Current Pediatric Reviews (v.7, #4)

One of the biggest challenges in modern medicine is the comprehension of programmed cell death (apoptosis) in the context ofpediatric diseases. Apoptosis is a highly regulated process that is critically important for cellular self-destruction in a variety ofnormal and disease situations. The term apoptosis was coined by Kerr and colleagues in 1972, after the Greek word meaningleaves falling from a tree in the autumn, to describe a tightly regulated cell suicide program under physiological conditions [1].Apoptotic cell is recognized by characteristic features [2] including chromatin condensation and nuclear fragmentation(pyknosis), plasma membrane blebbing, and cell shrinkage. The apoptotic cells break into apoptotic bodies, which are engulfedby phagocytosis without inflammatory response [3] (Fig. 1). Many of apoptotic changes are caused by the activation of a familyof intracellular cysteine proteases called caspases [4]. Now, almost four decades later, we understand much about the control ofthis machinery but need to translate this knowledge into clinical practice and drug discovery. Apoptosis eliminates unwanted orunnecessary cells and thus modulates pathological contexts [5]. Deregulation of apoptosis may cause diseases either byinsufficient or excessive programmed cell death [5-7]. The emerging advances in developmental biology, genomics & genetics,and cell immunology & biology, combined with the development of appropriate animal models, have offered tremendoussupport for the proposed roles for programmed cell death in pediatric disorders. Future studies using both animal models andclinical specimens hold the promise of scientific groundwork for medical interventions [8-17].Although advances in elucidating the regulation of apoptosis have laid the foundation for a deeper understanding of thepathophysiology of many pediatric diseases, we still need to know much about how the apoptotic machinery is connected tomany aspects of developmental and disease pathways in the human body [18-20].Apoptotic cell death occurs via tightly controlled and well-established extrinsic and intrinsic cellular diverse cascades [21-23].There are now growing lists of both upstream and downstream mediators of both extrinsic and intrinsic apoptosis. The extrinsicpathway of apoptosis is initiated by the binding of death ligands of the tumor necrosis factor (TNF) superfamily, e.g. Fasligand, TNF alpha or TNF-related apoptosis-inducing ligand (TRAIL), to their corresponding receptors on the surface of thecell, such as CD95 (Fas) or the TNF receptor [24-28]. This recruits the intracellular death inducing signaling complex (DISC).The DISC activates the initiator caspases, particularly caspase-8, leading to activation of downstream caspases, like caspases 9and 3, and eventually to apoptotic cell death. It is now well established that the extrinsic pathway plays important role incontrolling growth of cancer cells. The intrinsic (mitochondrial) pathway of apoptosis is regulated by pro and anti-apoptoticproteins of the Bcl-2 (B-cell lymphoma-2) family proteins [29, 30]. Upon apoptosis induction, cytochrome C is released fromthe mitochondria into the cytosol where it promotes the assembly of a caspase-activating complex termed the apoptosome [31-33]. The apoptosome is a multimeric protein complex containing Apaf-1, cytochrome c, and caspase-9. Upon binding to theapoptosome, caspase-9 is activated, and subsequently activates the downstream effector caspases, leading to proteolysis andapoptotic cell death.There are now various strategies that can be used to prevent or induce cell death or to develop drugs that can block pro- or antiapoptoticfactors. Furthermore, the promising new pharmaceutical strategies to treat deregulated gene-directed processes mayprovide advances in the control of various pediatric diseases including cancer, congenital malformations, immune systemdiseases, metabolic disorders and other diseases of various systems. Thus, the contribution of apoptosis to the pathogenesis ofvarious diseases and abnormalities is likely to be modulated by targeting specific factors involved in the entire process....

Resistance to undergo apoptosis is a characteristic feature of human malignancies, including childhoodleukemia. Apoptosis, or programmed cell death, is the most common intrinsic cell death programs that plays a critical rolein maintaining tissue homeostasis, for example in the hematopoietic system. Accordingly, too little apoptosis can promotetumor formation and also treatment resistance, since the anti-leukemic activity of most current treatment approaches, i.e.chemo-, radio- or immunotherapy critically depends on intact apoptosis signaling pathways in leukemia cells. Thus,defective apoptosis programs confer resistance to anti-leukemic therapy. The elucidation of the signaling componentsmediating apoptosis in leukemia cells has provided multiple targets for therapeutic intervention. These targets can beexploited to develop novel treatment strategies better directed at selective intervention points in the apoptotic machinery.Several of these approaches have already been translated into a clinical application. Thus, the exploitation of apoptosissignaling pathways for leukemia therapy opens new perspectives for effective treatment protocols for childhood leukemia.

Nephropathic cystinosis is a lethal autosomal recessive lysosomal storage disease that destroys kidney functionby ten years of age. It results from failure to transport cystine from lysosomes, leading to large accumulations of cystinewithin the lysosomes of almost all tissues. How the failure of function of cystinosin and the attendant lysosomal cystineaccumulation effects the severe phenotype is now being investigated on several fronts, chief among which is the increasein the rate of apoptosis observed in cultured cells and renal tissue whose lysosomes are cystine-laden.

Inhibition of Apoptosis in Pediatric Cancer by Survivin by Ayman Samkari, Rachel A. Altura (277-284).
An estimated 17.6 per 100,000 new cases of childhood cancer are diagnosed each year in the United States.The major subtypes of childhood cancer include leukemia, central nervous system tumors, lymphoma, neuroblastoma,rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms tumor, Germ cell tumors, and other rare tumors. Despiteimprovements in the diagnosis and treatment of these tumors over the last 30 years, subsets of children still have pooroutcomes and many others have significant morbidity. Dysregulation of apoptotic pathways has been shown to contributeto tumor formation as well as resistance to therapy in both pediatric and adult malignancies. Survivin, the smallestmember of the inhibitor of apoptosis protein (IAP) family, is highly expressed in diverse cancers and correlates withdecreased patient survival. Here, we review the current literature on Survivin expression in pediatric cancer, itsrelationship to clinical outcome and potential therapeutic options to target this protein in pediatric cancer.

Apoptotic Cell Death in Bronchopulmonary Dysplasia by Andreas A. Kroon, Martin Post (285-292).
Apoptosis plays an important role in normal lung development as well as repair after lung injury andcontributes to the pathophysiology of many lung diseases. Bronchopulmonary dysplasia (BPD) is a major cause ofneonatal pulmonary and non-pulmonary morbidity and is characterized by an arrest in alveolar development. Currentlythere is no specific treatment for BPD. Mechanical ventilation, exposure to high concentrations of oxygen andinflammation are important risk factors for the development of BPD. Emerging evidence links the pathophysiology ofBPD to an imbalance between anti-apoptotic and pro-apoptotic signaling pathways. Different apoptotic signalingpathways have been implicated, including Fas/FasL, caspase-dependent and -independent pathways, pro-survival Akt,transforming growth factor-β and p53. To what extent these pathways are involved in the pathogenesis of BPD is a hottopic of research. The aim of this review is to describe the timing and apoptotic events during lung development and thepathogenesis of BPD with particular focus upon apoptotic pathways activated by mechanical ventilation, hyperoxia andinflammation. An appreciation of these apoptotic pathways is essential for understanding the aetiology of and thedevelopment of treatments for pulmonary diseases such as BPD.

Triggers of Cell Death in the Developing Brain by Chrysanthy Ikonomidou (293-300).
Cell death occurs physiologically in the mammalian brain during the period of the growth spurt. In humans,this period starts in the 3rd trimester of gestation and ends by the third year of life. Environmental factors can interact withprogrammed cell death mechanisms to pathologically increase the numbers of neurons undergoing self elimination(apoptosis) and potentially lead to brain injury.It has been shown that classes of drugs which block glutamate N-methyl-D-aspartate (NMDA) receptors, activate γ-aminobutyric-acid (GABAA) receptors or block voltage gated sodium channels, when administered to immature rodentsduring susceptible developmental periods, trigger profound apoptotic cell death in the brain. Sedative, anesthetic andanticonvulsant drugs utilize these mechanisms to exert their actions. In addition, short exposures to non-physiologicoxygen levels can also trigger apoptotic cell death in the brains of infant rodents. Pathomechanisms involved in theneurotoxic actions of sedatives, anesthetics, anticonvulsants and oxygen include decreased expression of neurotrophins,inactivation of survival signaling proteins, activation of inflammatory cytokines as well as oxidative stress.These findings raise concerns regarding treatment of pregnant women, infants and young children with sedatives,anesthetics and anticonvulsants and premature infants with oxygen. Modified approaches should be developed for patientswithin these vulnerable age groups.

Longitudinal bone growth is a complex and tightly regulated process. A precise balance between proliferation,differentiation and cell death in growth plate chondrocytes is a key to normal bone growth in children. Indeed, decrease inproliferation/differentiation and increase in undesired cell death in growth plate cartilage are directly associated withimpaired bone growth. Proteasome inhibitors are currently undergoing phase I and II clinical trials to evaluate theirefficacy in the treatment of various childhood cancers. Effects of proteasome inhibitors on bone growth in children havenot yet been reported. However, recent preclinical observations suggest that proteasome inhibitors may selectively targetessential cell populations in growth plate cartilage, causing significant growth failure; an effect associated with increasedapoptosis and decreased proliferation of chondrocytes. The observed effects on apoptosis and proliferation in growth platechondrocytes appear to be mediated by several proteins including p53, apoptosis inducing factor (AIF), caspases, NF-.Band ß-catenine. These observations could have important implications for the use of proteasome inhibitors in the treatmentof childhood diseases.

Keratin 18, Apoptosis, and Liver Disease in Children by Yanci O. Mannery, Craig J. McClain, Miriam B. Vos (310-315).
Keratins, a major component of epithelial cell intermediate filaments, provide structural support to the cell andare important for the maintenance of structural integrity. Beyond its role of structural integrity in hepatocytes, keratin 18(K18) is a known marker of apoptosis and has been proposed as an indicator of progression in chronic liver diseases suchas nonalcoholic fatty liver disease (NAFLD). NAFLD is the most common cause of chronic liver disease in children andadolescents in the United States and throughout the world and comprises a wide spectrum of diseases ranging from simplesteatosis (fatty liver) to nonalcoholic steatohepatitis (NASH) and cirrhosis. While simple steatosis is typically benign innature, NASH is a more serious condition that may progress to end-stage liver disease and liver failure. Currently, liverbiopsy is considered the most reliable method of assessing the histological severity of disease and differentiating betweensimple steatosis and NASH. Because biopsy is invasive in nature, expensive, and subject to sampling error and/orvariability in interpretation, it is not suitable as a screening test. Therefore, it is necessary to examine known mechanismsassociated with the progression of liver disease, such as hepatocellular apoptosis, and identify potential biomarkers thatcould be used as a diagnostic tool in NASH. This review will focus on the 1) the role of keratins in health and disease, 2)K18 as a potential non invasive biomarker in liver disease, 3) overview of pediatric NAFLD and issues with diagnosis and4) potential role for K18 in assessing pediatric nonalcoholic fatty liver disease.

The Wilms’ tumor 1 (WT1) gene encodes a transcription factor that was among the first tumor suppressor genesto be identified. Dependent on the splice variant, some WT1 isoforms can function as transcriptional regulators, whereasother WT1 proteins are presumably involved in RNA processing. The mechanisms by which WT1 regulates transcriptionand the identification of bona fide target genes have been difficult to study, which is partially due to the complex nature ofthe gene and its context specific functions. While the role of WT1 as a tumor suppressor in Wilms’ tumor is widelyaccepted, considerable evidence points to an oncogenic function in other tumors. Recent studies have provided newinsights into the underlying mechanisms that lead to the development of Wilms’ tumor. In addition, a conditional Wt1knockout mouse model and RNAi-mediated screening approaches have uncovered new functions for WT1 indevelopment and tumorigenesis.

Apoptosis and Pediatric Idiopathic Neutropenia by Marco Garcia, Michael Jeng, Kari Nadeau (321-328).
Idiopathic neutropenia (IN) is a disorder that can lead to severe, life-threatening infections. IN in children isusually characterized by decreased neutrophil counts (<1500/ il) and can result in reduced monocyte count and phagocytefunction. Since no current therapies exist and the pathophyisiology is not fully understood, we sought to investigateimmune mechanisms of IN in children. We investigated the role of circulating Fas Ligand in mediating IN by comparingIN patients to healthy control (HC) patients. Our results suggest that high levels of FasL contribute to IN pathology.Additionally, the plasma from acute IN patients was found to have higher levels of soluble FasL than chronic IN patients.When incubating HC neutrophils with IN patient plasma, higher levels of apoptosis were seen. The plasma-derived factorin inducing apoptosis was found to be preferentially specific for neutrophils. Addition of anti-sFasL antibodies to INpatient plasma resulted in a significant decrease in neutrophil apoptosis. In summary, these data suggest that sFasL in INpatient plasma may reduce neutrophil cell death and that the Fas/FasL apoptotic pathway may play a role in the pathologyof idiopathic neutropenia.

Apoptosis in Anthracycline Cardiomyopathy by Jianjian Shi, Eltyeb Abdelwahid, Lei Wei (329-336).
Apoptosis is a tightly regulated physiologic process of programmed cell death that occurs in both normal andpathologic tissues. Numerous in vitro or in vivo studies have indicated that cardiomyocyte death through apoptosis andnecrosis is a primary contributor to the progression of anthracycline-induced cardiomyopathy. There are now severalpieces of evidence to suggest that activation of intrinsic and extrinsic apoptotic pathways contribute to anthracyclineinducedapoptosis in the heart. Novel strategies were developed to address a wide variety of cardiotoxic mechanisms andapoptotic pathways by which anthracycline influences cardiac structure and function. Anthracycline-induced apoptosisprovides a very valid representation of cardiotoxicity in the heart, an argument which has implications for the mostappropriate animal models of damaged heart plus diverse pharmacological effects. In this review we describe variousaspects of the current understanding of apoptotic cell death triggered by anthracycline. Differences in the sensitivity toanthracycline-induced apoptosis between young and adult hearts are also discussed.

The Death Pathways in the Neonatal Gut by Michal M. Godlewski (337-345).
Dynamic equilibrium between apoptosis and mitosis facilitates growth and development of the intestinalmucosa in mammalian neonates. Groups of enterocytes observed dying together on the villi in the gut of the neonatessuggest involvement of the paracrine factors in the propagation of the death signalling. The most potent death-inducing,paracrine factor for the enterocytes is the TGF-β. It is secreted from the macrophages and epithelial cells upon uptake ofapoptotic bodies. Through its receptor complex (TGF-R I and II) and SMAD cascade it regulates the intracellular balanceof the pro- and antiapoptotic proteins from the Bcl-2 family, sensitising enterocytes for other death signals (i. e. TNFβ)and directing them towards apoptosis. Just before and after birth mitosis-to-apoptosis ratio shifts towards proliferation.First ingestion of colostrum, initiates the major remodelling of the gut mucosa. Apoptosis is enhanced, facilitating theremoval of foetal-type enterocytes and closing of the gut barrier. Second major remodelling stage occurs after weaning,when the adaptation to the ingestion of solid foods takes place. Proliferation and cell death in the intestinal mucosa areunder the direct control of a variety of growth factors (EGF, IGFs, TGF-βs) and tissue hormones (leptin, ghrelin, GLP,CCK, etc.) provided either by mothers colostrum and milk or produced in the neonate organism. Finally, uncontrolledapoptosis may lead to the weakening of the gut barrier, which results in the increased susceptibility to the intestinaldisorders, pathogen infiltration and may lead to the development of the necrotising enterocolitis in the infants.

Apoptosis in Obstructive Nephropathy by Ethan I. Franke, Kirstan K. Meldrum (346-351).
Congenital urinary tract obstruction is an important cause of renal disease in children. Early animal studieshave shown a relationship between the timing of obstruction and the different pathological entities that collectivelycomprise renal maldevelopment. A multitude of molecular factors through both in-vivo and in-vitro studies have beenidentified that are involved in apoptosis and/or interstitial fibrosis as a result of urinary tract obstruction. These molecularfactors have been found to work in parallel and also occasionally divergent pathways. Apoptosis is a prominent feature ofearly in-utero urinary tract obstruction and a major factor in the loss of renal mass and renal dysfunction. Furthermore,there is an increasing body of evidence to suggest that apoptosis is an inciting event in the development of progressiveinterstitial fibrosis which further contributes to renal demise. Clearly, a better understanding of the mechanisms ofapoptosis and the subsequent development of targeted therapies directed against apoptosis may prove beneficial in theprevention of renal dysfunction associated with urinary tract obstruction.