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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

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Holland-Frei Cancer Medicine. 5th edition.

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Chapter 2Cell Proliferation, Differentiation, and Apoptosis

, MD, PhD, , MD, and , MD.

The biology of cell division, differentiation, and apoptosis is exceedingly similar in both normal and cancer cells. The cancer cell differs from its normal counterpart in that it is aberrantly regulated. Cancer cells generally contain the full complement of biomolecules that are necessary for survival, proliferation, differentiation, cell death, and expression of many cell-type-specific functions. Failure to regulate these functions properly, however, results in an altered phenotype and cancer.

Four cellular functions tend to be inappropriately regulated in a neoplasm. First, the normal constraints on cellular proliferation are ineffective. Second, the differentiation program can be distorted. The tumor cells may be blocked at a particular stage of differentiation, or they may differentiate into an inappropriate or abnormal cell type. Third, chromosomal and genetic organization may be destabilized such that variant cells arise with high frequency. Some variants may have increased motility or enzyme production that permits invasion and metastases. Finally, the tightly regulated cell death program (apoptosis) may be dysregulated.

To comprehend the biology of cancer, it is necessary to understand how these functions are controlled in normal cells and how they become uncontrolled in cancer cells. This chapter focuses on the biology of cell proliferation, differentiation, and apoptosis and how these functions are linked in the development of neoplasia.

Proliferation

Tumor Growth and Cell Proliferation In Vivo

Fundamentally, cancer is a disease of accumulation of clonal cells. Abnormal cell proliferation is necessary, although often insufficient, for tumorigenesis. It is the increase in tumor cell number, and thus tumor burden, that ultimately accounts for the adverse effects on the host. Indeed, the goal of most current cancer therapy is to reduce the number of tumor cells and to prevent their further accumulation. To better accomplish this goal, a more complete description of the unique characteristics of tumor cell proliferation is required. This task is made difficult by the fact that the mechanisms that underlie tumor and normal cell proliferation are very similar. In this section, we will review current understanding of the complex molecular mechanisms involved in the regulation of cell proliferation with particular emphasis on the aberrations in this system that occur in malignant cells.

From the perspective of biologic evolution, it is obvious that cells within a multicellular organism like humans have an intrinsic proliferative potential that is in vast excess of that required to meet the requirements of normal growth and development. Cellular life has evolved from single-celled organisms selected for their ability to replicate in a minimum amount of time, within the constraints imposed by the biochemical processes of cell division. Standard laboratory strains of bacteria, for example, can divide every 20 minutes under optimal conditions. The constraints of cell division imposed on multicellular organisms are greater since replication must be carried out with absolute fidelity to maintain the integrity of the organism. Even so, normal human cells can divide as often as once or twice a day in vivo. A cell dividing once a day would generate a cell number equal to the total number of cells in an adult human in less than 2 months. Clearly, then, human cells have inherited a surplus of proliferative capacity from their unicellular ancestors. Multicellular organisms must evolve mechanisms to restrain this proliferative capacity to appropriate times and places. The key in understanding tumor cell proliferation, then, is to characterize these mechanisms and to understand how they fail during tumorigenesis.

The rate of cell proliferation within any population of cells depends on three parameters: (a) the rate of cell division (Tc), (b) the fraction of cells within the population undergoing cell division (growth fraction), and (c) the rate of cell loss from the population due to terminal differentiation or cell death (see next section). Tc represents the time it takes to complete a cell division cycle. The cell division cycle can be divided into two functional phases, S and M phases, and two preparatory phases, G1 and G2 (Fig. 2.1). S phase is defined as the phase in which the DNA is replicated. Under normal circumstances, the time it takes a typical human cell to complete S phase is about 8 hours and is invariant. Fully replicated chromosomes are segregated to each of the two daughter nuclei by the process of mitosis during M phase. The length of M phase is about 1 hour and is also normally invariant. G1 phase precedes S phase, whereas G2 phase precedes M phase. G1 and G2 phases are required for the synthesis of cellular constituents needed to support the following phase and ultimately to complete cell division. In mammalian cells, the length of G2 phase is about 2 hours. The length of G1 phase is highly variable and can range from about 6 hours to several days or longer. The varying length of G1 phase accounts for most of the difference in Tc between different cell types or between cells growing under different conditions.

Figure 2.1. The cell cycle.

Figure 2.1

The cell cycle. When a cell is not synthesizing DNA (S phase) or completing mitosis (M phase), it is commonly termed as being a G (gap) phase. Normal cells are capable of resting in a nondividing state, called G0. They can begin one or more cycles of (more...)

Cell Cycle Control

A successful cell division cycle requires the orderly and unidirectional transition from one cell cycle phase to the next. Certain events must be completed before others are begun. For example, beginning mitosis before the completion of DNA replication would obviously be deleterious to the cell. In theory, the ordering of cell cycle events may be accomplished in a manner analogous to the substrate-product relationship of a metabolic, biochemical pathway. 1 The product of one reaction serves as the substrate and is thus required for the next reaction. Hence, regulation of the system is inherent in the biochemical events of the process itself. The prevailing view, however, is that the timing and ordering of cell cycle transitions is dependent on separate positive and negative regulatory circuits. The regulatory circuits enforce a series of checkpoints, allowing passage only after completion of critical cell cycle events. Two classes of regulatory circuits exist, intrinsic and extrinsic. Intrinsic regulatory pathways are responsible for the precise ordering of cell cycle events. Since the length of S, G2, and M phases in mammalian cells is relatively invariant, the transitions between these phases are controlled predominantly by intrinsic regulatory pathways. Extrinsic regulatory pathways function in response to environmental conditions or in response to detected cell cycle defects. Both types of regulatory circuits can use the same checkpoints. We will focus our attention on the extrinsic regulatory circuits where differences between normal and neoplastic cells are observed.

Passage of the cell cycle checkpoints ultimately requires the activation of intracellular enzymes known as cyclin-dependent kinases (CDKs). CDKs are extremely well conserved through evolution. CDKs exist in all eukaryotic cells from fungi to plants to mammals. In fact, CDKs from human cells can functionally substitute for the enzymes in yeast. The structural and functional conservation of these enzymes through evolution suggests that they are centrally important for the cell cycle in all eukaryotic cells. The requirement for these enzymes for cell cycle transitions has been amply documented, particularly in organisms like yeast that are amenable to genetic manipulation. 1 Since activation of CDKs is the central event in cell cycle transitions, it is not surprising that their activity is exquisitely regulated at several levels. 2 The active CDK holoenzyme is composed of a catalytic subunit and the cyclin regulatory subunit. One level of regulation is that each cyclin protein is synthesized at a particular stage of the cell cycle. For example, cyclin D is synthesized during G1, cyclin E is synthesized in late G1, cyclin A is synthesized during S and G2 phases, and cyclin B is synthesized in G2 and M phases (Fig. 2.2). Therefore, a given catalytic subunit cannot become active until an appropriate cyclin is synthesized. Upon synthesis, a cyclin can assemble with an appropriate catalytic subunit. However, this complex requires phosphorylation on threonine by another regulated kinase, the CDK-activating kinase or CAK. CAK is itself a CDK composed of cyclin H and CDK7 proteins. Hence the levels of CAK influence the activity of assembled CDKs. Another level of regulation is deactivation of the CDK by phosphorylation of its ATP binding site by yet another regulated kinase activity. This kinase activity is unusual in that it has dual specificity for both tyrosine and threonine. A CDK deactivated by phosphorylation of its ATP binding site can be reactivated by a dual specificity phosphatase of the Cdc25 family. In fact, dephosphorylation by these phosphatases may be the rate-limiting step in triggering cell cycle transitions. Another level of regulation is the presence of a diverse family of proteins known as cyclin-dependent kinase inhibitors, or CKIs, that can block activation of CDKs. Two distinct classes of CKIs have been described. One class inhibits multiple CDKs and includes p21CIP1, p27KIP1, and p57KIP2. The other class specifically inhibits cyclin D/CDK4 or 6 CDKs and includes p16INK4, p15INK4B, p18INK4C, and p19INK4D. The synthesis, degradation, and activity of these CKIs are regulated in response to both mitogenic and antimitogenic signals. For example, cell cycle regulation by cell-cell contact or transforming growth factor-𝛃 (TGF-𝛃) is mediated by p27KIP1. 3, 4 Once activated, the CDKs that drive the transition into a particular cell cycle phase often need to be deactivated before completion of that phase and transition to the ensuing phase. For example, the CDKs required for initiation of mitosis also prevent exit from mitosis and into G1 phase. The final level of CDK regulation involves their specific degradation in precise order. It is now generally understood that ubiquitin-mediated proteolysis is responsible for this regulation as well as the regulation of a host of other cell cycle regulators. 5 Hence, synthesis, post-translational modification, and programed degradation all contribute to the regulation of CDKs.

Figure 2.2. Cyclin-dependent kinase regulation of cell cycle transitions.

Figure 2.2

Cyclin-dependent kinase regulation of cell cycle transitions. A. The phases of the cell division cycle are shown. Transition from one phase to the next requires transit of a checkpoint, like the restriction point (R), during the G0/G1 to S phase transition. (more...)

The G0 to S Checkpoints

As discussed above, the time it takes to progress through the S, G2, and M phases of the cell cycle is relatively invariant. The length of G1 phase on the other hand is variable. In addition, cells can exit the cell cycle for extended periods of time and mammalian cells do so during the G1 phase of the cell cycle. Cells that have exited the cell cycle are said to be in a G0 state, or “quiescent.” Most cells in adults are in G0. This absence from the cell division cycle can be temporary or permanent, as is the case with terminally differentiated cells like neurons. Cells in G0 can be very active functionally and metabolically, and proliferation of G0 cells can be initiated by changes in cell density, the presence of mitogens or growth factors, or the supply of nutrients. These cells then enter the cell cycle, beginning a sequence of events that culminates in cell division. Hence, the G0/G1 to S phase transition is highly regulated, and the result of this regulation, by and large, determines the Tc and growth fraction of a population of cells.

Like other cell cycle transitions, the transition from G0 to S phase as cells re-enter the cell cycle is regulated by two major checkpoints: competence and the restriction point (R). These checkpoints are located approximately 12 and 2 hours before the start of the S phase, respectively. At least three growth factors, provided in serum, are required sequentially to transit these checkpoints following resumption of proliferation of fibroblasts: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF-1). As mentioned above, extracellular TGF-𝛃 has the opposite effect. It inhibits the growth of various epithelial cells by modulating expression of CKIs. Paracrine production of TGF-𝛃 could limit growth of both normal and cancer cells, and experimental models suggest that it may play a role in the regression of breast cancers in response to hormonal or drug therapies. 6 Once the R point has been passed, the cell is committed to a round of cell division. The cell now completes S, G2, and M phases without the need for growth factors or even additional protein synthesis. Once initiated, the cell cycle is not free running; the competence and restriction checkpoints must be passed in each subsequent G1 phase, thus requiring the continued presence of growth factors. The switching of cells back and forth between quiescence and cycling depends on extracellular conditions and is regulated differently in normal and tumor cells.

Growth factor receptors are complex, large proteins that span the plasma membrane. They have a specific domain that recognizes the growth factor on the outside of the cell, and their cytoplasmic portion may have an enzymatic function, such as a protein tyrosine kinase. Binding of a growth factor or ligand to its receptor can induce transmission of a signal to the cytoplasm through activation of the kinase. 7 The next step is a transduction of the cytoplasmic signal to the cell nucleus. This is accomplished by a heterogeneous group of molecules known as second messengers and includes various proteins that are phosphorylated by kinases, small molecules such as inositol phosphates and cyclic AMP, and ions, including Ca++, H+, and Zn++. Within the nucleus, genes are then activated in response to these second messengers. As an illustration of this general scheme, upon binding of EGF to the extracellular domains of its receptor, autophosphorylation occurs in the intracellular domain of the protein. This phosphorylated domain facilitates the formation of a protein complex containing Grb2 and Sos. This complex activates the Ras protein by catalyzing exchange of Ras-bound GDP for GTP. The GTP-bound form of Ras activates the c-Raf kinase. This kinase then triggers a phosphorylation cascade ultimately activating mitogen-activated protein kinase. This kinase may phosphorylate and regulate transcription factors such as Jun, Fos, and Myc.

A variety of proteins are produced during G1 after cells leave quiescence. Some are enzymes that expand metabolic functions lost by G0 cells, such as those providing energy, and more ribosomes are made for rapid protein synthesis. Others have so-called housekeeping functions that keep both quiescent and growing cells in metabolic balance. Only a few proteins appear to be key regulatory molecules. For example, enzymes are required for the synthesis of isoprenoids, which are necessary for activity of the Ras oncogene, and for the synthesis of polyamines, which have many functions including ionic binding to nucleic acids. The Ras oncogene product is synthesized as a precursor protein that requires post-translational processing to become biologically active and capable of transforming mammalian cells. Farnesylation appears to be a critical modification of the Ras protein, and drugs that inhibit farnesyl-protein transferase can block Ras-dependent transformation. These agents have been proposed as a potential new class of therapy for cancer. 8 Enzymes involved in the synthesis of DNA, such as thymidine kinase and DNA polymerase, as well as histones, are synthesized just prior to the S phase. These enzyme molecules relocate at the beginning of DNA synthesis, moving from the cytoplasm into the nucleus. A variety of experiments show that DNA is made by a high-molecular-weight, multienzyme complex. 9, 10 This complex contains many enzymes known to be involved in the process of DNA replication, but its size and other features are still a matter of debate. The onset of DNA replication has been investigated recently with in vitro systems, and these studies reveal that the synthesis of helicase enzymes, which possess DNA-unwinding ability, may provide the final factor for initiating the S phase. 11 After DNA synthesis has commenced, cell growth becomes relatively independent of external controls. The daughter cells, now in the G1 phase, will then either pass through another cycle or arrest in a quiescent G0 state, depending, once more, on external conditions. If these conditions are not adequate, the cell will become arrested before it reinitiates DNA synthesis.

How is re-entry into the cell cycle from G0 ultimately controlled? Like other cell cycle transitions, activation of CDKs are required. G0 cells are devoid of significant CDK activity. In the presence of mitogenic growth factors, expression of D-type cyclins (cyclins D1, D2, and D3) is stimulated and continues throughout G1 phase as long as the growth factors are present. 12 D-type cyclins complex with either CDK4 or CDK6 catalytic subunits to form a holoenzyme modified by CAK. All of the relevant substrates for cyclin D/CDK4 or 6 have probably not been enumerated. However, one important substrate is likely the retinoblastoma tumor-suppressor protein (Rb). Rb is constitutively expressed and constrains cells from progressing through the G1 phase of the cell cycle. 13 Rb complexes with many cellular proteins including the E2F transcription factors. When in complex with E2F, Rb represses transcription from E2F-dependent promoters. Upon phosphorylation by cyclin D/CDK4 or 6, 14 Rb loses its ability to restrain the cell cycle. 15 This response is presumably because it can no longer complex with E2F and represses E2F-dependent transcription. 16 The E2F family contains at least five members (E2F-1 through E2F-5). The E2F proteins function as transcriptional activators when in heterodimeric complex with one of the E2F-related proteins DP-1, 2, or 3. The heterodimeric complex binds a specific DNA sequence and activates transcription from the promoters of many genes important for S phase including dihydrofolate reductase, DNA polymerase α, and thymidine kinase. 17 Perhaps most importantly, E2F influences the expression of cyclin E. 18 In fact, of many E2F-dependent genes, cyclin E is the only one deregulated upon loss of Rb in normal cells. 19 Cyclin E expression begins in late G1 phase and complexes with CDK2. Cyclin E/CDK2 activity is necessary and sufficient for the start of S phase. 20 Forced expression of cyclin E/CDK2 activity can trigger the start of S phase in the absence of Rb phosphorylation and derepression of other E2F-dependent genes, suggesting that cyclin E is the primary target for regulation by cyclin D/CDK4 or 6, Rb, and E2F. 21, 22

DNA Damage-Induced Checkpoints

When nuclear DNA is damaged, normal cells initiate a response that includes cell cycle arrest, apoptotic cell death, and transcriptional induction of genes involved in DNA repair. Induction of apoptosis is an important response to DNA damage and is discussed in detail below. Normal cells in G1 phase prior to the R point will arrest in G1 phase upon sensing DNA damage. This arrest is presumably induced to prevent the replication of damaged DNA. Replication of damaged DNA can result in the incorporation of heritable genetic mutations. If cells are past the R point or within S phase, DNA replication is slowed, again to allow time for DNA repair. If cells sense DNA damage while in G2 phase, a G2 cell cycle arrest will occur. Different types of DNA damage can interfere with normal mitosis, resulting in heritable genetic mutations or cell death.

The tumor-suppressor genes ATM and p53 play an important part in responses to damaged DNA. 23, 24 For example, cells containing mutations in p53 fail to arrest in G1 or undergo apoptosis efficiently upon irradiation. Cells containing mutations in ATM are also deficient for cell cycle arrest as well as some forms of DNA repair. The p53 protein functions as a transcription factor by binding specific DNA sequences and regulating transcription from promoters containing those sequences. In normal cells, DNA damage induces an increase in p53 levels by inhibiting the normal rapid turnover of the protein. The p53 protein is normally targeted for ubiquitin-dependent proteolysis by association with the Mdm2 protein. This association is inhibited by phosphorylation of p53 on specific amino-terminal residues that is triggered by DNA damage. 25 Phosphorylation on these amino terminal residues also facilitates dephosphorylation or acetylation of p53 carboxy-terminal residues. These modifications increase the affinity of p53 for its DNA binding site by distinct mechanisms 26, 27 and hence increase its ability to activate transcription. The transcription of a number of genes can be affected by activation of p53. However, the ability of p53 to directly increase expression of p21CIP1 is probably important for p53-dependent G1 cell cycle arrest observed upon DNA damage. As discussed above, p21CIP1 is a CKI that can inhibit the activity of multiple CDKs, including cyclin D/CDK4 or 6 as well as cyclin E/CDK2. In summary, DNA damage generates a signal that can activate p53 by post-translational modification. Increased p53 activity upregulates p21CIP1, which prevents activation of CDKs, required for the G1 to S transition.

ATM is the gene whose mutation is responsible for ataxiatelangiectasia. Immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects, and cancer predisposition characterize this disease. ATM encodes a protein containing a phosphatidyl-inositol 3-kinase-like domain, implicating it in signal transduction. Like p53 mutant cells, mutant ATM cells are defective in the G1/S checkpoint activated after radiation-induced DNA damage. This defect is attributable to the lack of p53 activation that normally occurs, suggesting that ATM may participate in the same pathway as p53. ATM protein, and the related ATR protein, can, in fact, associate with and phosphorylate p53 at its amino-terminal sites. 28, 29 The p53-directed kinase activity of the ATM protein is itself activated by DNA damage. ATM protein, therefore, contributes to the activation and stabilization of p53 by phosphorylating amino-terminal sites during the radiation-induced DNA damage response. ATM protein may play a role in sensing DNA damage and generating the DNA damage signal.

Environmental agents like radiation or DNA-damaging chemicals most commonly induce DNA damage. Rarely, DNA damage can be generated by mistakes in the normal execution of the cell cycle. However, a form of DNA damage eventually occurs in all normal cells as they suffer replicative senescence. Normal cells have a limited replicative lifespan both in vivo and in vitro; a cell can undergo only a finite number of cell divisions. This limit is thought to be imposed, at least in part, by levels of telomerase activity. 30 Telomerase is an enzymatic activity within cells that is required to maintain the integrity of DNA ends. 31 DNA polymerases involved in DNA replication synthesize DNA in the 5′ to 3′ direction and require a primer and template. The requirement for a primer ensures that some genetic information will be lost from the 5′ end of DNA during each round of DNA replication. Telomerase adds DNA of a particular sequence to the ends of DNA without the need for a separate primer or template, thus protecting cells from loss of genetic information. Telomeric DNA also protects chromosomes from degradation or recombination. Without telomeric DNA, chromosomes become unstable. As normal cells become senescent, they lose telomerase activity and their cell division cycle is arrested. This cell cycle arrest may be mediated by the DNA damage checkpoint since shortened telomeric DNA is associated with DNA strand breaks that may be sensed as damaged DNA. Consistent with this hypothesis, shortening of telomeric DNA triggers a p53-dependent cell cycle arrest by accumulation of single stranded DNA. 32

Checkpoint Defects in Tumor Cells

To maintain tissue homeostasis and to support normal development, each organ maintains tight controls over Tc, growth fraction, and cell loss. Physiologic stimuli can alter these parameters in normal tissues, leading to increased tissue growth, but this growth will cease when the stimulus is withdrawn or a new steady state is achieved. In contrast to normal cells, however, tumor cells continue to proliferate even in the absence of proliferative signals. Although tumor cells proliferate under inappropriate conditions, they do not necessarily proliferate faster than normal cells. In fact, some normal tissues grow faster than cancers under physiologic conditions (Table 2.1). Biopsy samples from normal, inflammatory, and neoplastic lesions of the lung, cervix, vocal cord, or pharynx have been analyzed for the rate of cell proliferation; these studies showed that benign inflammatory lesions can grow over 20 times faster than cancer in a discrete time and place. 33– 35 Similarly, rapid proliferation of human lymphoid cells is induced by immunostimulants, and growth kinetics of these cells are similar to those observed in high-grade lymphomas. 36 So, it is not simply rapid growth at a single time and place that distinguishes neoplasia but rather growth that is not restrained to appropriate times and places.

Table 2.1. Growth Parameters of Human Neoplasms and Normal Tissues.

Table 2.1

Growth Parameters of Human Neoplasms and Normal Tissues.

It generally is believed that neoplastic cells multiply exponentially during the early phases of tumor cell growth. As the tumor mass increases, however, the rate of growth declines. Measuring tumor growth over time describes a curve with an exponential increase in the early period, then a flattening out of the growth rate over time (i.e., Gompertzian curve). 37 Several mechanisms have been invoked to explain this change in growth rate with larger tumors: (a) decrease in the growth fraction, (b) increase in cell loss (i.e., exfoliation, necrosis), (c) nutritional depletion of tumor cells resulting from outgrowth of available blood supply, or (d) lengthening of Tc. Experimental tumor models suggest that cell cycle time changes only slightly when tumor growth decreases. 38 Under adverse conditions, tumor cells often leave the growth fraction and enter a nongrowing state (G0 or prolonged G1) (see Fig. 2.1), although these same cells can re-enter the division cycle when conditions improve or when stimulated by growth factors. Therefore, the mass doubling time of tumors is correlated with the growth fraction (Table 2.2).

Table 2.2. Correlation between Mass Doubling Time and Growth Fraction.

Table 2.2

Correlation between Mass Doubling Time and Growth Fraction.

The biochemistry of growth appears to be very similar qualitatively in tumor and normal cells. 39 Despite numerous efforts, universal differences in biochemical machinery have not yet been discovered between normal and tumor cells. The fundamental difference probably lies in a relaxation of the regulation of cell growth. 9, 38 For example, normal cells generally are quiescent at physiologic levels of growth factors, whereas related tumor cells are able to proliferate under these conditions. In some experimental models, tumor cells proliferate in the absence of or at very low levels of growth factors. Further, fibroblast-derived tumor cells are less sensitive than normal cells to the presence of other cells in their immediate vicinity. Normal cells typically cease proliferation when the in vitro culture becomes confluent, but tumor cells can reach several-fold higher densities in culture. Also, cells of normal solid tissue lie on a secreted extracellular matrix (ECM) that is composed of various proteins that stimulate cell growth. 40 Tumor cells often are partly or completely independent of ECM for optimal growth, and they may secrete little matrix material. 41

What molecular defects bring about the relaxed growth requirements in neoplastic cells? Defects can occur at several levels. For example, limiting growth factors may not be needed because tumor cells inappropriately produce their own (i.e., an autocrine mechanism). Alternatively, receptors may be produced in excess, as is the case for EGF receptors in numerous clinical tumors, leading to adequate stimulation at the low growth factor concentrations found in vivo. Moreover, mutations that alter intracellular signaling mechanisms may bypass growth factor dependence. Mutated forms of proto-oncogenes and inactivated tumor-suppressor genes can activate growth in these ways. We will focus here on defects that occur in cell cycle regulatory proteins that enforce the checkpoints discussed above.

Like normal cells, the transit of cell cycle checkpoints in cancer cells ultimately requires the activation of CDKs. Due to the complexity of CDK regulation, defects leading to inappropriate activation of CDKs can occur at several levels. The overexpression of cyclin D1 has been detected in many human cancers due to gene amplification or translocation of the cyclin D1 gene. 42 The cyclin D1 gene is located on chromosome 11q13. This chromosomal region is amplified in a wide variety of human cancers including small-cell lung tumors (10%), primary breast cancers (13%), bladder cancer (15%), esophageal carcinoma (34%), and squamous cell carcinoma of the head and neck (43%) among others. 43 Of course, other potential oncogenes could be contained within the amplified region. However, cyclin D1 is likely important since its expression is consistently elevated in these tumors. Cyclin D1 overexpression can also be observed in tumors, such as sarcomas, colorectal tumors, and melanomas, without amplification of the gene. In some cases, cyclin D1 expression is activated by chromosomal translocation. In parathyroid adenoma, Motokura and colleagues. 44 have identified cyclin D1 as being translocated to the parathyroid hormone gene, thereby deregulating cyclin D1 expression. Translocation of the cyclin D1 gene with immunoglobulin heavy chain gene transcriptional control elements has also been observed in B cell lineage mantle cell lymphomas. Cyclin D1 is a growth factor responsive cyclin that plays an important role in regulating the G0/S checkpoint. Deregulated expression of cyclin D1 could inappropriately increase cyclin D1/CDK4 activity and drive transit of the checkpoint even in the absence of growth factors. Direct evidence that forced expression of cyclin D1 can facilitate tumorigenesis has been obtained from transgenic mice in which overexpression of cyclin D1 has been targeted to the mammary epithelium. These mice develop ductal hyperproliferation and eventual mammary tumor formation. 45

CDK activation can also be accomplished by inactivation of CKIs. Genetic mutation of CKI genes has also been observed frequently in human cancer. In this scenario, loss of a CKI relieves one constraint on the activation of CDKs and provides a proliferation stimulus. In particular, the INK4 locus within chromosomal region 9p21 is one of the most frequently mutated areas in human cancers. 46 This locus is also frequently methylated in some tumor types including bladder cancer and leukemia. Extensive methylation of DNA prevents efficient transcription of genes within the methylated region, thus silencing gene expression. Three proteins are encoded by the INK4 locus including the CKIs p16INK4a and p15INK4b as well as p19ARF (see below). It is likely that p16INK4a is a bona fide tumor-suppressor gene since many of the mutations detected in tumors specifically target expression of this protein, and because germline mutations that specifically map to p16INK4a have been detected in kindreds with familial melanoma and pancreatic adenocarcinoma. In addition, mutations in CDK4 that prevent binding with p16INK4a, thus relieving it of p16-mediated inhibition, have also been found in melanoma-prone families. Loss of p16INK4a may facilitate activation of cyclin D1/CDK4 or 6, which is likely to affect regulation of the G0/S checkpoint. Mutation of other CKIs in human cancer is rare, suggesting that they may be required for execution of the cell cycle. However, expression of p27KIP1 is inversely correlated with clinical outcome in a limited number of cancers, including melanoma and carcinoma of the oral cavity.

CDK activation also requires dephosphorylation of inhibitory threonine/tyrosine phosphorylation sites by the Cdc25 family of dual specificity phosphatases. In vitro evidence exists that the Cdc25 family memers are potential oncogenes. 47 Forced expression of Cdc25 can cooperate with Ha-Ras or loss of Rb to induce oncogenic transformation of primary cells. Overexpression of Cdc25 has also been detected in some primary human tumors. Cdc25A may be a direct transcriptional target for the myc oncogene. 48 Inappropriately high Cdc25 levels may provide an oncogenic stimulus by inappropriately activating CDK activity.

One of the most important genes involved in human cancer is the Rb tumor-suppressor gene. An interesting feature of retinoblastoma is that close to 40% of cases are hereditary, and susceptibility to retinoblastoma is inherited as a simple autosomal dominant trait with high (90%) penetrance. The simple genetics of retinoblastoma has provided the means to molecularly clone the gene responsible; mutational inactivation of both alleles of Rb is necessary and sufficient for retinoblastoma. 49 Mutation of Rb is observed at high frequency in osteosarcoma and soft-tissue sarcoma as well. Rb mutations can also be detected in a wide variety of clinically important cancers including carcinoma of the breast, prostate, bladder, kidney, liver, pancreas, cervix, and lung, as well as leukemia. Further, expression of wild-type Rb cDNA in cancer cells can inhibit their tumorigenicity. 50 As mentioned above, cyclin D/CDK4 or 6 phosphorylation, which, in turn, is regulated by p16INK4a, inhibits Rb function. This finding suggests that these three proteins function in the same biochemical pathway (Fig. 2.3). Support for this functional interrelation comes from the observation that deregulation of any one of these proteins greatly decreases the likelihood of detecting defects in the other proteins. For example, tumor cells that lose p16INK4a or overexpress cyclin D1 generally retain wild-type Rb. Cells lacking wild-type Rb typically express normal levels of cyclin D1 and p16INK4a. In addition, induction of cell cycle arrest by forced expression of p16INK4a only occurs in cells that contain functional Rb. If mutations in any of the members of this pathway are considered, disruption of this p16INK4a/cyclin D1/CDK4 or 6/Rb pathway may occur in most human cancers. Since this pathway is important for regulation of the G0 to S phase transition, it has a major influence on the growth fraction of normal tissues.

Figure 2.3. The Rb and p53 growth control pathways.

Figure 2.3

The Rb and p53 growth control pathways. Underphosphorylated and active Rb in complex with transcription factors like E2F represses the transcription of genes required for entry into S phase. Upon mitogenic stimulation, synthesis of cyclin D increases (more...)

The p53 gene is the most frequently mutated gene in human cancer. 51 Germ line p53 mutation is involved in the cancer-prone Li-Fraumeni syndrome. 52 Mice lacking p53 due to genetically engineered disruption are also cancer prone. Wild-type p53 is critically important for operation of the DNA damage-induced checkpoint (see above). Upon sensing DNA damage, p53 is activated, resulting in either G1 cell cycle arrest or apoptosis. These responses either allow time for the cell to repair the damage or to rid the body of cells with damaged DNA. Loss of p53 function, therefore, decreases genomic stability. Loss of genomic stability can increase the accumulation of additional genetic mutations required for neoplastic transformation. The Mdm2 gene encodes a protein that binds p53 and targets it destruction by the ubiquitin-proteosome pathway. Too much Mdm2 protein may be analogous to p53 inactivation since any p53 synthesized would be rapidly degraded. Mdm2 was originally identified as an oncogene amplified in a spontaneously transformed mouse cell line. Overexpression of Mdm2 mediated by gene amplification can also be detected in human cancer, particularly sarcoma. 53 Interestingly, the p19ARF protein encoded by the INK4a locus also regulates p53 function. 54 The p19ARF protein can bind Mdm2 and prevent Mdm2 from targeting p53 for degradation. Consistent with the ability of p19ARF to activate p53, forced expression of p19ARF can cause a p53-dependent cell cycle arrest. As discussed previously, mutations of the INK4 locus that inactivate p19ARF, as well as p16INK4a, are commonly observed in human cancer. Inactivation of p19ARF may contribute to tumorigenesis since Mdm2-mediated degradation of p53 would be unimpeded. The functional interrelation between p19ARF, Mdm2, and p53 defines another cell cycle checkpoint control pathway (see Fig. 2.3.) Deficiencies in this pathway also play a vital role in neoplastic transformation.

Although cancer cells use the same cell cycle machinery as normal cells, the cell cycle checkpoints in tumor cells are relaxed. Of the scores of proto-oncogenes and tumor-suppressor genes that have been identified to date, most function in signal transduction pathways that mediate mitogenic stimulation. These signal transduction pathways eventually converge on the cell cycle checkpoint that controlsthe G0/G1 to S phase transition and activate appropriate CDKs.Influencing the transit of this checkpoint has a major influence on the proliferation of normal and tumor cells by affecting both Tc and growth fraction. Despite the number and variety of these genes involved in signal transduction, relaxation of the G1/G0 to S checkpoint controls in tumor cells is mediated, for the most part, by disruption of two pathways, the Rb and p53 growth control pathways. These two genes, individually, are the most frequently mutated in human cancer cells. Disruption of the Rb or p53 pathways probably occurs in virtually every human cancer.

Differentiation

Most, if not all, tumor cells show abnormalities in differentiation (i.e., anaplasia). The anaplasia of tumors can provide insights into their etiology, degree of malignancy, prognosis, and sensitivity to therapeutic intervention by differentiation- or maturation-inducing agents. These differences in phenotype arise from differences in gene expression, not in gene content. The genes expressed by a particular cell only comprise approximately 10 to 20% of the coding capacity of the genome. In humans, there are over 100,000 genes that code for proteins; however, an individual cell generally expresses only 10,000 to 20,000 genes. Genes expressed by a particular cell depend on its embryonic lineage, developmental stage of the organism, tissue and cellular environment, and functions that the cell must fulfill. The mechanisms that regulate gene expression are incompletely understood; however, they most certainly entail the sequential action of cell-type-specific or cell-lineage-specific transcription factors that repress or activate the differentiation-specific genes. Programs of gene expression generally are instituted early in embryogenesis and sequentially altered as development proceeds. 55, 56

Some genes are expressed by many, if not all, cell types. These “housekeeping” genes generally encode proteins that participate in basic or universal cellular functions. Other genes that are expressed only in specific cell types and/or stages of development are said to be cell-type- or differentiation-specific genes. Thus, the expression of specific gene products marks both the cell lineage and the stage of differentiation.

Differentiation and Cell Proliferation

Differentiation begins shortly after the first few cell divisions that follow fertilization. Throughout development, and in adult organisms, the ability of a cell to proliferate is intimately connected to its state of differentiation. Adult tissues generally express a variety of factors that act to maintain both the proliferation and the differentiation status of the cells. These include secreted molecules, transmembrane receptors, intracellular signaling molecules, and transcription factors. For example, myoD 57 and c/EBP-a 58 are nuclear factors that activate the transcription of muscle- and adipocyte-specific genes, respectively; in addition, both proteins are potent inhibitors of cell proliferation.

In early embryos, cell proliferation is the primary means by which the cell mass increases. As the organism develops, however, proliferation becomes restricted. Some differentiated cells continue to proliferate, but others irreversibly lose this ability. Embryonic cells often display traits that confer on them a selective growth advantage over that of an adult cell. They proliferate vigorously, are capable of extensive migration, secrete factors that increase the local supply of blood, and produce enzymes capable of degrading basement membranes. These traits also are characteristic of tumor cells, including the ability to increase local blood supply (i.e., angiogenesis). Recent data suggest that tumor angiogenesis is an important, negative prognostic indicator for carcinomas and leukemias. 59, 60 Angiogenesis is now being investigated as a potential target for cancer therapy. 61 Thus, in adult organisms, mutations or conditions that activate portions of embryonic programs for gene expression or inactivate portions of the adult program can produce cells with many properties of malignant tumor cells. 62

Stem Cells

Stem cells have the capacity for both self-renewal (i.e., proliferation without a change in phenotype) and differentiation (i.e., changing into a new phenotype). Some stem cells have already undergone considerable differentiation, so further differentiation is restricted to a single cell type or lineage. Other stem cells are multipotent and differentiate into a variety of cell types (i.e., hematopoietic stem cells). It has been difficult to demonstrate cells in adults that are totipotent (i.e., capable of differentiating into most or all of the cell types that comprise the organism), but the recent cloning of animals from mature cells demonstrates the persistence of stem cell characteristics even in fully differential cells. 63 Also, recently, neuronal stem cells were shown to produce a variety of blood cell types 64 and adult human mesenchymal stem cells that are present in adult marrow were shown to have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. 65 Conversely, primitive hematopoietic stem cells can give rise to muscle cells. 68

In general, stem cell differentiation results in two types of changes: the expression of specialized, differentiation-specific gene products and a partial or complete restriction of the cell’s capacity for further proliferation. It then follows that another mechanism by which tumor cells might arise is through mutations that render a stem cell partly or wholly unable to differentiate.

Some cells, particularly in adults, are terminally differentiated. These cells are irreversibly blocked in their ability to proliferate, although they may perform specialized functions for a long period of time. Tumors of terminally differentiated cells are not found. Thus, tumors of mature muscle or nerve cells do not occur, although tumors of less differentiated myoblastic or neuronal stem cells do. Cell proliferation appears to be incompatible with the expression of a terminally differentiated program of gene expression. Thus, irreversible arrest of cell division and expression of the terminally differentiated phenotype are interdependent.

In many tissues, continuous proliferation is restricted to a subpopulation of cells, the stem cells, which undergo self-renewal, as well as differentiation, into cell types with a more restrictive proliferative potential. It then follows that mutations or conditions that interfere with the differentiation of stem cells will result in unbalanced proliferation and, thus, uncontrolled growth of the tissue. Mutations that drive proliferation are associated with an accumulation and overgrowth of less-differentiated cells in the tissue. A common feature of tumor cells is their failure to differentiate terminally under appropriate conditions either in vivo or in culture. 67– 69

Extracellular Factors That Control Differentiation

During embryogenesis and in a number of adult tissues, differentiation depends on external factors. These include insoluble factors such as ECM and both the proximity and type of neighboring cells as well as a growing list of soluble factors. In model systems, differentiation can be induced by a variety of biologic agents and drugs (Table 2.3.). Both the ECM and differentiation-promoting soluble factors may be produced in an autocrine or paracrine fashion.

Table 2.3. Induction of Differentiation in Culture.

Table 2.3

Induction of Differentiation in Culture.

Cell-cell and cell-ECM interactions are important for both the induction and maintenance of differentiation in several cell lineages. Although our understanding at a molecular level of insoluble factors is still incomplete, progress has been made in identifying key molecules and pathways through which these factors act. In the case of the ECM, specific cell surface receptors bind to particular components of the ECM. 70 It now appears that the binding of an ECM component to its cellular receptor activates an intracellular signal transduction pathway that is analogous to the signaling pathways that have been identified for polypeptide GFs and growth inhibitors. Tumor cells often lose their ability to sense the ECM or neighboring cells. 71

The soluble factors that regulate differentiation can be broadly classified into those that bind to cell surface receptors and those that freely cross the plasma membrane and bind to cytoplasmic or nuclear receptors. The first class includes molecules such as the fibroblast growth factors (FGFs) (TGF-𝛃 and TGF-𝛃) and hematopoietic factors such as colony-stimulating factor-1 (CSF-1), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Flt-3 Ligand, and the interleukins. These are all polypeptides, and many were first identified as GFs or growth inhibitors. It now is clear, however, that these factors have a multitude of effects depending on the target cells and the cellular microenvironment. 72

For example, basic FGF was identified as a fibroblast mitogen in brain and pituitary extracts, but recent data suggest that FGF induces mesodermal differentiation in early embryos, is angiogenic, and is a survival factor for endothelial cells. 73, 74 FGF also inhibits the differentiation of some cells. Terminal differentiation into mature myotubes cannot occur unless it is withdrawn from proliferating myoblasts. Similarly, TGF-𝛃 was first identified as a stimulator of anchorageindependent growth in mesenchymal cells and later as an inhibitor of epithelial cell proliferation. 75, 76 Like FGF, TGF-𝛃 stimulates the differentiation of some cells (i.e., keratinocytes or intestinal epithelial cells) but inhibits differentiation in others (i.e., myoblasts or preadipocytes). In some human tumor cells cultured in vitro or in athymic mice, TGF-𝛃 both inhibits tumor growth and promotes a more differentiated phenotype in the remaining cells. Other studies suggest that TGF-𝛃 induces the expression of one or more inhibitors of cyclin-dependent protein kinases (e.g., p21, p27, p16); inhibition of these kinases, in turn, prevents the phosphorylation and, thus, inactivation of the RB protein, thereby inhibiting cell proliferation. 77– 80

The membrane-permeable regulators of differentiation include retinoic acid (i.e., vitamin A) and its derivatives (RA). 81 There is strong evidence that concentration gradients of RA are critical for the morphogenesis of some tissues in the early embryo. 63 RA can stimulate or inhibit growth and differentiation depending on the cell type. In general, RA is required for the differentiation of many epithelial cells. It diffuses freely into cells, whereupon it binds to specific nuclear protein receptors (i.e., the retinoic acid receptors [RARs]). In addition, other nuclear proteins, called RAR coregulators, have been found that interact with RARs and modulate their actions in various cell types. 82 These differences may explain why specific cells and tissues differ in their responses to RA. Some differentiation-specific genes that are regulated by RA contain sequences specific to the initiation of transcription, 83 to which RA-RAR complexes bind and thereby activate transcription. 63, 84 The sex steroids, estrogen and testosterone, may regulate differentiation by similar mechanisms.

Tumor cells often produce factors that affect both growth and differentiation. Basic FGF can confer neoplastic properties when expressed in an inappropriate cell type (e.g., a fibroblast). In addition, inappropriate expression of FGF by one cell may stimulate the growth and affect the differentiation of neighboring cell types. 74

Intracellular Regulators

External factors and intrinsic programs of gene expression control cellular differentiation. In either case, the expression of differentiation-specific genes generally is under the control of a small number of master regulatory genes. Genes recently have been identified that are potential “master regulators” of developmental stages and differentiation-specific gene expression. The most globally acting master regulatory genes are known as homeotic genes, which were first identified as genetic loci that determined the developmental and spatial fates of cells in embryos of the fruit fly Drosophila. Similar genes have been identified in the genomes of higher organisms, including humans. Individual homeotic genes are expressed at different times during development and also are expressed in different adult tissues. Some homeotic genes code for extracellular factors, whereas others code for nuclear proteins that are probably transcriptional regulatory factors. Homeotic genes regulate programs of differentiation as opposed to individual differentiation-specific genes. They appear to act by initiating cascades of gene expression that involve regulatory genes having a more restricted range of actions. 85, 86 Some homeotic genes may function as tumor suppressors in normal tissues; others may promote tumorigenesis when mutated or deregulated. Mutations in homeotic genes may reactivate portions of an embryonic program of gene expression or suppress portions of an adult program of gene expression. 87– 89

DNA Methylation

In many cases, cells must go through one or more rounds of DNA replication before they can differentiate. This requirement may be because there often is a need to modify the pattern of DNA methylation before differentiation begins. DNA methylation in eukaryotes involves addition of a methyl group to the carbon/5 position of the cytosine ring. 90 Changes in DNA methylation commonly are introduced during DNA replication. The methylation of DNA on specific cytosine residues is believed to contribute to the changes in gene expression that occur during development. Presumably, DNA methylation affects gene expression because the transcriptional regulatory proteins that bind to methylated DNA differ from those that bind to unmethylated DNA. Many neoplastic tissues are hypomethylated relative to their normal counterparts, 91, 92 and, indeed, pharmacologic agents that alter the pattern of DNA methylation induce differentiation in a number of cultured cell lines. DNA methylation is probably not a universal mechanism for differentiation, however, and some cells can be induced to differentiate with either minimal or no change in cell cycle progression. 93

Differentiation and Cancer Therapy

Analysis of differentiation by tumor cells often provides valuable information for both the diagnosis and therapy of human cancers. As tumor cells grow and die, they can release glycoproteins and other products similar to those of fetal tissues, and these oncofetal products can be detected in serum or other body fluids to assist in diagnosis, follow-up, and selection of therapies. Examples include estrogen receptors and α-lactalbumin in breast cancer, prostate-specific antigen and prostatic acid phosphatase in prostate cancers, and myoglobin and desmin in sarcomas. 94

Elevations of these markers in the serum often predict relapse of the neoplasm before any sign by routine examination or radiographic tests. In general, the specificity of such markers for a given neoplasm is poor because minor elevations also occur with inflammatory and other benign conditions or with several types of neoplasms. In carcinoma of unknown primary site, tissue markers for neuroendocrine differentiation select for a subgroup of patients with improved response to chemotherapy. 95

Some tumor cells can be induced to differentiate terminally. This has been shown most extensively in cultured cell lines (see Table 2.3) but also in experimental animals. 80, 96, 97 After tumor cells have been induced to undergo terminal differentiation, their ability to grow as a tumor often is stably suppressed. In contrast to most anticancer drugs, which have nonspecific toxicity to both normal and cancer cells, drug-induced differentiation can be demonstrated with agents (or drug levels) that exert minimal effects on normal cells. 96 These observations have stimulated increased interest in clinical applications of differentiating agents to provide therapeutic gain with minimal toxicity. 139

A number of agents known to induce differentiation in various model systems have been used clinically (see Table 2.3). Some are useful only in a particular type of tumor; for example, estrogens and androgens have been useful in treating some breast, prostate, and gynecologic tumors, providing that tumor cells express the appropriate nuclear receptor. Other differentiation-inducing drugs have been more widely studied. For example, high doses of RA, hexamethylene bisacetamide (HMBA), or 5-azacytidine, which is an inhibitor of DNA methylation, can induce differentiation and inhibit the growth of several types of tumors in laboratory models. 97 HMBA was found to be active in patients with myelodysplastic syndromes, 98 and complete remission was achieved with direct evidence of terminal differentiation of leukemic cells to clonal granulocytes.

In fresh cultures of human promyelocytic leukemia, retinoid-induced differentiation, similar to the effects seen in passaged leukemia cell lines, has been observed. 99 All-trans-retinoic acid has been used in clinical therapy of promyelocytic leukemia with promising results, 100, 101 and retinoids have been used in combination with interferon-α to produce responses in patients with squamous cell cancer of the skin or cervix. 102 Because differentiating agents may inhibit tumor cell growth by multiple mechanisms, it is difficult to prove specific differentiating actions of these agents when used in patients. 101, 103 For example, retinoids not only induce differentiation in leukemia; they also downregulate anti-apoptotic genes. 104 An exception are studies in leukemias where sequential samples are readily available, differentiation markers are well established, and the clonality of differentiated cells can be ascertained by methods such as molecular cytogenetics (FISH). 99

Retinoids and other differentiating agents also are used in clinical trials to prevent cancer in patients with premalignant lesions or a high risk for developing cancer of the breast, cervix, colon, skin, lung, or oral cavity. Early results are encouraging, including the reversal of oral leukoplakia and prevention of second neoplasms in patients with treated squamous cell carcinoma of the head and neck. 103, 105 Greater knowledge about the molecular basis for the control of differentiation should lead to more accurate predictions, however, as well as rational design of therapies for controlling tumor growth by manipulating the state of differentiation. 97, 106, 107

Apoptosis

Programmed cell death (PCD), also termed apoptosis, is the necessary mechanism complementary to proliferation that ensures homeostasis of all tissues. It has been estimated that 50 to 70 billion cells perish each day in the average adult because of PCD,108 a process by which, in a year, each individual will produce and eradicate a mass of cells equal to its entire body weight. This process needs to be highly regulated since defects in the apoptotic machinery will lead to extended cell survival and may contribute to neoplastic cell expansion. Extended cell survival also creates a permissive environment for genetic instability and accumulation of mutations. Furthermore, defects in apoptotic pathways confer resistance to chemotherapy, radiation, and immune-mediated cell destruction.

Three major pathways have been elucidated so far, which all result in the activation of caspase-3, a cysteine proteinase that cleaves substrates after aspartic acid (asp) residues. One is the mitochondrial/cytochrome C pathway, largely mediated through Bcl-2 family members, which results in activation of Apaf-1, caspase-9, and then caspase-3 (Fig. 2.4). The second signals ligation of members of the TNF-receptor family (e.g., Fas, TRAIL receptors) and activates caspase-8 and subsequently caspase-3. Finally, granzyme B (a cytolytic T-cell product) directly cleaves and activates several caspases, resulting in apoptosis.

Figure 2.4. Mitochondrial (MC)/Bcl-2 (right) and death receptor pathways to apoptosis.

Figure 2.4

Mitochondrial (MC)/Bcl-2 (right) and death receptor pathways to apoptosis.

Apoptosis is a genetically determined process. Cell death during development of the nematode C. elegans involves the molecules CED-3 and CED-4, which are required for cell death, and CED-9, which protects cells from death. In mammals, CED-3 homologs constitute a family of cystein proteases with aspartate specificity, formerly called the ICE (interleukin-1alpha-converting enzyme) family and now designated caspases,109 which are the key effector proteins of apoptosis in mammalian cells.110 The discovery that human Bcl-2 has functional and structural similarity to CED-9 demonstrated that programed cell death in mammalian cells occurs by a highly conserved mechanism as apoptosis in the nematode. 111, 112

The PCD cascade can be divided into several stages (see Fig. 2.4). Multiple signaling pathways lead from death, triggering extracellular or intracellular agents to a central control and an execution stage. In this stage, the activation of CED-3/caspases occurs, which leads to the characteristic “apoptotic” structural lesions accompanying cell death: cytoplasmic and chromatin condensation and DNA fragmentation. Many environmental, pharmacologic, or physiologic stimuli can trigger apoptosis, a selection of which is listed in Table 2.4.

Table 2.4. Proteins Involved in the Regulation of Apoptosis.

Table 2.4

Proteins Involved in the Regulation of Apoptosis.

Central Role of Caspases in Apoptosis

Caspases are zymogens: they exist as inactive polypeptides that can be activated by removal of the regulatory prodomain and assembled into the active heteromeric protease. Currently, the caspase family consists of 13 members (see Table 2.4). They encompass a death domain (DD), a death effector domain (DED), and a caspase-recruitment domain (CARD). 113 The DD is present in members of the TNF receptor family and is involved in the early events of the signaling pathway. The DED and CARD are critical in the downstream portion of the pathways by recruiting caspases to the plasma membrane before their activation. Recent studies have shown that the apoptotic cascade triggered by cytochrome C and dATP is mediated by binding of caspase-9 to Apaf-1 through CARD/CARD interactions. 114 Caspase-9 becomes activated and, in turn, activates and cleaves caspase-3. NMR spectorscopy data provide evidence that basic/acidic surface polarity in the CARD domain is highly conserved and may represent a general mode for CARD/CARD interaction. 115 Caspase-9 deletion in knockout mice prevents activation of caspase-3 in embryonic brains in vivo, leading to perinatal death with a markedly enlarged and malformed cereburm. 116 Caspase-9-deficient thymocytes show resistance to dexamethasone but not to Fas-mediated apoptosis, implicating a functional diversification of caspase cascades, depending on the external stimulus.

Caspases can be grouped into three subfamilies based on their specificities. Group I, or ICE, subfamily of caspases (caspase-1, -4, and -5) prefer the tetrapeptide sequence WEHD and are believed to play a role mainly in inflammation, whereas members of group II (caspases-2, -3, and -7) and group III (caspases-6, -8, -9, and -10) display specificity for DEXD and (I/L/V)EXD, respectively, and are mainly involved in apoptosis. 117– 119 The finding that caspases-8 and -10 each contain two N-terminal located DEDs that enable them to associate with death receptors has placed these two caspases upstream in the apoptotic activation pathway. 120– 122 In turn, caspase-3 appears to be a downstream central executioner 123– 125 that can directly process pro-caspases-2, -6, -7, and -9. 126, 127 Findings by several groups have revealed that the activation of caspase-3 requires the Ced-4 homolog, Apaf-1, and pro-caspase-9, as well as dATP and cytochrome C. 128– 130 Hence, caspase-9 is upstream in the pathway and is regulated by Bcl-2 family genes. For murine caspase-11 and -12, no human counterparts have been described so far. Recently, the isolation of human caspase-13 (ERICE) from the ICE subfamily was reported. 131 The demonstration that activation of ERICE is mediated by caspase-8 has supported a potential downstream role for active ERICE in caspase-8–mediated cell death.

There is also evidence that cell death can proceed in the absence of caspases, perhaps through alterations in the mitochondrial membrane permeability transition (PT). 132, 133

Substrates of caspases

Activated caspases cleave numerous targets resulting in the so-called “death of a thousand cuts.” Caspase targets include cytoskeleton proteins (nuclear lamins, actin, gelsolin), regulators of DNA repair (poly ADP-ribose polymerase [PARP]), degradation of nuclear DNA by activation of DNA-dependent protein kinase, and deactivation of the inhibitor of caspase-activated deoxyribonuclease protein (ICAD) 134 ; RNA splicing (U1 protein), nuclear mitotic apparatus protein (NuMA), and cell cycle proteins (including Rb and p21-activated kinase [PAK]). 135 Caspases are also involved in extracellular apoptotic events including the cleavage of apoptotic bodies and exposure of phosphatidylserine on the cell surface. Caspase-dependent cleavage of p21 136 and Rb 137 during DNA-damage-induced apoptosis provides one of the potential links between apoptosis and cell cycle progression. Recent studies demonstrated that Bcl-2, Bcl-XL, and XIAP proteins are also substrates for caspase cleavage. 138– 140 Cleavage of these proteins releases a C-terminal product that lacks the BH4 domain and acts as a death effector. Hence, once caspase activation has been initiated, proteins that inhibit apoptosis are functionally inactivated and converted into peoapoptotic efectors. Since drug resistance is associated with an inability of tumor cells to undergo apoptosis, direct activation of caspases in cancer cells may be an effective strategy to kill resistant cells. One of the proposed approaches is to induce intracellular cleavage of caspase-1 or caspase-3 by a nontoxic, lipid-permeable, dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. 141 Using this chemically induced dimerization, it was possible to induce rapid apoptosis in a Bcl-XL-independent manner. Obviously, caspase activators should be selective for cancer cells. Whether these approaches can be realized is presently uncertain.

Natural Inhibitors of Caspases

The function of caspases, even after activation by cleavage, is subject to inhibition by other physiologic caspase inhibitors, thereby preventing unwanted or accidental proteolysis. Alterations in the expression or function of these proteins may confer resistance of tumor cells to the apoptotic stimuli. Viral proteins including CrmA (inhibitor of caspase-1 and -8) and p35 (which inhibits almost all caspases) were the first described caspase inhibitors. 142 The decoy protein, FLICE-inhibitory protein (FLIP), which prevents the binding of FLICE to its cofactor FADD, inhibits caspase-8 FADD-like interleukin-1B-converting enzyme (FLICE), which is required for its activation. The recent discovery of the CARD domain-containing protein ARC (apoptosis repressor with caspase recruitment domain) suggests the existence of decoy proteins for other caspases. A new family of proteins known as IAPs (for inhibitors of apoptosis proteins) was identified via homology with the baculovirus IAP genes and includes IAP1, IAP2, NAIP, XIAP, and survivin. 142– 147 Survivin seems to preferentially target caspase-9. XIAP, c-IAP1, and c-IAP2 prevent the proteolytic processing of pro-caspases-3, -6, and -7 by preventing the conformational changes of pro-caspase-9 required for downstream activation. 148, 149 Additionally, active caspase-3 function is directly inhibited by the binding of cleaved caspase-3 or -7 by XIAP, c-IAP1, c-IAP2, and survivin. These findings suggest that the ratio of caspases to IAPs is likely to be critical. However, since IAPs function by blocking caspase activation, they may not be able to prevent cell death induced by caspase-independent mechanisms.

Loss of IAP-related genes may cause cell death in mammalian cells and certain disorders, that is, NAIP mutations are observed in two-thirds of patients with spinal muscular atrophy 144 c-IAP2 and a novel gene MLT are rearranged in the t(11,18) 150 found in MALT lymphomas, potentially conferring a survival advantage to lymphoma cells and in some cases rendering them immune to Fas-induced apoptosis. 151

A new human gene survivin, has been described that encodes a structurally unique IAP apoptosis inhibitor that is undetectable in terminally differentiated adult tissues but prominently expressed in transformed cell lines and in all of the most common human cancers of lung, colon, pancreas, prostate, and breast and in high-grade non-Hodgkin’s lymphomas. 152 Survivin is the first apoptosis inhibitor that is selectively expressed in the G2-M cell cycle-phase and directly associates with mitotic spindle microtubules. 153 Inhibition of the survivin-tubulin interaction by microtubule-disrupting agents such as vincristine or nocodazole or mutagenesis of the caspase-binding BIR domain 154 results in increased caspase-3 activity and induction of apoptosis in G2-M-synchronized cells. Therefore, survivin appears to be a novel apoptotic guardian of a cell cycle checkpoint. High levels of survivin expression are associated with poor clinical outcome in neuroblastoma and colon and gastric cancers. 155– 157 Intriguingly, the coding strand of survivin is extensively complementary to that of effector cell protease receptor-1 (EPR-1), 158 although they are coded from separate genes located at 17q25. 159 The finding that downregulation of survivin by overexpression of ERP-1 in vitro increases apoptosis and inhibits growth of transformed cells has supported a potential role for endogenous ERP-1 as a natural antisense 160 and survivin as a potential new target for apoptosis-based therapy.

Death Receptors

Cells require both internal and external means of regulating the activation of caspases and the death machinery. Cell surface death receptors can, depending on other contextual events, transmit apoptosis signals in response to external stimuli such as death ligands, growth factor withdrawal, or chemotherapeutic agents. Death receptors belong to the tumor necrosis factor (TNF) receptor family and have a characteristic cysteine-rich extracellular domain 161 and a homologous cytoplasmic “death domain” 162 that initiates apoptotic signaling inside the cell. These receptors can induce apoptotic cell death within hours after ligand binding and may exert their apoptogenic effects differentially in diverse cell types, 163 depending on downstream signaling.

Fas/Fas Ligand

Fas ligand (FasL) is a type II membrane protein predominantly expressed in activated T cells. It is cleaved by a metalloproteinase to produce a soluble form. Recent data indicate that the membrane-bound form of FasL is functional, whereas shedding of soluble FasL inhibits cytotoxicity and may prevent the killing of healthy bystander cells by cytotoxic T cells. 164 Downregulation of Fas receptors and killing of activated T-lymphocytes through the constitutive expression of Fas-ligand on tumor cells has been suggested as a mechanism for pathologic suppression of immune surveillance. 165 Such “immune privilege” has been demonstrated in melanomas and colon cancers. 166, 167 Binding of FasL to Fas (CD95) or cross-linking Fas with agonistic antibodies results in receptor trimerization. 162, 168 Adapter proteins (FADD/MORT1 and RAIDD) bind to DD via their own DDs. 169– 172 A separate DED (of FADD/MORT1) binds to the prodomain of the caspase-8 (FLICE/MACH) and thereby links of the Fas death inducing signaling complex (DISC) with proteases 173, 174 and thereby apoptosis. Another pathway involves the Fas DD 175 binding protein Daxx, which, in turn, activates the c-Jun NH2-terminal kinase (JNK), the JNK kinase kinase ASK1 (apoptosis signal-regulating kinase 1), and Bcl-2; however, the importance of this pathway is uncertain. Observations from several studies 176– 178 suggest that a functional Fas pathway requires intact p53 and thus provide a potential mechanism for p53-mediated resistance of cancer cells to chemotherapy. A p53-binding sequence has been identified in the Fas promoter 179 and gene restoration therapy with p53 results in upregulation of Fas. 178

The CD95 system is an important regulator of T-cell cytotoxicity that is involved in the killing of mature T cells after immune response and killing of targets by cytotoxic T cells and natural killer cells. A frame shift mutation that renders cells resistant to Fas-mediated apoptosis has been found in adult T-cell leukemia. This finding has suggested that mutation of Fas gene may be one of the mechanisms in the progression of ATL. 180 Enthusiasm for the clinical use of Fas as a target is dampened by the observation that anti-Fas antibody induces rapid (within hours) death of mice from fulminant hepatic toxicity. 181, 182 Soluble Fas L may be less liver toxic but induces less apoptosis. This finding may explain why high soluble FasL levels found in many cancers are not associated with toxicity. 183 As a consequence, no trials in humans are underway.

Inhibitors of death receptor signaling

Downstream regulatory factors can suppress Fas/FasL death signaling. FLIP (for FLICE-inhibitory proteins) and several viral homologs v-FLIP 184 interact with the adapter protein FADD, inhibiting the interaction of FLICE with death receptors (CD95 death receptor 169 TRAMP 185– 187 and TRAIL-R) and thereby protecting cells against death-receptor-induced apoptosis. This finding may contribute to the oncogenicity of several FLIP-encoding viruses. The human cellular homologue, designated FLIP, 188 is predominantly expressed in muscle and lymphoid tissues. High levels of FLIP protein are also detectable in melanoma cell lines and in primary malignant melanomas but not in normal melanocytes, indicating that FLIP upregulation probably occurs during tumorigenesis. Downregulation of FLIP with actinomycin D correlates with acquisition of TRAIL sensitivity in resistant melanomas. 189 A novel cell-surface expressed gene toso appears to interfere with caspase-8 processing, 190 therefore inhibiting Fas-mediated apoptosis in T cells. The FAIM (Fas inhibitory molecule) isolated from B-cells 191 is another molecule that produces substantial, but not complete, resistance against Fas-mediated apoptosis. Fas was also recently implicated in the development of the multidrug resistance phenotype 192 involving the MDR1 gene product P-glycoprotein (P-gp).

TRAIL and Its Receptors

TRAIL (“TNF-related apoptosis inducing ligand” or APO2-L) is a molecule that binds to a different family of death-inducing receptors DR4, DR5. These receptors bind to and activate caspases through FLICE2 (FADD-like interleukin-1𝛃-converting enzyme2). Subsequently, nonsignaling decoy receptors (DcR1, DcR2) were identified in normal human tissues but not in most cancer cell lines examined. Their recognition of TRAIL may prevent TRAIL from binding to functional TRAIL receptors, therefore blocking and not transducing the cell death signal. At this point in time, the definitive role(s) of TRAIL in apoptosis remains to be determined since the presence of “protective” TRAIL receptors does not correspond to resistance or sensitivity to TRAIL-mediated apoptosis in some systems. 189 The fact that DR4 and DR5 are expressed in many tumors, whereas DcR1 and DcR2 are expressed predominantly in normal tissues, suggests that TRAIL could differentially induce apoptosis in tumor cells, but exceptions to this paradigm already exist.

TRAIL has been evaluated as a possible therapeutic agent and appears to have more promise than Fas. Distinguishing TRAIL from FasL is the observation that TRAIL seems to only induce apoptosis in malignant cell lines and not normal cell lines. In melanoma cell lines, TRAIL induces apoptosis. 193, 194 Recombinant soluble TRAIL induced significant apoptosis in myeloid and lymphoid cell lines and decreases in viability were observed in 20% of samples from patients with hematologic malignancies. 195 Among glioma cell lines, which preferentially express DR4 and 5, but not the decoy receptors, 10/12 cell lines were sensitive to TRAIL. 196 In breast cancer, TRAIL induced >90% apoptosis in only 1/8 cell lines. 197

A variety of factors may affect TRAIL sensitivity. There does not appear to be synergism with FasL, and neither ATRA nor MDR1 affects sensitivity. 195 P53 status also appears unrelated to TRAIL sensitivity; however, high Bcl-2 levels inhibit sensitivity. 196 Among sensitive melanoma cell lines, the levels of DR4/DR5 correlated with sensitivity in one study 193 but not in another. 194 Resistance to TRAIL was shown to be secondary to loss of cell surface expression secondary to either gene loss (4/9 lines) or because it was trapped in the cytoplasm. 198 Resistance has also been correlated with high levels of expression of FLIP, the TRAIL inhibitor, in resistant melanoma cell lines; 194 however, a correlation was not observed in all studies. Expression of the inhibitory receptor TRID was also reported. 197 Stimulation of cells with CD40-CD40L leads to downregulation of TRAIL and upregulation of TNF and Fas to promote B cell survival. 199 Combined, these data suggest that TRAIL is capable of inducing apoptosis in malignancies, including those of hematologic origin, but that multiple mechanisms of resistance likely affect sensitivity to TRAIL. Modification of the molecule by the introduction of a leucine zipper promotes multimerization. This modified molecule, LZ-TRAIL, increases killing of human breast cancer cell lines and mouse cell lines and confers a survival advantage to mice injected with the breast cancer cell line MDA-231. In an important distinction from FasL, no hepatotoxicity was observed. 200

Similar to many of the other apoptosis-inducing molecules discussed below, preclinical data suggest that the greatest efficacy with TRAIL may be in its combination with conventional chemotherapy.

Structure and Function of BCL-2-Related Proteins

The Bcl-2 family of proteins consists of both inhibitors and promoters of PCD. There are four important Bcl-2 structural homology motifs: BH1, BH2, and BH3, present in both the anti- and pro-survival subfamilies, and BH4, present only among antiapoptotic proteins. 201– 204 Most antiapoptotic proteins contain BH1 and BH2, and those closely resembling Bcl-2 contain all four domains. The proapoptotic proteins form two subfamilies. The Bax group includes Bax, Bak, and Bok (Mtd), resembles Bcl-2, and contains BH1, 2, and 3 domains. The BH3 domain group encompasses seven family members (Bik, Blk, Hrk, BNIP3, BimL, Bad, Bid, EGL-1) that possess only the BH3 domain, which is essential for their function.

Many Bcl-2-family proteins can associate with each other through a complex network of homo- and heterodimers 205 that depend on interactions between the BH1, BH2 or BH3 domains. 201, 206 Bcl-2 forms homodimers or heterodimers with Bax, Bcl-XL, Bcl-XS, Mcl-1, and BAD. 205– 208 The ratio of antiapoptotic versus proapoptotic dimers is important in determining resistance of a cell to apoptosis, but, in most cases, the functional significance of these interactions has not been explored. However, mutational analysis studies and concerns about the methods used to determine these interactions have raised questions about the requirement for direct protein-protein interactions between the anti- and proapoptotic BCL2 family members. 209– 211 Also, recent analyses of cells expressing various levels of Bcl-2 and Bax have revealed that the degree of protection against apoptosis correlates with the amount of Bcl-2 that is free of Bax, rather than the number of Bcl-2-Bax heterodimers. 212 Deletion mutants of Bcl-2 lacking the BH4 domain, which permits interaction with proteins, including Bag-1, Raf-1, calcineurin, p53-binding protein, and Nip1-3, exhibit either loss of function or dominant-inhibitory activity and thereby paradoxically promote apoptosis. 213– 215

Post-transcriptional modifications: Phosphorylation of BAD and Bcl-2.

Post-translational changes of Bcl-2 and BAD can affect protein-protein interactions and subsequent activity. Phosphorylation of BAD by PKA or Akt 216 results in decreased apoptosis. 217 Alternatively, dephosphorylation of BAD by the Ca2+-activated protein phosphatase calcineurin enhances BAD heterodimerization with Bcl-XL and promotes apoptosis. 218 The function of Bcl-2 also appears to be modulated by phosphorylation; however, the consequence of this effect remains controversial. Studies have demonstrated increased resistance to apoptosis when Bcl-2 is phosphorylated on serine 70 (by PKCα) in response to IL-3, erythropoietin, or bryostatin. 219– 222 Furthermore, enforced phosphorylation of Bcl-2 by bryostatin in low-Bcl-2-expressing REH cells induced a >10-fold increase in resistance to drug-induced cell death. 221 In contrast, other studies have suggested that phosphorylation results in the loss of function and proapoptotic ability. In this context, the administration of taxol or other microtubule-damaging drugs to leukemia, lymphoma, and breast and prostate cancer cell lines induces serine phosphorylation of Bcl-2 and cell death. 223– 226 The finding that the c-Jun N-terminal kinase may also be a Bcl-2 kinase 227 provides a hypothetical mechanism for the downstream events following JNK/SAPK pathway activation by several proapoptotic stimuli. 228, 229 Intriguingly, overexpression of the kinase ASK-1 or the Fas-binding protein Daxx leads to the activation of JNK/SAPK and apoptosis. 175, 230 These findings have supported a link between Fas-induced apoptosis and Bcl-2 checkpoint control. Since there are five serine and three threonine amino acids present in the loop domain of Bcl-2 where phosphorylation occurs, differences in the site of phosphorylation or the kinase acting on Bcl-2 may account for the diametrically opposed effects of Bcl-2 phosphorylation on apoptosis induction that have been reported. 231– 233

Other means of modulating the function of Bcl-2 have been observed. Cleavage of the loop domains of Bcl-2 and Bcl-XL by caspases 138, 234 following Fas triggering, growth factor withdrawal, alphavirus infection, and etoposide treatment 139, 235– 237 has been associated with degradation by the ubiquitin-dependent proteasome complex and loss of antiapoptotic potency. 238 Therefore, the ratio of cleaved versus uncleaved protein may be another mechanism for regulating the decision of cells to undergo apoptosis that could be exploited therapeutically. In addition, the ratio may affect the susceptibility of tumor cells to attempts to induce apoptosis. The demonstration that mimicking phosphorylation of certain Bcl-2 phosphorylation sites abolishes ubiquitin-dependent degradation and confers resistance against induction of apoptosis has supported the inability of the proteasome to degrade phosphorylated Bcl-2. 239

Mitochondria and Apoptosis

Mitochondria isolated from cells induced to undergo apoptosis can stimulate apoptosis-like destruction of naive nuclei, whereas mitochondria purified from Bcl-2 overexpressing cells fail to confer this effect. 240 Interestingly, chemical inducers of mitochondrial megapore opening can induce mitochondria derived from normal cells to liberate factors that result in the apoptosis-like destruction of nuclei. 240 Recently, this effect has been attributed to the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria and thereby activation of proteases (see Fig. 2.4). 241, 242 Two general mechanisms for release of caspase-activating proteins from mitochondria have been proposed: one involves an osmotic dysequilibrium that leads to an expansion of the matrix space, organelle swelling, rupture of the outer membrane, and eventually necrosis; the other envisions opening of channels in the outer membrane, release of cytochrome c into the cytosol, and activation of caspases resulting in apoptosis. 132 A key function of Bcl-2-like proteins is to somehow retain cytochrome c in the mitochondria. 243 Models using synthetic lipid membranes in vitro support this idea, but at present there is no direct evidence for in vivo channel formation or ion-channel activity of Bcl-2 or Bcl-XL. Also, it remains to be determined whether the suppression of cytochrome c release by Bcl-2 reflects the ability of Bcl-2 either to block Bax channels or to transport cytochrome c back to the mitochondria. 243, 244 However, the demonstration that Bcl-2 mutants that lack their C-terminal TM domain, and therefore inefficiently associate with mitochondria, retain partial antiapoptotic activity has suggested that membrane targeting of Bcl-2 is not absolutely critical for its function. 207

Although caspase activation usually occurs downstream of mitochondrial permeability transition, recent data indicate that caspases can also induce dissipation of the mitochondrial inner transmembrane potential and therefore act upstream of mitochondria. 245, 246 These observations suggest that caspases and mitochondria can engage in a circular self-amplification loop that could accelerate or coordinate the apoptotic response. Studies in yeast have provided compelling evidence that Bax-like proteins mediate caspase-independent death via channel-forming activity, which could promote the mitochondrial permeability transition or puncture the mitochondrial outer membrane. 132 Bax can form pores in artificial membranes in vitro. 247 In contrast, recombinant Bcl-2 or Bcl-XL inhibits opening of the purified megachannel reconstituted into liposomes as well as megachannel opening in cells and isolated mitochondria. 248 Bax triggers a rapid caspase-dependent apoptosis, but in the presence of caspase inhibitors, a slower nonapoptotic cell death without DNA fragmentation occurs. 249 Bax-induced cytochrome c release could result in disruption of electron transport with loss of ATP and generation of reactive oxygen species that cause caspase-independent cell death.

The proapoptotic protein Bid is cleaved by activated caspase-8 in Fas signaling pathway (see Fig. 2.4). 250 The finding that truncated Bid translocates from the cytosol to mitochondria and induces cytochrome c release and loss of membrane potential has provided a link between death-receptor-mediated and mitochondrial pathways. 133

In conclusion, mitochondrial damage is likely to be critical in controlling cell death, either by release of proteins that trigger caspase activation or by disruption of electron transport followed by slow, nonapoptotic cell death.

Apoptosis Activating Factors (Apafs)

Of the Bcl-2/Bcl-XL-binding proteins in C elegans, CED-4, which serves to recruit caspases via its N-terminus CARD 251 domain and activate them, 252 is probably the most important. 252– 258 Zou and colleagues have isolated apoptosis activating factors (Apafs) 259 that result in cleavage and activation of caspase-9. Apaf-1 resembles CED-4 with an amino-terminal CARD domain that can bind directly to caspases 251 and a large domain containing 12 WD-40 repeats at the C-terminus thought to interact with Apaf-2/cytochrome c. ATP may activate Apaf-1 by binding to the nucleotide-binding p-loop motif. An essential role for Apaf-1 in the Bcl-2-regulated pathway is supported by observations of reduced apoptosis in the brain and striking craniofacial abnormalities with impaired processing of caspases-2, -3 and -8 in Apaf-1 knockout mice. 260, 261 Remarkably, cells from Apaf-1 –/– mice are refractory to apoptotic stimuli controlled by Bcl-2 but respond normally to signals from “death receptors.”

In conclusion, CED-4 or human Apafs are likely to be critical for Bcl-2 function in regulation of the caspases.

Taken together, an intriguing network of genes controlling proliferation, differentiation, and apoptosis has evolved that provides new targets for cancer therapy that were unimaginable only a few years ago.

References

1.
Hartwell L H, Weinert T A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989;246:629–634. [PubMed: 2683079]
2.
Morgan D O. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261–291. [PubMed: 9442875]
3.
Polyak K, Lee M -H, Erdjument-Bromage H, Koff A, Roberts J M, Tempst P, Massague J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994;78:59–66. [PubMed: 8033212]
4.
Polyak K, Kato J Y, Solomon M J, Sherr C J, Massague J, Roberts J M, Koff p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994;8:9–22. [PubMed: 8288131]
5.
Koepp D M, Harper J W, Elledge S J. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell. 1999;97:431–434. [PubMed: 10338207]
6.
Zugmaier G, Lippman M E. Effects of TGF beta on normal and malignant mammary epithelium. Ann N Y Acad Sci. 1990;593:272–275. [PubMed: 2375597]
7.
Druker B J, Mamon H J, Roberts T M. Oncogenes, growth factors, and signal transduction. N Engl J Med. 1989;321:1383–1391. [PubMed: 2682241]
8.
Gibbs J B, Oliff A, Kohl N E. Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell. 1994;77:175–178. [PubMed: 8168127]
9.
Pardee A B. G1 events and regulation of cell proliferation. Science. 1989;246:603–608. [PubMed: 2683075]
10.
Tubo R A, Berezney R. Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration. J Biol Chem. 1987;262:1148–1154. [PubMed: 3027082]
11.
Challberg M D, Kelly T J. Animal virus DNA replication. Ann Rev Biochem. 1989;58:671–717. [PubMed: 2549858]
12.
Matsushime H, Roussel M F, Ashmun R A, Sherr C J. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell. 1991;65:701–713. [PubMed: 1827757]
13.
Goodrich D W, Wang N P, Qian Y W, Lee E Y, Lee W H. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell. 1991;67:293–302. [PubMed: 1655277]
14.
Matsushime H, Ewen M E, Strom D K. et al. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell. 1992;71:323–334. [PubMed: 1423597]
15.
Connell-Crowley L, Harper J W, Goodrich D W. Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell. 1997;8:287–301. [PMC free article: PMC276080] [PubMed: 9190208]
16.
Nevins J R, Leone G, DeGregori J, Jakoi L. Role of the Rb/E2F pathway in cell growth control [review] J Cell Physiol. 1997;173:233–236. [PubMed: 9365528]
17.
Wu C L, Zukerberg L R, Ngwu C, Harlow E, Lees J A. In vivo association of E2F and DP family proteins. Mol Cell Biol. 1995;15:2536–2546. [PMC free article: PMC230484] [PubMed: 7739537]
18.
Geng Y, Eaton E N, Picon M. et al. Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. Oncogene. 1996;12:1173–1180. [PubMed: 8649818]
19.
Hurford R K J Jr, Cobrinik D, Lee M H, Dyson N. pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. Genes Dev. 1997;11:1447–1463. [PubMed: 9192872]
20.
Ohtsubo M, Theodoras A M, Schumacher J, Roberts J M, Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995;15:2612–2624. [PMC free article: PMC230491] [PubMed: 7739542]
21.
Leng X, Connell-Crowley L, Goodrich D, Harper J W. S-Phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation. Curr Biol. 1997;7:709–712. [PubMed: 9285720]
22.
Lukas J, Herzinger T, Hansen K. et al. Cyclin E-induced S phase without activation of the pRb/E2F pathway. Genes Dev. 1997;11:1479–1492. [PubMed: 9192874]
23.
Amundson S A, Myers T G, Fornace A J, Jr Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress. Oncogene. 1998;17:3287–3299. [PubMed: 9916991]
24.
Hoekstra M F. Responses to DNA damage and regulation of cell cycle checkpoints by the ATM protein kinase family. Curr Opin Genet Dev. 1997;7:170–175. [PubMed: 9115420]
25.
Unger T, Juven-Gershon T, Moallem E. et al. Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2. EMBO J. 1999;18:1805–1814. [PMC free article: PMC1171266] [PubMed: 10202144]
26.
Sakaguchi K, Herrera J E, Saito S. et al. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev. 1998;12:2831–2841. [PMC free article: PMC317174] [PubMed: 9744860]
27.
Waterman M J, Stavridi E S, Waterman J L, Halazonetis T D. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nature Genet. 1998;19:175–178. [PubMed: 9620776]
28.
Banin S, Moyal L, Shieh S. et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 1998;281:1674–1677. [PubMed: 9733514]
29.
Khanna K K, Keating K E, Kozlov S. et al. ATM associates with and phosphorylates p53: mapping the region of interaction. Nature Genet. 1998;20:398–400. [PubMed: 9843217]
30.
de Lange T. Activation of telomerase in a human tumor. Proc Natl Acad Sci U S A. 1994;91:2882–2885. [PMC free article: PMC43476] [PubMed: 8159672]
31.
Buchkovich K J. Telomeres, telomerase, and the cell cycle. Prog Cell Cycle Res. 1996;2:187–195. [PubMed: 9552395]
32.
Saretzki G, Sitte N, Merkel U, Wurm R E, von Zglinicki T. Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments. Oncogene. 1999;18:5148–5158. [PubMed: 10498864]
33.
Fabrikant J I, Cherry J. The kinetics of cellular proliferation in normal and malignant tissues. V. Analysis of labeling indices and potential tissue doubling times in human tumor cell populations. J Surg Oncol. 1969;1:23–47. [PubMed: 5406356]
34.
Fukuda K, Iwasaka T, Hachisuga T. et al. Immunocytochemical detection of S-phase cells in normal and neoplastic cervical epithelium by anti-BrdU monoclonal antibody. Anal Quant Cytol Histol. 1990;12:135–138. [PubMed: 2350389]
35.
Teodori L, Trinca M L, Goehde W. et al. Cytokinetic investigation of lung tumors using the anti-bromodeoxyuridine (BUdR) monoclonal antibody method: comparison with DNA flow cytometric data. Int J Cancer. 1990;45:995–1001. [PubMed: 2161804]
36.
Carlsson M, Totterman T H, Matsson P, Nilsson K. Cell cycle progression of B-chronic lymphocytic leukemia cells induced to differentiate by TPA. Blood. 1988;71:415–421. [PubMed: 3257396]
37.
Tannock I. Cell kinetics and chemotherapy: a critical review. Cancer Treat Rep. 1978;62:1117–1133. [PubMed: 356975]
38.
Fingert HJ, Campisi J, Pardee AB. Molecular biology and biochemistry of cancer. In Gynecologic Oncology. Edited by R Knapp, R Berkowitz. New York: Macmillan, 1991: p 30.
39.
Weber G. Biochemical strategy of cancer cells and the design of chemotherapy: G. H. A. Clowes Memorial Lecture. Cancer Res. 1983;43:3466–3492. [PubMed: 6305486]
40.
Juliano R. Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation. Bioessays. 1996;18:911–917. [PubMed: 8939069]
41.
Liotta L A. Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial Award lecture. Cancer Res. 1986;46:1–7. [PubMed: 2998604]
42.
Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv Cancer Res. 1996;68:67–108. [PubMed: 8712071]
43.
Sherr C J. Cancer cell cycles. Science. 1996;274:1672–1677. [PubMed: 8939849]
44.
Motokura T, Bloom T, Kim H G. et al. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature. 1991;350:512–515. [PubMed: 1826542]
45.
Wang T C, Cardiff R D, Zukerberg L. et al. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature. 1994;369:669–671. [PubMed: 8208295]
46.
Kamb A. Cyclin-dependent kinase inhibitors and human cancer. Curr Top Microbiol Immunol. 1998;227:139–148. [PubMed: 9479829]
47.
Galaktionov K, Lee A K, Eckstein J. et al. CDC25 phosphatases as potential human oncogenes. Science. 1995;269:1575–1577. [PubMed: 7667636]
48.
Galaktionov K, Chen X, Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature. 1996;382:511–517. [PubMed: 8700224]
49.
Goodrich D W, Lee W H. The molecular genetics of retinoblastoma. Cancer Surveys. 1990;9:529–554. [PubMed: 2101724]
50.
Lee W -H. The molecular basis of cancer suppression by the retinoblastoma gene. Princess Takamatsu Symp. 1989;20:159–170. [PubMed: 2488231]
51.
Prives C, Hall P A. The p53 pathway. J Pathol. 1999;187:112–126. [PubMed: 10341712]
52.
Akashi M, Koeffler H P. Li-Fraumeni syndrome and the role of the p53 tumor suppressor gene in cancer susceptibility. Clin Obstet Gynecol. 1998;41:172–199. [PubMed: 9504235]
53.
Oliner J D, Kinzler K W, Meltzer P S, George D L, Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature. 1992;358:80–83. [PubMed: 1614537]
54.
Sharpless N E, DePinho R A. The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev. 1999;9:22–30. [PubMed: 10072356]
55.
Gurdon J B. Embryonic induction—molecular prospects. Development. 1987;99:285–306. [PubMed: 3308408]
56.
Jan Y N, Jan L Y. HLH proteins, fly neurogenesis, and vertebrate myogenesis. Cell. 1993;75:827–830. [PubMed: 8252617]
57.
Sorrentino V, Pepperkok R, Davis R L, Ansorge W, Philipson L. Cell proliferation inhibited by MyoD1 independently of myogenic differentiation. Nature. 1990;345:813–815. [PubMed: 2359457]
58.
Umek R M, Friedman A D, McKnight S L. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991;251:288–292. [PubMed: 1987644]
59.
Weidner N, Carroll P R, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401–409. [PMC free article: PMC1887042] [PubMed: 7688183]
60.
Weidner N, Semple J P, Welch W R, Folkman J. Tumor angiogenesis and metastasis —correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1–8. [PubMed: 1701519]
61.
Bernstein L R, Liotta L A. Molecular mediators of interactions with extracellular matrix components in metastasis and angiogenesis. Curr Opin Oncol. 1994;6:106–113. [PubMed: 7515692]
62.
Mintz B, Fleischman R A. Teratocarcinomas and other neoplasms as developmental defects in gene expression. Adv Cancer Res. 1981;34:211–278. [PubMed: 7025592]
63.
Dolle P, Ruberte E, Kastner P. et al. Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nature. 1989;342:702–705. [PubMed: 2556642]
64.
Bjornson C R, Rietze R L, Reynolds B A, Magli M C, Vescovi A L. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo [see comments] Science. 1999;283:534–537. [PubMed: 9915700]
65.
Pittenger M F, Mackay A M, Beck S C. et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–147. [PubMed: 10102814]
66.
Jackson K A, Mi T, Goodell M A. Hematopoietic potential of stem cells isolated from murine skeletal muscle [in process citation] Proc Natl Acad Sci U S A. 1999;96:14482–14486. [PMC free article: PMC24462] [PubMed: 10588731]
67.
Rheinwald J G, Beckett M A. Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes. Cell. 1980;22(2 Pt 2):629–632. [PubMed: 6160916]
68.
Wille J J Jr, Maercklein P B, Scott R E. Neoplastic transformation and defective control of cell proliferation and differentiation. Cancer Res. 1982;42:5139–5146. [PubMed: 6291749]
69.
Yuspa SH, Lichti U, Strickland J, et al. Aberrant regulation of differentiation in epidermal carcinogenesis. In Growth Factors, Tumor Promoters and Cancer Genes. Edited by NH Colburn, HL Moses, EJ Stanbridge. New York: Liss, 1988: pp 183–189.
70.
Ekblom P, Vestweber D, Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Biol. 1986;2:27–47. [PubMed: 3548769]
71.
Petersen O W, Ronnov-Jessen L, Howlett A R, Bissell M J. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells Proc Natl Acad Sci U S A 1992. 899064–9068. [published erratum appears in Proc Natl Acad Sci U S A 1993;90:2556]. [PMC free article: PMC50065] [PubMed: 1384042]
72.
Varticovski L, Druker B, Morrison D, Cantley L, Roberts T. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature. 1989;342:699–702. [PubMed: 2556641]
73.
Lemmon S K, Riley M C, Thomas K A. et al. Bovine fibroblast growth factor: comparison of brain and pituitary preparations. J Cell Biol. 1982;95:162–169. [PMC free article: PMC2112374] [PubMed: 6183268]
74.
Rifkin D B, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol. 1989;109:1–6. [PMC free article: PMC2115467] [PubMed: 2545723]
75.
Rozengurt E. Early signals in the mitogenic response. Science. 1986;234:161–166. [PubMed: 3018928]
76.
Todaro G J, De Larco J E, Fryling C, Johnson P A, Sporn M B. Transforming growth factors (TGFs): properties and possible mechanisms of action. J Supramol Struct Cell Biochem. 1981;15:287–301. [PubMed: 6267317]
77.
Moses H L, Yang E Y, Pietenpol J A. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell. 1990;63:245–247. [PubMed: 2208284]
78.
Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle [comment] Cell. 1994;79:181–184. [PubMed: 7954786]
79.
Sherr C J. G1 phase progression: cycling on cue [see comments] Cell. 1994;79:551–555. [PubMed: 7954821]
80.
Twardzik D R, Ranchalis J E, McPherson J M. et al. Inhibition and promotion of differentiated-like phenotype of a human lung carcinoma in athymic mice by natural and recombinant forms of transforming growth factor-beta. J Natl Cancer Inst. 1989;81:1182–1185. [PubMed: 2746671]
81.
Sporn MB, Roberts AB, Goodman DS. The Retinoids. New York: Academic, 1984: p 56.
82.
Glass C K, Devary O V, Rosenfeld M G. Multiple cell type-specific proteins differentially regulate target sequence recognition by the alpha retinoic acid receptor. Cell. 1990;63:729–738. [PubMed: 2171781]
83.
Bennington J L. Cellular kinetics of invasive squamous carcinoma of the human cervix. Cancer Res. 1969;29:1082–1088. [PubMed: 5781100]
84.
Vasios G W, Gold J D, Petkovich M, Chambon P, Gudas L J. A retinoic acid-responsive element is present in the 5’ flanking region of the laminin B1 gene. Proc Natl Acad Sci U S A. 1989;86:9099–9103. [PMC free article: PMC298441] [PubMed: 2556699]
85.
Gehring W J. Homeo boxes in the study of development. Science. 1987;236:1245–1252. [PubMed: 2884726]
86.
Holland P W, Hogan B L. Expression of homeo box genes during mouse development: a review. Genes Dev. 1988;2:773–782. [PubMed: 2905315]
87.
Castronovo V, Kusaka M, Chariot A, Gielen J, Sobel M. Homeobox genes: potential candidates for the transcriptional control of the transformed and invasive phenotype. Biochem Pharmacol. 1994;47:137–143. [PubMed: 7906121]
88.
Deschamps J, Meijlink F. Mammalian homeobox genes in normal development and neoplasia. Crit Rev Oncogenesis. 1992;3(1–2):117–173. [PubMed: 1372520]
89.
McCormick A, Campisi J. Cellular aging and senescence. Curr Opin Cell Biol. 1991;3:230–234. [PubMed: 1883615]
90.
Singal R, Ginder G D. DNA methylation. Blood. 1999;93:4059–4070. [PubMed: 10361102]
91.
Feinberg A P, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983;301:89–92. [PubMed: 6185846]
92.
Goelz S E, Vogelstein B, Hamilton S R, Feinberg A P. Hypomethylation of DNA from benign and malignant human colon neoplasms. Science. 1985;228:187–190. [PubMed: 2579435]
93.
Carlsson M, Totterman T H, Matsson P, Nilsson K. Cell cycle progression of B-chronic lymphocytic leukemia cells induced to differentiate by TPA. Blood. 1988;71:415–421. [PubMed: 3257396]
94.
Gorstein F, Thor A. Tumor markers in diagnostic pathology. In Clinics in Laboratory Medicine. Edited by F Gorstein, A Thor. Philadelphia: WB Saunders, 1990.
95.
Hainsworth J D, Johnson D H, Greco F A. Poorly differentiated neuroendocrine carcinoma of unknown primary site. A newly recognized clinicopathologic entity. Ann Intern Med. 1988;109:364–371. [PubMed: 2841895]
96.
Reiss M, Gamba-Vitalo C, Sartorelli A C. Induction of tumor cell differentiation as a therapeutic approach: preclinical models for hematopoietic and solid neoplasms. Cancer Treat Rep. 1986;70:201–218. [PubMed: 3510735]
97.
Waxman S, Rossi GB, Takaku F. The Status of Differentiation Therapy of Cancer. New York: Raven, 1988.
98.
Andreeff M, Stone R, Michaeli J. et al. Hexamethylene bisacetamide in myelodyplastic syndrome and acute myelogenous leukemia: A Phase II clinical trial with a differentiation-inducing agent. Blood. 1992;80:2604–2609. [PubMed: 1421378]
99.
Breitman T R, Collins S J, Keene B R. Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid. Blood. 1981;57:1000–1004. [PubMed: 6939451]
100.
Castaigne S, Chomienne C, Daniel M T. et al. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results [see comments] Blood. 1990;76:1704–1709. [PubMed: 2224119]
101.
Warrell R P Jr, Frankel S R, Miller W H Jr. et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid) N Engl J Med. 1991;324:1385–1393. [PubMed: 1850498]
102.
Chabner B A. Biological basis for cancer treatment [comment] Ann Intern Med. 1993;118:633–637. [PubMed: 8452330]
103.
Cheson B D, Jasperse D M, Chun H G, Friedman M A. Differentiating agents in the treatment of human malignancies. Cancer Treat Rev. 1986;13:129–145. [PubMed: 3536087]
104.
Andreeff M, Jiang S, Zhang X. et al. Expression of bcl-2-related genes in normal and AML progenitors: changes induced by chemotherapy and retionic acid. Leukemia. 1999;13:1881–1892. [PubMed: 10557066]
105.
Hong W K, Lippman S M, Itri L M. et al. Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck [see comments] N Engl J Med. 1990;323:795–801. [PubMed: 2202902]
106.
Meyskens F L Jr. Coming of age — the chemoprevention of cancer [editorial; comment] [see comments] N Engl J Med. 1990;323:825–827. [PubMed: 2202903]
107.
Rowley J D, Aster J C, Sklar J. The clinical applications of new DNA diagnostic technology on the management of cancer patients. JAMA. 1993;270:2331–2337. [PubMed: 8230596]
108.
Reed J C. Dysregulation of apoptosis in cancer. J Clin Oncol. 1999;17:2941–2953. [PubMed: 10561374]
109.
Alnemri E S, Livingston D J, Nicholson D W. et al. Humanf ICE?CED-3 protease nomenclature. Cell. 1996;87:171. [PubMed: 8861900]
110.
Yuan J, Shaham S, Ledoux S. et al. The c. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-convecting enzyme. Cell. 1993;75:641–652. [PubMed: 8242740]
111.
Hengartner M O, Horvitz H R. Programmed cell death in Caenorhabditis elegans. Curr Opin Genet Dev. 1994;4:581–586. [PubMed: 7950327]
112.
Vaux D L, Weissman I L, Kim S K. Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science. 1992;258:1955–1957. [PubMed: 1470921]
113.
Hofmann K, Bucher P, Tschopp J. The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci. 1997;22:155–156. [PubMed: 9175472]
114.
Li P, Nijhawan D, Budihardjo I. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479–489. [PubMed: 9390557]
115.
Chou J J, Matsuo H, Duan H, Wagner G. Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell. 1998;94:171–180. [PubMed: 9695946]
116.
Kuida K, Haydar T F, Kuan C Y. et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking Caspase 9. Cell. 1998;94:325–337. [PubMed: 9708735]
117.
Nicholson D W, Thornberry N A. Caspases: killer proteases. Trends Biochem Sci. 1997;22:299–306. [PubMed: 9270303]
118.
Talanian R V, Quinlan C, Trautz S. et al. Substrate specificities of caspase family proteases. J Biol Chem. 1997;272:9677–9682. [PubMed: 9092497]
119.
Thornberry N A, Rano T A, Peterson E P. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997;272:17907–17911. [PubMed: 9218414]
120.
Fernandes-Alnemri T, Armstrong R C, Krebs J. et al. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci. 1996;93:7464–7469. [PMC free article: PMC38767] [PubMed: 8755496]
121.
Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803–815. [PubMed: 8681376]
122.
Muzio M, Chinnaiyan A M, Kischkel F C. et al. FLICE, a novel FADDl-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:817–827. [PubMed: 8681377]
123.
Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 1997;16:2271–2281. [PMC free article: PMC1169829] [PubMed: 9171342]
124.
MacFarlane M, Cain K, Sun X M, Alnemri E S, Cohen G M. Processing/activation of at least four interleukin-1 beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. J Cell Biol. 1997;137:469–479. [PMC free article: PMC2139780] [PubMed: 9128256]
125.
Takahashi A, Hirata H, Yonehara S. et al. Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and Crm-A-sensitive caspase 8. Oncogene. 1997;14:2741–2752. [PubMed: 9190889]
126.
Srinivasula S M, Fernandes-Alnemri T, Zangrilli J. et al. The CED-3/interleukin-1 beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2 alpha are substrates for the apoptotic mediator CPP32. J Biol Chem. 1996;271:27099–27106. [PubMed: 8900201]
127.
Fernandes-Alnemri T, Takahashi A, Armstrong R. et al. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 1995;55:6045–6052. [PubMed: 8521391]
128.
Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic programin cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157. [PubMed: 8689682]
129.
Yang J, Liu X, Bhalla K. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked [see comments] Science. 1997;275:1129–1132. [PubMed: 9027314]
130.
Zou H, Henzel W J, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 [see comments] Cell. 1997;90:405–413. [PubMed: 9267021]
131.
Humke E W, Ni J, Dixit V M. ERICE, a novel FLICE-activatable caspase. J Biol Chem. 1998;273:15702–15707. [PubMed: 9624166]
132.
Green D R, Reed J C. Mitochondria and apoptosis. Science. 1998;281:1309–1312. [PubMed: 9721092]
133.
Green D R. Apoptotic pathways: the roads to ruin. Cell. 1998;94:695–698. [PubMed: 9753316]
134.
Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis [see comments] Nature. 1998;391:96–99. [PubMed: 9422513]
135.
Enari M, Sakahira H, Yokoyama H. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [see comments] Nature 1998. 39143–50. [published erratum appears in Nature 1998;393:396]. [PubMed: 9422506]
136.
Fujita N, Nagahashi A, Nagashima K, Rokudai S, Tsuruo T. Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases. Oncogene. 1998;17:1295–1304. [PubMed: 9771973]
137.
Zhang Y, Fujita N, Tsuruo T. Caspase-mediated cleavage of p21Waf1/Cip1 converts cancer cells from growth arrest to undergoing apoptosis. Oncogene. 1999;18:1131–1138. [PubMed: 10022118]
138.
Cheng E H, Kirsch D G, Clem R J. et al. Conversion of bcl-2 to a Bax-like death effector by caspases. Science. 1997;278:1966–1968. [PubMed: 9395403]
139.
Grandgirard D, Studer E, Monney L. et al. Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2. EMBO J. 1998;17:1268–1278. [PMC free article: PMC1170475] [PubMed: 9482724]
140.
Fattman C L, An B, Dou Q P. Characterization of interior cleavage of retinoblastoma protein in apoptosis. J Cell Biochem. 1997;67:399–408. [PubMed: 9361194]
141.
MacCorkle R A, Freeman K W, Spencer D M. Synthetic activation of caspases: artificial death switches. Proc Natl Acad Sci U S A. 1998;95:3655–3660. [PMC free article: PMC19891] [PubMed: 9520421]
142.
Clem R J, Miller L K. Control of programmed cell death by the baculovirus genes p35 and iap. Mol Cell Biol. 1994;14:5212–5222. [PMC free article: PMC359040] [PubMed: 8035800]
143.
Duckett C S, Nava V E, Gedrich R W. et al. A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 1996;15:2685–2694. [PMC free article: PMC450204] [PubMed: 8654366]
144.
Roy N, Mahadevan M S, McLean M. et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell. 1995;80:167–178. [PubMed: 7813013]
145.
Rothe M, Pan M G, Henzel W J, Ayres T M, Goeddel D V. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell. 1995;83:1243–1252. [PubMed: 8548810]
146.
Uren A G, Pakusch M, Hawkins C J, Puls K L, Vaux D L. Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors. PNAS. 1996;93:4974–4978. [PMC free article: PMC39390] [PubMed: 8643514]
147.
Hay B A, Wassarman D A, Rubin G M. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell. 1995;83:1253–1262. [PubMed: 8548811]
148.
Stennicke H R, Deveraux Q L, Humke E W. et al. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 1999;274:8359–8362. [PubMed: 10085063]
149.
Deveraux Q L, Roy N, Stennicke H R. et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 1998;17:2215–2223. [PMC free article: PMC1170566] [PubMed: 9545235]
150.
Dierlamm J, Baens M, Wlodarska I. et al. The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21)p6ssociated with mucosa-associated lymphoid tissue lymphomas. Blood. 1999;93:3601–3609. [PubMed: 10339464]
151.
Greiner A, Seeberger H, Knorr C, Starostik P, Muller-Hermelinck H K. MALT-type B-cell lymphomas escape the sensoring FAS-mediated apoptosis. Blood. 1998;92:484a.
152.
Ambrosini G, Adida C, Altieri D C. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3:917–921. [PubMed: 9256286]
153.
Li F, Ambrosini G, Chu E Y. et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998;396:580–584. [PubMed: 9859993]
154.
Tamm I, Wang Y, Sausville E, Scudiero D A, Vigna N, Oltersdorf T, Reed J C. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 1998;58:5315–5320. [PubMed: 9850056]
155.
Adida C, Berrebi D, Peuchmaur M. et al. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma [letter] Lancet. 1998;351:882–883. [PubMed: 9525374]
156.
Lu C D, Altieri D C, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998;58:1808–1812. [PubMed: 9581817]
157.
Kawasaki H, Altieri D C, Lu C D. et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998;58:5071–5074. [PubMed: 9823313]
158.
Altieri D C. Xa receptor EPR-1. FASEB J. 1995;9:860–865. [PubMed: 7615156]
159.
Ambrosini G, Adida C, Sirugo G, Altieri D C. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem. 1998;273:11177–11182. [PubMed: 9556606]
160.
Adida C, Crotty P L, McGrath J. et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998;152:43–49. [PMC free article: PMC1858132] [PubMed: 9422522]
161.
Smith C A, Farrah T, Goodwin R G. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell. 1994;76:959–962. [PubMed: 8137429]
162.
Nagata S. Apoptosis by death factor. Cell. 1997;88:355–365. [PubMed: 9039262]
163.
Consoli U, El-Tounsi I, Sandoval A. et al. Differential induction of apoptosis by fludarabine monophosphate in leukemic B and normal T cells in chronic lymphocytic leukemia. Blood. 1998;91:1742–1748. [PubMed: 9473241]
164.
Tanaka M, Itai T, Adachi M, Nagata S. Downregulation of Fas ligand by shedding [see comments] Nat Med. 1998;4:31–36. [PubMed: 9427603]
165.
Strand S, Hofmann W J, Hug H. et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells — a mechanism of immune evasion? Nature Med. 1996;2:1361–1366. [PubMed: 8946836]
166.
Hahne M, Rimoldi D, Schroter M. et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science. 1996;274:1363–1366. [PubMed: 8910274]
167.
O’Connell J, O’Sullivan G C, Collins J K, Shanahan F. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med. 1996;184:1075–1082. [PMC free article: PMC2192789] [PubMed: 9064324]
168.
Nagata S, Golstein P. The Fas death factor. Science. 1995;267:1449–1456. [PubMed: 7533326]
169.
Boldin M P, Varfolomeev E E, Pancer Z. et al. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem. 1995;270:7795–7798. [PubMed: 7536190]
170.
Chinnaiyan A M, O’Rourke K, Tewari M, Dixit V M. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995;81:505–512. [PubMed: 7538907]
171.
Chinnaiyan A M, Tepper C G, Seldin M F. et al. FADD/MORT1 is a common mediator of CD95 (Fas/APO1) and tumor necrosis factor recreptor-induced apoptosis. J Biol Chem. 1996;271:4961–4965. [PubMed: 8617770]
172.
Duan H, Dixit V M. RAIDD is a new ‘death’ adaptor molecule. Nature. 1997;385:86–89. [PubMed: 8985253]
173.
Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803–815. [PubMed: 8681376]
174.
Muzio M, Chinnaiyan A M, Kischkel F C. et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:817–827. [PubMed: 8681377]
175.
Yang X, Khosravi-Far R, Chang H Y, Baltimore D. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell. 1997;89:1067–1076. [PMC free article: PMC2989411] [PubMed: 9215629]
176.
Muller M, Wilder S, Bannasch D. et al. p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med. 1998;188:2033–2045. [PMC free article: PMC2212386] [PubMed: 9841917]
177.
Fukazawa T, Fujiwara T, Morimoto Y. et al. Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells. Oncogene. 1999;18:2189–2199. [PubMed: 10327065]
178.
Owen-Schaub L B, Zhang W, Cusack J C. et al. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol Cell Biol. 1995;15:3032–3040. [PMC free article: PMC230534] [PubMed: 7539102]
179.
Owen-Schaub L B, Radinsky R, Kruzel E, Berry K, Yonehara S. Anti-Fas on nonhematopoietic tumors: levels of Fas/APO-1 and bcl- 2 are not predictive of biological responsiveness. Cancer Res. 1994;54:1580–1586. [PubMed: 7511047]
180.
Maeda T, Yamada Y, Moriuchi R. et al. Fas gene mutation in the progression of adult T cell leukemia. J Exp Med. 1999;189:1063–1071. [PMC free article: PMC2193006] [PubMed: 10190897]
181.
Ogasawara J, Watanabe-Fukunaga R, Adachi M. et al. Lethal effect of the anti-fas antibody in mice. Nature. 1993;364:806–809. [PubMed: 7689176]
182.
Andreeff M, Hansen H, Cirrincione C. et al. Cellular RNA content: a major prognostic factor in adult acute leukemia and non-Hodgkin lymphoma. Proc Intl Conf Analyt Cytol. 1984;10:25.
183.
Schneider P, Holler N, Bodmer J L. et al. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med. 1998;187:1205–1213. [PMC free article: PMC2212219] [PubMed: 9547332]
184.
Senkevich T G, Bugert J J, Sisler J R. et al. Genome sequence of a human tumorigenic poxvirus: predication of specific host response-evasion genes. Science. 1996;273:813–816. [PubMed: 8670425]
185.
Bodmer J L, Burns K, Schneider P. et al. TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas (Apo-1/CD95) Immunity. 1997;6:79–88. [PubMed: 9052839]
186.
Kitson J, Raven T, Jiang Y P. et al. A death-domain-containing receptor that mediates apoptosis. Nature. 1996;384:372–375. [PubMed: 8934525]
187.
Chinnaiyan A M, O’Rourke K, Yu G L. et al. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science. 1996;274:990–992. [PubMed: 8875942]
188.
Irmler M, Thome M, Hahne M. et al. Inhibition of death receptor signals by cellular FLIP. Nature. 1997;388:190–195. [PubMed: 9217161]
189.
Griffith T S, Chin W A, Jackson G C, Lynch D H, Kubin M Z. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells [in process citation] J Immunol. 1998;161:2833–2840. [PubMed: 9743343]
190.
Hitoshi Y, Lorens J, Kitada S I. et al. Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells. Immunity. 1998;8:461–471. [PubMed: 9586636]
191.
Schneider T J, Fischer G M, Donohoe T J, Colarusso T P, Rothstein T L. A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes. J Exp Med. 1999;189:949–956. [PMC free article: PMC2193037] [PubMed: 10075978]
192.
Johnstone R W, Cretney E, Smyth M J. P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death. Blood. 1999;93:1075–1085. [PubMed: 9920858]
193.
Thomas W D, Hersey P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells. J Immunol. 1998;161:2195–2200. [PubMed: 9725211]
194.
Griffith T S, Chin W A, Jackson G C, Lynch D H, Kubin M Z. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol. 1998;161:2833–2840. [PubMed: 9743343]
195.
Snell V, Clodi K, Zhao S. et al. Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies. Br J Haematol. 1997;99:618–624. [PubMed: 9401075]
196.
Andreeff M, Wong G, Koziner B, Espiritu E, Clarkson B. Non B-Non T acute lymphoblastic leukemia (ALL): evidence for complete b cell differentiation of a quiescent subpopulation and their response to induction therapy. Proc Am Assoc Cancer Res. 1985;26:28.
197.
Keane M M, Ettenberg S A, Nau M M, Russell E K, Lipkowitz S. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res. 1999;59:734–741. [PubMed: 9973225]
198.
Golstein P. Cell death: trail and its receptors. Curr Biol. 1997;7:R750–R753. [PubMed: 9382834]
199.
Ribeiro P, Renard N, Warzocha K. et al. CD40 regulation of death domains containing receptors and their ligands on lymphoma B cells. Br J Haematol. 1998;103:684–689. [PubMed: 9858217]
200.
Walczak H, Miller R E, Ariail K. et al. Tumoricidal activity of tumor necrosis factor-related apoptosis- inducing ligand in vivo [see comments] Nat Med. 1999;5:157–163. [PubMed: 9930862]
201.
Yin X M, Oltvai Z N, Korsmeyer S J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature. 1994;369:321–323. [PubMed: 8183370]
202.
Chittenden T, Flemington C, Houghton A B. et al. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J. 1995;14:5589–5596. [PMC free article: PMC394673] [PubMed: 8521816]
203.
Zha H, Aime-Sempe C, Sato T, Reed J C. Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem. 1996;271:7440–7444. [PubMed: 8631771]
204.
Gibson L, Holmgreen S P, Huang D C. et al. Bcl-w, a novel member of the bcl-2 family, promotes cell survival. Oncogene. 1996;13:665–675. [PubMed: 8761287]
205.
Sato T, Irie S, Krajewski S, Reed J C. Cloning and sequencing of a cDNA encoding the rat Bcl-2 protein. Gene. 1994;140:291–292. [PubMed: 8144041]
206.
Sedlak T W, Oltvai Z N, Yang E. et al. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. PNAS. 1995;92:7834–7838. [PMC free article: PMC41240] [PubMed: 7644501]
207.
Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619. [PubMed: 8358790]
208.
Yang E, Zha J, Jockel J. et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995;80:285–291. [PubMed: 7834748]
209.
Hsu Y T, Wolter K G, Youle R J. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci U S A. 1997;94:3668–3672. [PMC free article: PMC20498] [PubMed: 9108035]
210.
Hsu Y T, Youle R J. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem. 1998;273:10777–10783. [PubMed: 9553144]
211.
Wang K, Gross A, Waksman G, Korsmeyer S J. Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing [in process citation] Mol Cell Biol. 1998;18:6083–6089. [PMC free article: PMC109194] [PubMed: 9742125]
212.
Otter I, Conus S, Ravn U. et al. The binding properties and biological activities of Bcl-2 and Bax in cells exposed to apoptotic stimuli. J Biol Chem. 1998;273:6110–6120. [PubMed: 9497329]
213.
Hanada M, Aime-Sempe C, Sato T, Reed J C. Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J Biol Chem. 1995;270:11962–11969. [PubMed: 7744846]
214.
Hunter J J, Bond B L, Parslow T G. Functional dissection of the human Bcl2 protein: sequence requirements for inhibition of apoptosis. Mol Cell Biol. 1996;16:877–883. [PMC free article: PMC231068] [PubMed: 8622689]
215.
Borner C, Martinou I, Mattmann C. et al. The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J Cell Biol. 1994;126:1059–1068. [PMC free article: PMC2120115] [PubMed: 8051205]
216.
del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997;278:687–689. [PubMed: 9381178]
217.
Zha J, Harada H, Yang E, Jockel J, Korsmeyer S J. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-X. Cell. 1996;87:619–628. [PubMed: 8929531]
218.
Alnemri E S, Robertson N M, Fernandes T F, Croce C M, Litwack G. Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells. Proc Natl Acad Sci U S A. 1992;89:7295–7299. [PMC free article: PMC49696] [PubMed: 1502141]
219.
Ito T, Deng X, Carr B, May W S. Bcl-2 phosphorylation required for anti-apoptosis function. J Biol Chem. 1997;272:11671–11673. [PubMed: 9115213]
220.
May W S, Tyler P G, Ito T. et al. Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis. J Biol Chem. 1994;269:26865–26870. [PubMed: 7929424]
221.
Ruvolo P P, Deng X, Carr B K, May W S. A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem. 1998;273:25436–25442. [PubMed: 9738012]
222.
Poommipanit P B, Chen B, Oltvai Z N. Interleukin-3 induces the phosphorylation of a distinct fraction of bcl-2. J Biol Chem. 1999;274:1033–1039. [PubMed: 9873048]
223.
Haldar S, Jena N, Croce C M. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A. 1995;92:4507–4511. [PMC free article: PMC41973] [PubMed: 7753834]
224.
Haldar S, Chintapalli J, Croce C M. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res. 1996;56:1253–1255. [PubMed: 8640809]
225.
Haldar S, Basu A, Croce C M. Bcl2 is the guardian of microtubule integrity. Cancer Res. 1997;57:229–233. [PubMed: 9000560]
226.
Blagosklonny M V, Schulte T, Nguyen P, Trepel J, Neckers L M. Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res. 1996;56:1851–1854. [PubMed: 8620503]
227.
Maundrell K, Antonsson B, Magnenat E. et al. Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J Biol Chem. 1997;272:25238–25242. [PubMed: 9312139]
228.
Chen Y R, Wang X, Templeton D, Davis R J, Tan T H. The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J Biol Chem. 1996;271:31929–31936. [PubMed: 8943238]
229.
Graves J D, Draves K E, Craxton A. et al. Involvement of stress-activated protein kinase and p38 mitogen- activated protein kinase in mIgM-induced apoptosis of human B lymphocytes. Proc Natl Acad Sci U S A. 1996;93:13814–13818. [PMC free article: PMC19435] [PubMed: 8943018]
230.
Ichijo H, Nishida E, Irie K. et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 1997;275:90–94. [PubMed: 8974401]
231.
May W S, Tyler P G, Ito T, Armstrong D K, Qatsha K A, Davidson N E. Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis. J Biol Chem. 1994;269:26865–26870. [PubMed: 7929424]
232.
Haldar S, Jena N, Croce C M. Inactivation of Bcl-2 by phosphorylation. PNAS. 1995;92:4507–4511. [PMC free article: PMC41973] [PubMed: 7753834]
233.
Chang B S, Minn A J, Muchmore S W, Fesik S W, Thompson C B. Identification of a novel regulatory domain in Bcl-XL and Bcl-2. EMBO J. 1997;16:968–977. [PMC free article: PMC1169697] [PubMed: 9118958]
234.
Fadeel B, Hassan Z, Hellstrom-Lindberg E. et al. Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells. Leukemia. 1999;13:719–728. [PubMed: 10374876]
235.
Wesselborg S, Engels I H, Rossmann E, Los M, Schulze-Osthoff K. Anticancer drugs induce caspase-8/FLICE activation and apoptosis in the absence of CD95 receptor/ligand interaction. Blood. 1999;93:3053–3063. [PubMed: 10216102]
236.
Landowski T H, Shain K H, Oshiro M M. et al. Myeloma cells selected for resistance to CD95-mediated apoptosis are not cross-resistant to cytotoxic drugs: evidence for independent mechanisms of caspase activation. Blood. 1999;94:265–274. [PubMed: 10381522]
237.
Eischen C M, Kottke T J, Martins L M. et al. Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. Blood. 1997;90:935–943. [PubMed: 9242521]
238.
Clem R J, Cheng E H, Karp C L. et al. Modulation of cell death by Bcl-XL through caspase interaction. PNAS. 1998;95:554–559. [PMC free article: PMC18458] [PubMed: 9435230]
239.
Dimmeler S, Breitschopf K, Haendeler J, Zeiher A M. Dephosphorylation targets Bcl-2 for ubiquitin-dependent degradation: a link between the apoptosome and the proteasome pathway. J Exp Med. 1999;189:1815–1822. [PMC free article: PMC2193081] [PubMed: 10359585]
240.
Zamzami N, Susin S A, Marchetti P. et al. Mitochondrial control of nuclear apoptosis (see comments) J Exp Med. 1996;183:1533–1544. [PMC free article: PMC2192517] [PubMed: 8666911]
241.
Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157. [PubMed: 8689682]
242.
Susin S A, Zamzami N, Castedo M. et al. Bcl-2 inhibits the mitochondrial relapse of an apoptogenic protease. J Exp Med. 1996;184:1331–1341. [PMC free article: PMC2192812] [PubMed: 8879205]
243.
Kluck R M, Bossy-Wetzel E, Green D R, Newmeyer D D. The release of cytochrome c from mitochondria: a primary site for bcl-2 regulation of apoptosis. Science. 1997;275:1132–1136. [PubMed: 9027315]
244.
Yang J, Liu X, Bhalla K. et al. Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129–1132. [PubMed: 9027314]
245.
Marzo I, Brenner C, Zamzami N. et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med. 1998;187:1261–1271. [PMC free article: PMC2212234] [PubMed: 9547337]
246.
Marzo I, Susin S A, Petit P X. et al. Caspases disrupt mitochondrial membrane barrier function. FEBS Lett. 1998;427:198–202. [PubMed: 9607311]
247.
Pan G, O’Rourke K, Dixit V M. Caspase-9, Bcl-XL, and Apaf-1 form a ternary complex. J Biol Chem. 1998;273:5841–5845. [PubMed: 9488720]
248.
Yang J C, Cortopassi G A. dATP causes specific release of cytochrome C from mitochondria [in process citation] Biochem Biophys Res Commun. 1998;250:454–457. [PubMed: 9753651]
249.
Xiang J, Chao D T, Korsmeyer S J. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci U S A. 1996;93:14559–14563. [PMC free article: PMC26172] [PubMed: 8962091]
250.
Li H, Zhu H, Xu C J, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998;94:491–501. [PubMed: 9727492]
251.
Hofmann K, Bucher P, Tschopp J. The CARD domain: a new apoptotic signaling motif. Trends Biochem Sci. 1997;22:155–156. [PubMed: 9175472]
252.
Chinnaiyan A M, O’Rourke K, Lane B R, Dixit V M. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science. 1997;275:1122–1126. [PubMed: 9027312]
253.
Shaham S, Horvitz H R. Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities. Genes Dev. 1996;10:578–591. [PubMed: 8598288]
254.
Shaham S, Horvitz H R. An alternatively spliced C. elegans ced-4 RNA encodes a novel cell death inhibitor. Cell. 1996;86:201–208. [PubMed: 8706125]
255.
Irmler M, Hofmann K, Vaux D, Tschopp J. Direct physical interaction between the Caenorhabditis elegans ‘death proteins’ CED-3 and CED-4. FEBS Lett. 1997;406(1–2):189–190. [PubMed: 9109415]
256.
Spector M S, Desnoyers S, Hoeppner D J, Hengartner M O. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature. 1997;385:653–656. [PubMed: 9024666]
257.
Wu D, Wallen H D, Nunez G. Interaction and regulation of subcellular localization of CED-4 by CED-9. Science. 1997;275:1126–1129. [PubMed: 9027313]
258.
Hu Y, Benedict M A, Wu D, Inohara N, Nunez G. Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. PNAS. 1998;95:4386–4391. [PMC free article: PMC22498] [PubMed: 9539746]
259.
Zou H, Henzel W J, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997;90:405–413. [PubMed: 9267021]
260.
Yoshida H, Kong Y Y, Yoshida R. et al. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell. 1998;94:739–750. [PubMed: 9753321]
261.
Cecconi F, Alvarez-Bolado G, Meyer B I, Roth K A, Gruss P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell. 1998;94:727–737. [PubMed: 9753320]
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