) clearly compromise this checkpoint function. A second more rapid but transient checkpoint is also induced by DNA damage through Chk I phosphorylation of the Cdc25A phosphatase and the inhibition of cyclin E—Cdk2 and cyclin A—Cdk2 complexes. This later checkpoint works independently of p53.

Control of the S phase checkpoint is in part mediated by the Cdc25A phosphatase inhibiting Cdk2 activity and the loading of Cdc45 onto chromatin. Failure to load Cdc45 onto chromatin prevents the recruitment of DNA polymerase a and replicon initiation. A second mechanism for S phase arrest is signaled by phosphorylation of NBS by ATM. The importance of the S phase checkpoint is in protecting replication forks from trying to replicate through DNA strand breaks.

The arrest of cells in G2 following DNA damage is observed readily in mammalian cells and was studied by radiation biologists for decades before checkpoints were understood at the molecular level. The arrest occurs after the levels of cyclin A increase in quantity but before cyclin B increases. The function of this checkpoint in normal cells is to prevent cells with damaged chromosomes from attempting the complex process of mitosis: they are arrested in G2 to allow DNA repair to be completed. It follows, therefore, that cells lacking the G2 checkpoint are radiosensitive, because they cannot repair all of their damaged chromosomes before entering mitosis. At the molecular level, multiple kinase signaling pathways have been implicated in regulating this checkpoint. For example, ATM and Chk target the Cdc25C phosphatase and prevent cyclin B/A—Cdkl activation. In addition, other regulatory proteins have been implicated in G2 arrest, such as polo-like kinases, Brcal and p53bpl .lt appears that the G2 checkpoint is the most regulated of all checkpoints and probably the most important in preventing the inappropriate entry of damaged cells into mitosis. Consequently, targeting the inhibition of key components of the G2 checkpoint could increase radiosensitization.

The hallmark of cancer is a lack of the ability to respond to signals that normally would cause the cell to stop progressing through the cycle and dividing. Checkpoint proteins provide an important mechanism by which a cell can temporarily halt its transit through the cell cycle and attempt to restore chromosome integrity.

GENOMIC IMPRINTING

With the notable exception of the sex chromosomes, both parents contribute equally to the genetic makeup of their offspring. Although two copies of each gene are present, there are certain instances in which only one allele is expressed. This is termed genomic imprinting.

The imprinting that results in the inhibition of gene expression results from either DNA methylation or a chromatin structure change. It is parent-of- origin specific, and it is erased if passed through the opposite sex. Imprinting varies with the species, the individual, the tissue, and the time. It plays a critical role in fetal development; it can be turned on and off at different times.

Genomic imprinting is important in oncology because it leads to loss of heterozygosity and therefore to the inheritance of cancer susceptibility in a non-Mendelian fashion. Presently, about 20 imprinted genes are known in the human, but it is suspected that there are many more. Several are implicated in carcinogenesis, including one oncogene and several tumor-suppressor genes. For example, in the ease of a paternally imprinted, maternally expressed gene (such as the one involved in Wilms’ tumor), males always would pass on an inactive gene; females always would pass on an expressed gene.