Apoptosis in Normal Development and Cancer

The Role of Apoptosis in Normal and Abnormal Embryonic Development Principles of abnormal development. Past, present . Germ cell cancer of the testis.
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In the nematode Caenorhabditis elegans the key event in programmed cell death is activation of a protease termed CED-3 and cleavage of its substrates 13 , No programmed cell death occurs in worms with mutations to ced Mammals have about a dozen homologs of CED-3 that are collectively termed caspases 15 , While some believe that caspase activation will also turn out to be the key essential event in apoptosis in mammalian cells, the majority of researchers currently believe that in most cases of apoptosis the key event is mitochondrial failure due to loss of cytochrome c from the mitochondrial intermembrane space 17 , During programmed cell death in C.

The mammalian protein caspase 9 most closely resembles CED-3 and caspase 9 can be activated by Apaf-1, an adaptor protein that resembles CED-4, the worm adaptor required for activation of CED-3 19 — In mammalian cells Apaf-1 is activated by cytochrome c after it is released from the mitochondria Caspase 9 can cleave and activate other caspases such as caspase 3, which cleaves many proteins within the cell, including ICAD, an inhibitor of an endonuclease CAD that cleaves the cell's DNA 24 , which can be detected as the classical ladder pattern by electrophoresis 25 , Although Apaf-1 resembles C.

Experiments in cell lines derived from mice lacking the gene for Apaf-1 or caspase-9 show that although they are needed for rapid exhibition of the apoptotic phenotype, they do not affect clonagenic potential of factor-starved or chemotherapy-treated cells 7. Thus, even though lack of Apaf-1 and caspase-9 delayed appearance of the apoptotic phenotype in cell death caused by retinoblastoma protein or cytotoxic drugs 30 , it did not necessarily affect the number of cells that ultimately died.

Although activation of these caspases can cause secondary damage to the mitochondria, in most cells they can cause cell death independently of the mitochondria 33 , Mammals have many CED-9 like proteins, known collectively as the Bcl-2 family, but none of these proteins interacts directly with Apaf-1, the mammalian protein most similar to CED-4 While there is general agreement that Bcl-2 family members, including Bcl-2 itself, Mcl-1, Bcl-x and Bcl-w, can inhibit apoptosis, provide clonagenic protection and act upstream of the mitochondria, exactly how they work is not known Gene deletion experiments provide evidence that the apoptosis pathways that can be inhibited by Bcl-2 require either Bax or Bak, two pro-apoptotic Bcl-2 family members, to function Caenorhabditis elegans does not have any pro-apoptotic Bcl-2 family members, so provides few clues as to how they function.

One model is that when an apoptotic signal is received, Bax and Bak aggregate on the mitochondrial outer membrane and form channels that allow cytochrome c to escape These are pro-apoptotic proteins. The BH3-only proteins are controlled by transcription, phosphorylation, sequestration and cleavage. For example, the tumor suppressor gene p53 induces apoptosis by transcriptionally activating the gene for PUMA, which can bind to and antagonize anti-apoptotic Bcl-2 family members 41 , Bmf and Bim are kept inactive in healthy cells by sequestration on microtubules and myosin, respectively 43 , Bid is activated following cleavage by caspase 8 45 and Bad is regulated by phosphorylation In addition to proteins such as Bcl-2 that inhibit cell death upstream of the mitochondria and prior to activation of the caspases, there is another family of proteins termed IAPs that act after caspases become activated by binding to them and preventing them from cleaving their substrates Mammals also have a number of IAP-binding proteins that have similar N-termini, but unlike the Drosophila IAP antagonists, which are cytoplasmic, most of the mammalian ones reside in the mitochondria in healthy cells Experiments using recombinant proteins have shown that inhibition of caspases by IAPs can be relieved by addition of these IAP antagonists in vitro.

The evidence that inhibition of cell death can lead to cancer comes mainly through accidents of nature, such as translocations in lymphomas and leukemias. For some cell death genes knockout and transgenic mice have provided evidence confirming that failure of cell death can cause cancer. Although there have been a very large number of studies determining the levels of expression of genes for cell death inhibitors in various types of cancer, these studies only provide correlative evidence.

The most common cancer of the blood cells in humans is the B cell neoplasia follicular lymphoma.


  1. Apoptosis: A Review of Programmed Cell Death?
  2. The Role of Apoptosis in Normal and Abnormal Embryonic Development.
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  4. Apoptosis in the development and treatment of cancer | Carcinogenesis | Oxford Academic;
  5. The Role of Apoptosis in Normal and Abnormal Embryonic Development.
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The bcl-2 gene was identified because it lies at the breakpoint of the t 14;18 translocation that is found in most cases. Experiments in vitro showed that Bcl-2 can prevent apoptosis of cells starved of cytokine and revealed it to be the first oncogene that acts by inhibiting cell death rather than by stimulating cell proliferation 3.

Expression of Bcl-2 in transgenic mice confirmed that inhibition of apoptosis can lead to cancer, as these mice develop B cell lymphomas and leukemias 6 , 52 — However, although Bcl-2 is a very potent inhibitor of apoptosis, bcl-2 transgenic mice only develop cancers when they are very old. These results suggest that inhibition of cell death is only very weakly oncogenic or, put the other way, apoptosis of potential cancer-forming cells is not a potent suppressor of tumorigenesis, at least in mice.


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    • Although on its own expression of a bcl-2 transgene in the lymphoid compartment of transgenic mice did not lead to the rapid development of tumors, when combined with a c- myc transgene leukemias developed extremely rapidly, much more rapidly than in mice bearing either transgene alone 6. This potent synergy between a growth-inducing oncogene and a cell death inhibitor may indicate that apoptosis is important in preventing survival of cells that already have activating mutations in growth-promoting oncogenes.

    These results imply that cells can detect disturbances caused by an activated oncogene and engage the apoptotic mechanism. Blocking apoptosis by overexpression of Bcl-2 or loss of a component of the signal transduction pathway such as p53 that connects the damage sensors to the cell death mechanism would greatly facilitate development of malignancy. Humans heterozygous for loss of function mutations to p53 Li—Fraumeni syndrome and mice with one or both alleles of p53 deleted develop cancers at an early age in many different tissues 55 , Whether induction of apoptosis or cell cycle arrest is important for the tumor suppressor activity of p53 is unclear.

    Although lymphoid cells in bcl-2 transgenic mice are just as resistant to apoptosis due to ionizing radiation and DNA mutagens as those from p53 mutant mice 59 , the bcl-2 transgenics have a far lower incidence of lymphoma Similarly, mice lacking genes for PUMA or both Bax and Bak do not develop cancer with anything approaching the frequency seen in p53 null mice, even though p53 requires PUMA to induce apoptosis in response to DNA damage and all pmediated apoptosis is thought to require Bax and or Bak.

    This implies that the ability of p53 to induce apoptosis is relatively unimportant for its tumor suppressor activity. On the other hand, the incidence of tumors is also not markedly elevated in p21 deleted mice 60 , so this activity of p53 also seems to be relatively unimportant. These findings suggest that there may be an additional activity of p53 that accounts for its ability to act as a tumor suppressor. Recently it has been suggested that p53 may also act independently of transcriptional regulation to directly bind to and inhibit Bcl-x at the mitochondria, leading to apoptosis Because the gene for PUMA must be induced by p53 following DNA damage and PUMA is required for pmediated apoptosis 42 , this alternative activity of p53 seems unlikely to represent a major aspect of the pro-apoptotic activity of p53 and, therefore, would not be important for pmediated tumor suppression.

    Experiments studying established tumors expressing c-myc or studying the transformation of cells from mice engineered to overexpress myc give support to the idea that the ability of p53 to cause apoptosis may be important to prevent progression of a tumor after the initial oncogenic event Thus, non-apoptotic activities of p53, such as causing cell cycle arrest, might be necessary to prevent the first oncogenic mutation, whereas its ability to induce apoptosis might allow it to retard subsequent transforming events.

    This translocation results in production of a fusion protein with an N-terminal half containing the BIR domains of cIAP2 and the C-terminal half containing the protease-like parts of paracaspase Translocations resulting in increased expression of Bcl10 are also associated with MALT lymphoma 67 , For almost all of the pro- and anti-apoptotic proteins there have been studies demonstrating correlations between their expression and various types of cancer.

    For example, in addition to the follicular lymphomas that bear bcl-2 translocations, elevated Bcl-2 expression has been associated with progression in melanoma 69 and both it and Mcl-1 have been observed in some myelomas 70 , The problem with these observations is that detection of a protein in cancer cells does not prove that it was involved in causing the cancer or is required for its persistence. The protein Survivin, which was wrongly assumed to be a cell death inhibitor because it bears a BIR domain, is required for chromosome segregation during cell division 77 , That Survivin can be detected in many cancers but few normal tissues may simply reflect the fact that some cells in all cancers are dividing, whereas most cells in normal adult tissues rest in G 0 Nevertheless, because cells cannot divide without Survivin, it might turn out to be an excellent target for novel cancer chemotherapeutics: One can have much more confidence that an inhibitor of apoptosis can act as an oncogene or that a promoter of cell death can act as a tumor suppressor based on findings obtained from transgenic and knockout mouse studies.

    If transgenic mice made to overexpress an apoptosis inhibitor have an increased incidence of cancer, the oncogenic potential of such a gene can be verified. Increased cancer incidence in mice with a targeted deletion of a gene suspected to be a promoter of cell death confirms the ability of such a gene to act as a tumor suppressor. First, clinical studies typically examine single alterations e.

    This makes it virtually impossible to determine negative results. It is possible that extracellular survival factors—influenced by cell density or microenvironment—can affect drug-induced death Anticancer agents induce apoptosis in normal tissues as well as in tumors. In fact, many of the pathologists who identified apoptosis in tumors realized that apoptotic cell death was induced in a subset of normal tissues e.

    Studies using mouse models provide strong support for this idea.

    For example, moderate doses of radiation and chemotherapy induce apoptosis in the murine thymus, spleen, bone marrow and intestine, the same tissues that account for the deleterious side-effects of chemotherapy. Similarly, ectopic expression of Bcl-2 in bone marrow cells achieves a similar effect Together, these studies strongly suggest that drug-induced apoptosis causes loss of normal cells and contributes to the side effects of cancer therapy. The studies described above highlight the fact that disruption of apoptosis can promote tumor initiation, progression and treatment resistance.

    Apoptosis in Normal Development and Cancer - CRC Press Book

    Indeed, it is remarkable that the same genetic alterations that influence apoptosis during tumorigenesis also modulate treatment sensitivity. For example, c-Myc enhances apoptosis in low concentrations of survival factors or oxygen and following treatment with diverse cytotoxic agents 38 , 82 , , conversely, loss of p53 and overexpression of Bcl-2 suppress apoptosis induced by oncogenes, depletion of survival factors, hypoxia and cytotoxic drugs 17 , , As a result, anti-apoptotic mutations arising during the course of tumor development can simultaneously select for chemoresistant cells.

    This pattern of co-selection may explain the phenomenon of de novo drug resistance, i. Studies on c-Myc-induced lymphomagenesis support directly link disruption of apoptosis during tumor development to de novo resistance Perhaps this explains why many advanced solid tumors are inherently difficult to treat. In addition to cell death, other programmed responses contribute to the deletion of potentially cancerous cells.

    Senescence is an irreversible program of cell-cycle arrest that is disrupted in many tumors or tumor-derived lines Replicative senescence was originally defined by the observation that primary cells have a genetically determined limit to their proliferative potential in cell culture, after which they permanently arrest with characteristic features.

    However, other stimuli can induce phenotypes suggestive of senescence, including mitogenic oncogenes and ionizing radiation — These observations imply that cellular senescence can be induced by diverse stimuli leading to the engagement of a common cell-cycle arrest program. In this view, senescence is conceptually similar to apoptosis, which is induced by diverse stimuli leading to the engagement of a common cell death program.

    Consequently, the biological roles of cellular senescence may go beyond the control of cellular or organismal aging, and reflect a global anti-proliferative response to a variety of cellular stresses. Recent evidence suggests that anticancer agents induce cellular senescence in human tumor-derived lines treated in culture or as xenographs , Since apoptotic programs can be manipulated to produce massive changes in cell death, the genes and proteins controlling apoptosis are potential drug targets.

    As indicated above, many empirically derived cytotoxic drugs already may target apoptosis, albeit indirectly and non-exclusively. They are also mutagenic and toxic to normal tissues. In contrast, agents that directly induce apoptosis may provide less opportunity for acquired drug resistance, decrease mutagenesis and reduce toxicity.

    Two observations suggest that such strategies are feasible. First, most anti-apoptotic mutations act relatively upstream in the program e.

    Apoptosis in Normal Development and Cancer

    Secondly, tumor-specific alterations in apoptotic programs provide opportunities to target cell death in a selective manner. Several current strategies are discussed below. In instances where apoptosis is disabled by dominant oncogenes, agents that disrupt their anti-apoptotic function can produce remarkable increases in cell death. Overexpression of anti-apoptotic Bcl-2 family members can promote tumorigenesis and chemoresistance, suggesting that functional inhibition of these proteins might be lethal to cancer cells.

    Adenovirus-mediated gene transfer of Bcl-x S , a dominant-negative repressor of Bcl-2 and Bcl-x L , can synergize with chemotherapy and promote tumor regression in xenographs , , but produces minimal apoptosis when introduced into normal epithelial cells Some current anticancer agents may unwittingly target the Bcl-2 family; for example, taxanes may induce phosphorylation and inactivation of Bcl-2 As more is learned about Bcl-2 structure and function, small molecule inhibitors of Bcl-2 action might become feasible.

    Hyperactivation of cell survival signaling may accompany tumor development, and these pathways are particularly exciting targets for small molecule inhibition. In principle, small molecule inhibition of any of these molecules might restore apoptosis to tumor cells, or synergize with more classic agents to induce cell death Oncogenic ras mutations deregulate normal growth control but can also signal cell survival Therefore, agents that interfere with Ras function might be cytostatic or cytotoxic.

    To be biologically active, Ras must be modified by a farnysltransferase, and many groups have developed farnysltransferase inhibitors as anti-tumor agents. Although these agents were predicted to be cytostatic, they can induce massive apoptosis and tumor regression of mammary carcinomas arising in ras transgenic mice In addition, inhibition of Ras-GAP induces apoptosis specifically in tumor, but not in normal cells, suggesting Ras-GAP as a novel target for cancer therapy Why would these agents induce apoptosis and, moreover, why would they be selective?

    The answer is unknown, but it is possible that tumor cells become dependent on survival signaling through the PI-3 kinase pathway, which could be suppressed by Ras inhibition. In circumstances where apoptosis is lost by a recessive mutation, restoring the dysfunctional gene or activity can promote massive cell death. For example, reintroduction of p53 into p53 mutant tumor cells can directly induce apoptosis or enhance treatment sensitivity in tumor cell lines or in xenographs , Indeed, strategies using this approach are currently in clinical trials As a general rule, tumor cells are inherently more sensitive to p53 inhibition than normal cells, perhaps because mitogenic oncogenes can activate p53 to promote apoptosis Hence, there is some rationale for selectivity of this approach.

    Of note, strategies to counter recessive anti-apoptotic mutations need not rely on gene or protein therapy. In some instances, inactivation of a pro-apoptotic gene might actually promote cell survival by relieving inhibition of a downstream death suppressor e. PTEN loss de-represses Akt. In such instances, the downstream effector may be a more suitable drug target Although mutations in death receptors are rare events in human tumors, changes accompanying tumorigenesis can alter the regulation of these pathways. Through an unknown mechanism, the expression of the decoy receptor 1 seems to be widely lost on tumor cells, making them exquisitely susceptible to TRAIL-mediated cell death.

    Because DR5 is a presponsive gene, combination therapy with TRAIL and classic cytotoxic agents may be particularly effective in treating tumors with functional p53 This protein is responsible for the cytopathic effects of CAV and induces apoptosis in tumor cells but not normal cells Although its mechanism of action remains unknown, apoptin-induced apoptosis is independent of p53 and is enhanced by Bcl-2 , At the very least, the remarkable effects of apoptin underscore the point that selective induction of apoptosis in tumor cells is achievable.

    Basic studies suggest that it is possible to directly harness the pro-apoptotic forces produced by certain oncogenic mutations to selectively kill tumor cells. As mentioned earlier, oncogenes like c- myc and inactivation of tumor suppressors such as Rb force proliferation but at the same time promote apoptosis, presumably as a cellular safe-guard against tumorigenesis.

    In principle, these E1A mutants, or small molecules which mimic their action, may provide tumor-specific anticancer agents by exploiting the fact that the Rb pathway is disrupted in the majority of human cancers. Rb mutations lead to increased E2F activity, and this promotes both proliferation and apoptosis 91 — It has been suggested that simultaneous inactivation of Rb and cdk2 would be particularly pro-apoptotic, and produce a synthetic lethal effect in cells with a mutant Rb pathway Consistent with this hypothesis, cell-permeable peptides that interfere with cdk2 activity readily induce apoptosis in tumor cells while having little effect on normal cell lines Interestingly, although E2F-induced apoptosis is potentiated by p53 91 , these peptides can induce apoptosis in pdeficient tumor cell lines.

    Therefore, this strategy has widespread potential. As indicated above, the propensity of certain tissues e. Since most advanced solid tumors have lost p53 function, these inhibitors should not interfere with cell death of most tumor cells. Several caveats exist with respect to p53 inhibition in patients. First, the premise is based on animal studies, and it is not yet known the extent to which p53 contributes to anticancer agent toxicity in humans.

    Secondly, although the protective effects in mice are striking, the magnitude of the protective effect over repeated doses and time is unknown. Thirdly, p53 inhibition may promote mutations by allowing the survival of mutated cells. However, transient inhibition of p53 may be less mutagenic In any case, most current anticancer agents are directly mutagenic, and it is not clear whether p53 inhibitors which would only indirectly promote mutations would be worse. At the very least, this strategy warrants further consideration.

    The last decade has seen an extraordinary increase in our understanding of apoptosis, and its contribution to cancer and cancer therapy. Furthermore, the molecular mechanisms that control and execute apoptotic cell death are coming into focus. Although there is much more to learn, our current understanding of apoptosis provides new avenues for cancer diagnostics, prognosis and therapy. In the coming years, it seems likely that rational strategies to manipulate cell suicide programs will produce new therapies that are less toxic and mutagenic than current treatment regimens.

    Oncogenes such as E1A and c- myc induce apoptosis through pdependent and independent pathways, and both pathways may facilitate cytochrome c release from mitochondria.

    Introduction

    Components of the oncogene-induced cell-death program that are mutated in human tumors are shown in black, candidate tumor suppressors are shown in gray. Anticancer agents induce apoptosis. Hematoxylin and eosin staining of lymph nodes from lymphoma-bearing mice B cell lymphoma left untreated A or isolated 5 h after treatment with cyclophosphamide B. Apoptotic cells are identified by their reduced size and by the presence of highly condensed chromatin. Therefore understanding the mechanisms involved in physiological as well as in disturbed or dysregulated apoptosis may lead to the development of new methods of preventive treatment of various developmental abnormalities.

    The present review summarizes data on the mechanisms of programmed cell death and concentrates on apoptosis involved in normal or disturbed gametogenesis and in normal and abnormal embryonic development.

    1st Edition

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    Major DNA fragmentation is a late event in apoptosis. Thornberry NA, Lazebnik Y. Ashkenazi A, Dixit VM. Evan G, Littlewood T. A matter of life and cell death. Cell cycle regulation and apoptosis. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis.

    Adams JM, Cory S. The Bcl-2 protein family: BCL-2 family members in the development and degenerative pathologies of the nervous system. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Apoptosis pattern elicited by several apoptogenic agents on the seminiferous epithelium of the adult rat testis. Apoptosis of germ cells during human prenatal oogenesis.

    Apoptosis in different stages of human oogenesis. Human spermatogenesis in vitro: Respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J Clin Endocrinol Metab. Fas regulates germ cell apoptosis in the human testis in vitro. The Fas system is a key regulator of germ cell apoptosis in the testis. Expression of Fas and Fas ligand in the testes and testicular germ cell tumors: Involvement of Fas in the apoptosis of mouse germ cells induced by experimental cryptorchidism.

    DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Expression of p53 protein in spermatogenesis is confined to the tetraploid pachytene primary spermatocytes.