Mentre gli scienziati rimettono ordine nel complesso origami biochimico attraverso il quale un foglio in bianco di cellule indifferenziate viene rinchiuso all'interno di una creatura completamente sviluppata, essi stanno imparando una sconcertante lezione: il contesto è tutto. Ciò che rappresenta una deformità in un organismo, per un altro può rappresentare un dono dell'evoluzione.

Si prenda il caso della sindattilia, cioè  la presenza di membrana interdigitale alla mano e al piede. Ciò che in una persona viene considerata una lieve deformità, nell'anatra espande il suo territorio tanto da farle abbracciare sia l'acqua che il cielo.

Come mai le estremità degli arti di alcune creature sfociano in mani o piedi con dita ben definite, mentre altre si traducono in pagaie? In un rapporto pubblicato sul numero del 3 maggio della rivista Science, alcuni ricercatori del Memorial Sloan-Kettering Cancer Center di Manhattan hanno detto di aver trovato una buona parte della risposta: si tratta di un segnale biochimico che pare controlli il manifestarsi o meno della membrana interdigitale.

In un lampante esperimento il Dr. Hongyan Zou e la Dsa Lee Niswander hanno bloccato tale segnale e hanno trovato che così i polli sviluppano piedi di anatra. "L'effetto è veramente molto drammatico", ha detto la Dsa Niswander in un'intervista. Nel suo lavoro ha messo in evidenza che bloccando il segnale molecolare rappresentato dalle proteine morfogenetiche dell'osso – BMP, bone morphogenetic proteins – ha prodotto la deformità in "praticamente il 100%" degli arti sui quali stava conducendo l'esperimento.

Altri laboratori hanno trovato che le BMP sono importanti in altri distretti di un organismo in via di sviluppo, in quanto dicono a un gruppo di cellule se formare un pollice o un mignolo, e sono pure importanti nell'orchestrare lo sviluppo di reni, occhi e altri tessuti. In passato erano interessate a queste molecole soprattutto le società di biotecnologie nella speranza di guadagni con farmaci che stimolano la crescita dell'osso, ma queste proteine stanno dimostrando di essere così ubiquitarie durante lo sviluppo embrionale che alcuni ricercatori desiderano poter dar loro un nome più eloquente.

"È veramente un gruppo affascinante di molecole" ha detto la Dsa Brigid Hogan, biologa dello sviluppo presso l'Howard Hughes Medical Institute della Vanderbilt University. "Probabilmente sono importanti per lo sviluppo di qualunque organo."

L'anno scorso il laboratorio della Dsa Hogan ha scoperto che se in un embrione in via di sviluppo il gene che codifica per un certo tipo di molecola di BMP viene cancellato, l'organismo morirà senza formare lo strato medio delle cellule, cioè il mesoderma, che dà origine a una varietà di tessuti come muscolo, rene, cartilagine e osso. Più recentemente, in un'indagine in corso non ancora pubblicata, il suo laboratorio ha scoperto che mettendo fuori combattimento il gene per un altro tipo di BMP si impedisce ai topi di produrre sperma.

Un'altra ricercatrice, la Dsa Elizabeth Robertson, biologa all'Università di Harvard, ha trovato che mettendo fuori combattimento i geni per le BMP si sono prodotte delle deformità dell'occhio e del rene nonché delle malformazioni ai piedi.

"La domanda è: come fa un tipo di molecola giocare tutti questi differenti ruoli?" ha detto la Dsa Cheryll Tickle, una studiosa di biologia dello sviluppo presso l'University College di Londra. Ha detto che lo studio della Dsa. Niswander sarebbe importante nel districare i vari percorsi chimici coi quali le BMP pilotano la trasformazione di un uovo in un neonato. "Potrebbe trattarsi di un equilibrio molto complicato" ha detto. In alcuni stadi di sviluppo le BMP sono implicate nella morte cellulare programmata, cioè la rimozione di tessuto indesiderato. Ma esse hanno pure altri ruoli.

Come se i congegni della natura non fossero già abbastanza complessi, alcuni ricercatori hanno trovato che altri geni sono pure implicati nello sviluppo di un arto. Nel numero del 26 aprile di Science i Dottori. Yasuteru Muragaki, Stefan Mundlos, Joseph Upton e Bjorn R. Olsen della Harvard Medical School hanno descritto come avevano ricollegato una deformità umana chiamata sinpolidattilia – membrana interdigitale accompagnata da dita soprannumerarie - a una mutazione del gene detto homeobox. Si è visto per la prima volta che geni homeobox controllano lo sviluppo del moscerino della frutta, la Drosophila melanogaster: una mutazione in uno di tali geni può far sì che le zampe spuntino sulla testa là dove dovrebbero esserci le antenne. A partire dalla scoperta dei geni homeobox, geni corrispondenti sono stati trovati in tutto il regno animale.

Il Dr. Mundlos ha detto che il suo gruppo di ricercatori potrebbe aiutare nel rispondere ad alcune intricate domande sui modi molto diversi con cui gli arti possono svilupparsi. "Perché il pesce ha le pinne, mentre il pollo ha dei  piedi con tre dita davanti e uno dietro, mentre noi abbiamo cinque dita?" ha chiesto. "Noi crediamo che la mutazione che abbiamo trovato getti luce su questo quesito."

I geni homeobox producono molecole denominate fattori di trascrizione, che attivano e inattivano altri geni. Le dita addizionali della mano e del piede della sinpolidattilia sembrano scaturire quando una mutazione fa sì che il gene homeobox inserisca parecchie copie di un aminoacido, l'alanina, in una regione del fattore di trascrizione che sembra essere implicato con le dita in accrescimento. I topi - e gli esseri umani con le loro mani a cinque dita – hanno una sequenza di 15 alanine nella proteina dell'homeobox. I polli ne hanno 9, mentre il pesce zebra non ne ha neppure una.

Il Dr Mundlos ha detto che non era ancora chiaro come i risultati ottenuti dal suo gruppo sullo sviluppo di un arto fossero in relazione con le recenti scoperte sulle BMP. "Tutte queste molecole sono in stretta relazione una con l'altra", ha detto. "Abbiamo un'evidenza piuttosto buona che tutte quante hanno un qualcosa a che fare con un modello molto precoce."

Mentre i geni homeobox producono fattori di trascrizione che operano all'interno del nucleo della cellula, le molecole BMP vengono usate per inviare segnali da cellula a cellula. Un segnale homeobox potrebbe dire a un gene di fabbricare una BMP. Oppure, di converso, una BMP potrebbe far sì che una cellula dia delle istruzioni al suo nucleo per attivare un gene homeobox. Il tutto fa parte della complessa orchestrazione dello sviluppo.

Il trucco della Dsa Niswander di trasformare piedi di pollo in piedi di anatra fu inspirato dalla ricerca degli ultimi cinque anni, la quale indica che i geni sono impegnati nel costruire molecole segnale di BMP nel tessuto interdigitale di mani e piedi. Gli scienziati di molti laboratori hanno stabilito ciò attraverso una tecnica chiamata ibridazione dell'RNA in situ. Dopo che un gene è attivato, le sue istruzioni genetiche vengono copiate in molecole di RNA messaggero che mandano le informazioni ai ribosomi extranucleari dove vengono sintetizzate le proteine. Per determinare se un gene è attivo, si deve semplicemente cercare il suo RNA messaggero.

Per effettuare la ricerca di geni attivi per le BMP, gli sperimentatori hanno confezionato degli agglomerati  di RNA in modo che le loro sporgenze e gli incavi chimici formassero un'immagine speculare delle corrette molecole di RNA messaggero per le BMP. All'RNA fabbricato fu quindi assegnata un'etichetta chimica in modo da poter essere facilmente rintracciato e identificato. Quando gli scienziati immersero un embrione di pulcino o di topo nell'RNA fabbricato, esso ha messo in luce e ha chiarito i suoi ruoli complementari, o immagini speculari. Allora i ricercatori impiegarono degli anticorpi contro l'etichetta chimica per concentrare l'attenzione proprio dove a livello somatico le molecole di RNA stavano congregandosi. Questi erano i punti dove il gene della BMP era stato attivato. Gli scienziati sospettarono che nella maggior parte degli animali le molecole di BMP impedissero il formarsi delle membrane interdigitali scatenando la morte cellulare programmata. Quando ricevono un segnale dalle BMP, le cellule interdigitali compiono un suicidio di massa, permettendo lo sviluppo di dita indipendenti. Nelle anatre - e a quanto pare, anche nelle persone con sindattilia - ciò non si verifica.

La Dsa Niswander decise di vedere cosa accadrebbe se avesse bloccato il segnale del suicidio cellulare. Molecole come le BMP liberano il loro messaggio legandosi a una molecola recettrice che si fissa all'esterno della membrana esterna di una cellula. Quando la molecola di BMP si lega al recettore, ha inizio una cascata di reazioni chimiche interne che in ultima analisi dice al nucleo di creare gli enzimi necessari per eseguire una mansione specifica.

Per eliminare il segnale BMP, la Dsa Niswander e i suoi colleghi usarono un tipo di terapia genica inversa. Dapprima provocarono una mutazione nel gene che fabbrica i recettori BMP. Inserirono il gene mutato in un retrovirus che inserì il gene alterato nelle cellule di embrioni di pulcino. Gli embrioni col gene mutato produssero dei recettori BMP defettivi che erano sordi al segnale del suicidio. Come era prevedibile, i pulcini svilupparono tra le dita del piede delle membrane interdigitali simili a quelle delle anatre.

Quindi, usando la tecnica di ibridazione per scoprire dove i geni BMP erano attivi, gli scienziati rivolsero la loro attenzione alle anatre e trovarono che mentre i geni si erano attivati in altre parti del corpo, non si erano attivati  nei tessuti interdigitali.

Gli scienziati trovarono pure che la mancanza di BMP portò nei pulcini a un altro cambiamento: essi tendevano a sviluppare delle penne ai piedi invece di squame. Gli scienziati sono dell'avviso che durante lo sviluppo certe cellule sono in grado di assumere identità differenti. Se abbandonate a se stesse, diventano penne. Se stimolate delicatamente dalle BMP diventano squame.

Gli scienziati ritengono che il sistema di segnalazione scoperto nei pulcini è attivo anche negli esseri umani. Quando la natura va a inciampare in un trucco utile, tende a conservarlo. "Sia il segnale cellulare che molecolare sembrano essere molto ben conservati nel pulcino e nel topo", ha detto la Dsa Niswander. "Presumibilmente questo è  vero anche negli esseri umani."

I dettagli su come veramente la morte cellulare programmata sia in grado di plasmare gli arti rimane un mistero, ha detto il Dr Matthew P. Scott, professore di biologia dello sviluppo e genetica all'Università di Stanford e ricercatore dell'Howard Hughes Medical Institute. "Adesso abbiamo bisogno di sapere come i segnali BMP si producono nelle giuste aree", ha detto. "Ma non potremmo porci questa domanda senza il lavoro di Zou e Niswander".

Le implicazioni dell'esperimento possono andare al di là del campo dell'embriologia. La morte cellulare programmata è importante anche nel modellare altre parti del corpo, incluso il cervello, dove nel feto e nel neonato una profusione di neuroni è spazzata via per lasciar posto al circuito dei neuroni. Si è ipotizzato che anche la BMP è coinvolta in tale processo.

Ma le molecole giocano anche dei ruoli che sembrano non aver nulla a che fare col suicidio cellulare. "La cosa interessante a proposito delle BMP è che possono svolgere azioni diverse in  stadi diversi di sviluppo embrionale" ha detto il Dr. Hogan Vanderbilt. "In alcuni contesti promuovono la morte cellulare, in altri la sopravvivenza, a seconda di dove e quando vengono espresse."

Homeobox

Pit-1 homeobox-containing protein bound to DNA

A homeobox (or homoeobox) is a DNA sequence found within genes that are involved in the regulation of development (morphogenesis) of animals, fungi and plants. Genes that have a homeobox are called homeobox genes and form the homeobox gene family.

A homeobox is about 180 base pairs long; it encodes a protein domain (the homeodomain) which can bind DNA. Homeobox genes encode transcription factors which typically switch on cascades of other genes, for instance all the ones needed to make a leg. The homeodomain binds DNA in a specific manner. However, the specificity of a single homeodomain protein is usually not enough to recognize only its desired target genes. Most of the time, homeodomain proteins act in the promoter region of their target genes as complexes with other transcription factors, often also homeodomain proteins. Such complexes have a much higher target specificity than a single homeodomain protein.

A particular subgroup of homeobox genes are the Hox genes, which are found in a special gene cluster, the Hox cluster (also called Hox complex). Hox genes function in patterning the body axis. Thus, by providing the identity of particular body regions, Hox genes determine where limbs and other body segments will grow in a developing fetus or larva. Mutations in any one of these genes can lead to the growth of extra, typically non-functional body parts in invertebrates, for example aristapedia complex in Drosophila, which results in a leg growing from the head in place of an antenna and is due to a defect in a single gene (this mutation is also known as Antennapedia). Mutation in vertebrate Hox genes usually results in spontaneous abortion.

The homeobox genes were first found in the fruit fly Drosophila melanogaster and have subsequently been identified in many other species, from insects to reptiles and mammals. The diagram to the right is a structural model of the Rattus norvegicus Pit-1 homeobox-containing protein (purple) bound to DNA. Pit-1 is a regulator of growth hormone gene transcription. Pit-1 is a member of the POU DNA-binding domain family of transcription factors so it can bind to DNA using both the POU domain and the homeodomain. Homeobox genes have even been found in fungi, for example the one-cellular yeasts, and plants. The well known homeotic genes in plants (MADS-box genes) are not homologous to Hox genes in animals. Plants and animals do not share the same homeotic genes, and this suggests that homeotic genes were evolved once in the early evolution of animals and once again in the early evolution of plants. Humans generally contain homeobox genes in four clusters, called HOXA (or sometimes HOX1), HOXB, HOXC, or HOXD, on chromosomes 7, 17, 12, and 2, respectively.

Mutations to homeobox genes can produce easily visible phenotypic changes. Two examples of homeobox mutations in the above-mentioned fruit fly are legs where the antennae should be, and a second pair of wings. Duplication of homeobox genes can produce new body segments, and such duplications are likely to have been important in the evolution of segmented animals. Interestingly, there is one insect family, the xyelid sawflies, in which both the antennae and mouthparts are remarkably leg-like in structure.

Programmed Cell Death in Development
by Dr. Michael Wride and Dr. Leon Browder
Department of Biochemistry and Molecular Biology, University of Calgary
December 4, 1998

Why must some cells die for the good of the embryo?

Programmed cell death (PCD) is an important mechanism in both development and homeostasis in adult tissues for the removal of either superfluous, infected, transformed or damaged cells by activation of an intrinsic suicide program.

One form of PCD is apoptosis, (an ancient Greek word used to describe the "falling off" of petals from flowers or leaves from trees and was proposed by Kerr, Wyllie and Currie in 1972 to refer to the peculiar morphology of physiologically occurring cell death which plays a complementary but opposite role to mitosis in the regulation of animal cell populations).

Apoptosis is characterized by maintenance of intact cell membranes during the suicide process so as to allow adjacent cells to engulf the dying cell so that it does not release its contents and trigger a local inflammatory reaction.

Cells undergoing apoptosis usually exhibit a characteristic morphology, including fragmentation of the cell into membrane-bound apoptotic bodies, nuclear and cytoplasmic condensation and endolytic cleavage of the DNA into small oligonucleosomal fragments (Steller, 1995). The cells or fragments are then phagocytosed by macrophages.

Signals that can trigger apoptosis can include:

- lineage information

- damage due to ionizing radiation or viral infection

- extracellular signals.

Extrinsic signals may either suppress or promote apoptosis, and the same signals may promote survival in one cell type and invoke the suicide program in others (Steller, 1995).

For example, death receptors that are members of the tumour necrosis factor receptor (TNFR) family sit in cell membranes, but their intracellular domains have direct access to the cell death machinery that lies ready and waiting within the cell (Ashkenazi and Dixit, 1998).

When members of the TNF ligand family bind to their receptors some of the pathways activated can include those that bring about cell death. Conversely, there are other factors, such as the neurotrophins, that bind to cell surface receptors and which act to prevent cell death!

Invocation of the suicide program involves the synthesis of specific messenger RNA molecules and their translation. PCD can sometimes be suppressed by inhibiting transcription or translation (Steller, 1995), which provides evidence that cell death is mediated by intrinsic cellular mechanisms.

I'm interested in life! Why do I want to think about death?

Cell death is currently the subject of considerable research activity. This interest stems, in part, from the potential for understanding oncogenesis and the possibility of exploiting the cell death program for therapeutic purposes. For example, inhibition of cell death might contribute to oncogenesis by promoting cell survival instead of death. Likewise, triggering cell death might provide the means for eliminating unwanted cells (e.g., tumor cells).

What on Earth has death got to do with development?

Recognition of PCD as a developmental mechanism dates back well over 100 years (Clark and Clark, 1996; this is an excellent account of old descriptions of "apoptosis" before there was "apoptosis"). Developmental processes that involve PCD include:

- Elimination of transitory organs and tissues. Examples include phylogenetic vestiges (pronephros and mesonephros in higher vertebrates), anuran tails and gills and larval organs of holometabolous insects.

- Tissue remodeling. Vertebrate limb bud development (Fig. 11.42, Saunders, 1982; Fig. 1, Saunders, 1966) is an example. If PCD fails, in formation of the digits, digits remain joined by soft tissue. Compare, for example, the situation in the chick and duck hind limbs. If chick limb mesoderm is combined with duck ectoderm, PCD fails and the digits remain joined (Saunders, 1966). This observation implicates the ectoderm in providing the signal to trigger PCD. Another example is formation of heart loops during vertebrate development. Depletion of cells in spinal ganglia occurs during development of the chick embryo. As shown in Table 11.1 (Saunders, 1982), there is precise chronological and spatial control over this process. Interestingly, injections of nerve growth factor reduce the frequency of cell death in the spinal ganglia. This observation provides a link between growth control and PCD.

Worm work!

Much of our current knowledge about the molecular genetics of PCD comes from work on Caenorhabditis elegans. The adult hermaphrodite C. elegans forms 1090 somatic cells, of which 131 die by apoptosis. There are four stages in apoptosis in worms, which are equally applicable to the sequence of apoptotic events in vertebrates (Steller, 1995):

- decision whether a cell should die or assume another fate;

- death;

- engulfment of the dead cell by phagocytes;

- degradation of the engulfed corpse.

A number of genes have been identified that regulate these processes in worms (see Fig. 1, Steller, 1995). Mutations affecting the final three stages affect all somatic cells, whereas genes affecting the death verdict affect very few cells. Execution itself is mediated by ced-3, ced-4 and ced-9 (ced = cell death defective).

Cloning of the apoptotic genes of C. elegans and their characterization have led to considerable understanding of the molecular events of apoptosis and to the identification of mammalian homologues of the apoptotic effectors. For example, ced-9 and ced-3 are homologous to the protooncogene bcl-2 and the cytokine processing enzyme ICE (interleukin-1ß-converting enzyme) respectively, while ced-4 is homologous to the apoptotic protease activating factor1 (Apaf1) gene. A further outline of these factors is presented below.

The Bcl-2 Family, Caspases, and Apaf1: Tools for Suicide.

The Bcl-2 family: homologues of ced-9

The bcl-2 gene was initially cloned and characterized as a candidate proto-oncogene involved in the t(14:18) translocation that is characteristic of the human B-cell malignancy follicular lymphoma (Tsujimoto et al. 1984) and is the vertebrate homologue of the C. elegans ced-9 gene. Bcl-2 facilitates survival of cells in which it is expressed and possesses a hydrophobic tail that allows it to associate with various cellular membranes, including those of the mitochondrial outer membrane, the endoplasmic reticulum and the nuclear envelope.

Opposing factors exist in the bcl-2 family

A number of ced-9/bcl-2 family members have been identified. Some of which, like bcl-2, protect cells from apoptosis (these include bcl-xl, bcl-w, and mcl-1) while others actually promote apoptosis (these include bax, bcl-xs, bad, and bak).

The members of the bcl-2 family are characterized by conserved amino acid sequence motifs. These are called bcl-2 homology (BH) domains and there are at least 4 different BH domains (BH 1-4) some of which confer pro-apoptotic activity, while others confer anti-apoptotic activity to the proteins in which they are present.

It is apparent from a number of studies that the various bcl-2 family members are able to dimerize with each other to reinforce or cancel out their cell death or cell survival promoting activities. During development, the expression of these molecules will influence whether cells in particular tissues at certain stages of development will survive or die.

A full discussion of bcl-2 family members can be found in Adams and Cory (1998).

Caspases: homologues of ced-3

Caspases are the vertebrate homologues of the product of the C. elegans ced-3 gene. The vertebrate prototype member of this family is ice (interleukin-1ß-converting enzyme). At least eleven vertebrate ICE-like proteins have been identified in vertebrates and these have subsequently been named caspases to signify the fact that they are proteolytic enzymes that specifically cleave at aspartate residues in target proteins within the cell during apoptosis.

For example, caspases cleave proteins called lamins that are associated with the nuclear matrix and which are essential for the structural support of the nucleus. As soon as the lamins are removed by caspases, the nucleus undergoes the characteristic morphological changes that are associated with apoptosis (described above). Although caspases do not directly degrade DNA (they are proteases), they can degrade a protein called DFF45, which is an inhibitor of the nuclease (the DNA munching molecule) responsible for the DNA fragmentation that generally accompanies apoptosis (Mitamura et al.,1998). The various caspases act in a cascade to activate each other (also by proteolytic cleavage). In fact, the involvement of caspases in apoptosis has been called "death by a thousand cuts" because proteins and DNA are rapidly sliced up into bits after the initial apoptosis-inducing stimulus!

A full discussion of caspases can be found in Thornberry and Lazebnik (1998).

Apafs: homologues of ced-4

For a long time there was no identified vertebrate homologue of the nematode ced-4 gene. However, recently a human homologue has been identified (Zou et al 1997). Various forms of cellular stress that promote apoptosis result in activation of pro-apoptotic bcl-2 family members like bax. Bax moves from the cytoplasm to the mitochondria and punctures holes in the outer mitochondrial membrane. This results in the release of cytochrome c from mitochondria and subsequently the binding of cytochrome c to Apaf1 to create an active complex, which in turn activates caspase-9 and finally caspase-3 (reviewed by Green, 1998).

The mouse Apaf1 gene has recently been identified and transgenic mice have been made that lack Apaf1 (Cecconi et al. 1998; Yoshida et al. 1998). These mice exhibited dramatic developmental defects associated with alterations in apoptosis during development and did not survive past about day 16 of development. The phenotype of these mice included severe craniofacial malformations, brain overgrowth, the persistance of the interdigital webs, and dramatic alterations in the development of the lens and retina! An important gene indeed!

When fibroblasts from these mice lacking Apaf1 were grown in culture, they showed an enhanced potential to survive in the presence of drugs such as staurosporine which would normally cause mouse fibroblasts to die. However, Apaf -/- thymocytes and T lymphocytes could be killed by drugs in culture showing that different systems for apoptosis exist in different tissues.

Finally, another fascinating and important finding from these studies is that it was shown that the human Apaf1 gene maps to a region of human chromosome 12 that has been implicated in a human genetic syndrome called Noonan syndrome. Individuals that possess this syndrome have many of the characteristic defects exhibited by the Apaf1 -/- mice, including craniofacial, limb, and retinal abnormalities.

How do Cells in Flies Die?

Recently, Drosophila has entered the apoptosis field (McCall and Steller, 1997) and it is certain that the combined use of the powerful genetic, molecular, biochemical, and cell biological techniques available in Drosophila will provide important insights into the genes involved in and the molecular mechanisms of apoptosis.

Cell death is important during embryonic development and metamorphosis in Drosophila. During embryonic development, apoptosis occurs in the head, epidermis and central nervous system. Further molecular characterization of mutants with defects in cell death has led to the identification of a region of the Drosophila genome (cytological region 75C) which contains a number of genes involved in cell death: reaper, head involution defective (hid), and grim.

reaper

This gene is specifically expressed in cells destined to die and its pattern of expression anticipates the pattern of cell death in the Drosophila embryo. Furthermore, the reaper gene can be induced by X-ray irradiation in cells of the Drosophila embryo that do not normally undergo cell death. The reaper gene encodes a protein that contains a putative sequence that has been called the death domain and this appears to be essential for conferring the cell death-promoting activity of the reaper protein.

Interestingly, a number of vertebrate proteins containing death domains have been identified, including the cell death promoting members of the tumour necrosis factor-receptor (TNFR) family and proteins that are essential for cell death signaling that associate with TNFRs, including the Fas Associated Death Domain (FADD) and the TNFR Associated Death Domain (TRADD; reviewed by Ashkenazi and Dixit, 1998).

head involution defective (hid)

hid mutants have low levels of cell death, particularly in the head region. hid is predicted to encode a novel protein with limited homology to the putative amino acid sequence of the reaper protein. hid is also expressed in cells that live as well as dying cells, but co-expression of hid and reaper in the midline glia of the Drosophila nervous system results in a dramatic increase in cell death when compared to that seen due to reaper alone. Thus, it appears that, despite the fact that the putative hid protein does not contain a death domain, hid acts in synergy with reaper to regulate Drosophila cell death.

grim

grim appears to have a similar role to hid in modulating reaper activity, since it too does not contain a death domain.

Fly Eyes!

Insights into the effects of the reaper, hid, and grim genes in Drosophila have been provided in studies in which these genes have been ectopically expressed in the Drosophila eye under the control of the eye-specific pGMR promoter. The effect of expression of these genes is complete eye ablation (removal) at high doses, while at lower doses of gene expression various intermediate eye phenotypes are observed.

The phenotype of the eyes is very sensitive to genes acting downstream of reaper, hid, and grim, so this has allowed for screening for mutations that act as genetic modifiers of reaper, hid, and grim activity; i.e. mutations that promote apoptosis are enhancers of eye defects, while mutations that inhibit cell death suppress these phenotypes.

One Drosophila gene identified in this way is the thread gene, which encodes a Drosophila homologue of the baculovirus Inhibitor of Apoptosis (IAP) protein (viral infected cells will try to kill themselves to prevent viral amplification and spread, but through the course of evolution viruses have developed the ability to produce proteins like IAP that inhibit apoptosis in cells that they infect).

Human homologues of IAP have been identified and these include the Neuronal Apoptosis Inhibitory Protein (NAIP), which has been shown to be involved in the muscle wasting disease Spinal Muscular Atrophy (SMA; Roy et al. 1995). Roy et al. showed that in individuals with SMA, mutations in the NAIP locus lead to a failure of normally occurring inhibition of motor neuron apoptosis and this contributes towards the death of motor neurons and brings about the SMA phenotype.

Drosophila caspases

Drosophila homologues of caspases have been identified using degenerate PCR amplification from embryonic cDNA libraries. These Drosophila caspases have been termed DCP-1 and DrICE and they induce apoptosis in cultured cells to which they have been introduced. Mutations to these genes result in defects in the development of the trachea and a complete lack of imaginal discs. So far, no Drosophila homologues of bcl-2 gene family members have been identified, although it seems highly likely that they exist.

Why do some cells die and yet others survive?

There is evidence from work on higher organisms that extrinsic signals may protect cells from apoptosis by suppressing the suicide program (Raff, 1992). For example, the survival of developing neurons may depend upon neurotrophic factors secreted by their targets; a failure to receive sufficient stimulation results in death.

What is the advantage to the organism
of using extrinsic signaling to sustain cell survival?

One possibility is that it could provide a simple system to eliminate cells that end up in the wrong place; without a signal to sustain them, rogue cells would be eliminated (Raff, 1992). Consider primordial germ cells, for example. In mammals, they originate in the hindgut and must migrate to the genital ridges, where they form the gametes. Those that fail to reach the genital ridges are eliminated, presumably because they are deprived of the signal (Steel factor) that is required for their survival in the genital ridges (De Felici and Pesce, 1994).

A disadvantage to the organism of the mechanism that necessitates signaling to prevent apoptosis is that its failure by mutation can lead to the survival of unwanted cells, which - paradoxically - can lead to death of the organism itself. On the other hand, an opportunity is presented by such a mechanism to allow investigators to devise means for targeting unwanted cells for destruction. This might be accomplished by harnessing tumor necrosis factor (TNF), which triggers apoptosis in some target cells. Prostate cancer is an example. The survival of prostate cells is dependent upon androgens; androgen depletion leads to a reduction in cell number by apoptosis. Recently, the dependence of prostate cells on androgens to avoid cell death has been exploited therapeutically by the use of androgen ablation to invoke apoptosis in prostate cancer cells and prolong survival in men with prostate cancer.

Significantly, resistance to androgen depletion correlates with overexpression of bcl-2, the human ced-9 homologue that acts as a brake on prostate cancer cell apoptosis (Raffo et al., 1995). Thus, escape from androgen sensitivity by overexpression of bcl-2 in a subset of prostate cancer cells leads to proliferation of these cells and, ultimately, death of the patient. If bcl-2 could be down-regulated, apoptosis of these cells could be invoked and the cancer controlled.

Ironically, there may be an inverse correlation between cell death and survival of the organism. Exploitation of this relationship holds much promise for therapeutic control of diseases such as cancer.

Patologie genetiche umane
associate a sindattilia

Sindrome di Timothy
del Q-T lungo con sindattilia

La Sindrome di Timothy è una malattia genetica che interessa diversi organi e sistemi. Tutti i soggetti affetti presentano aumentato rischio di morte improvvisa da aritmie (causato da intervallo QT molto allungato all’elettrocardiogramma) e sindattilia delle dita delle mani e dei piedi (le dita sono attaccate tra di loro, da tessuto mucocutaneo). Gli altri sintomi non sono presenti in tutti i soggetti affetti e possono essere i seguenti: crisi di ipoglicemia (improvviso e grave abbassamento degli zuccheri nel sangue), aumentato rischio di infezioni, problemi di linguaggio (difficoltà nell’articolazione di alcune frasi), malformazioni del cuore (dotto di Botallo pervio), problemi comportamentali (talvolta autismo), lievi dismorfismi del viso, problemi nella crescita dei capelli, problemi dentali.

La malattia è determinata su base genetica ed è stato identificato nel 2004 il gene le cui alterazioni (mutazioni) causano la malattia. Questo gene si trova sul cromosoma 12 ed è indicato con la sigla Cav1.2. Il gene è importante per l’eccitazione e la contrazione del cuore. Il suo ruolo in altri tessuti non è ancora chiarito in modo definitivo, ma sembra chiaro che l’alterazione trovata con i pazienti affetti dalla TS, produce una corrente di Ca⁺⁺ sostenuta che entra nelle cellule, causando un sovraccarico intracellulare di calcio in differenti tipi di cellule. A livello cardiaco una corrente prolungata del calcio, prolunga la ripolarizzazione dei cardiomiociti, aumentando il rischio di aritmia, che può essere causa di morte in questa patologia.

La TS si trasmette come carattere autosomico. Sappiamo con sicurezza che i genitori di pazienti affetti possono essere portatori del difetto genetico solo in alcune cellule (questa condizione si chiama mosaicismo), senza manifestare alcun segno di malattia o manifestando manifestazioni molto lievi (brevi crisi di ipoglicemia).

La diagnosi si basa sull’esecuzione dell’elettrocardiogramma, eseguito alla nascita in bambini che manifestino la sindattilia delle dita delle mani. Da qui l’importanza di eseguire un controllo dell’ECG nei bambini che nascono con questa anomalia.

La terapia farmacologica classica del QT lungo (farmaci betabloccanti), può non garantire una protezione massima per la prevenzione degli episodi aritmici. Sono in studio farmaci che blocchino l’ingresso di calcio nelle cellule, per il ridurre il rischio aritmico dei pazienti. Per dare la massima protezione antiaritmica è possibile impiantare un "defibrillatore automatico" (ICD), un apparecchio simile ad un "pacemaker" in grado di riconoscere e correggere eventuali episodi di fibrillazione ventricolare. Il defibrillatore è quindi in grado di salvare la vita dei portatori di TS, qualora si verifichino episodi di aritmie potenzialmente fatali. Si raccomanda di evitare i farmaci che allungano l’intervallo QT (elenco già riportato nella scheda del QT lungo). Per la prevenzione delle crisi ipoglicemiche si raccomanda di evitare periodi di digiuno prolungato e sono in studio farmaci che blocchino l’ingresso di calcio nelle cellule, per il trattamento di urgenza delle crisi ipoglicemiche, che non rispondono alla terapia classica. I problemi di eventuali malformazioni cardiache vengono corretti con elevata percentuale di successo e lo stesso vale per la correzione della sindattilia, dal momento che essa è solo mucocutanea. Si raccomanda di intraprendere subito terapia antibiotica mirata in caso di infezioni anche banali, Infine per quanto riguarda i problemi del linguaggio ed i problemi comportamentali è fondamentale i supporto del neuropsichatra infantile e del logopedista.

Redazione a cura di Telethon
con la consulenza scientifica della Dsa R. Bloise e della Prof.sa S.G.Priori
IRCCS Fondazione Salvatore Maugeri, Cardiologia Molecolare, Pavia
Ultimo aggiornamento: Settembre 2004

Sindrome di Apert

La sindrome di Apert (ACS I) è caratterizzata da craniosinostosi e sindattilia simmetrica (cutanea e/o ossea) delle mani e dei piedi. La prevalenza alla nascita è stimata intorno a 1:65000 nati vivi. La maggior parte dei casi sono sporadici anche se sono stati descritti casi familiari in cui la trasmissione è di tipo autosomico dominante.

In base al tipo di sindattilia, si distingue in Sindrome di Apert di tipo 1 (sindattilia del 2°,3° e 4° dito), di tipo 2 (del 2°,3°,4° e 5° dito) e di tipo 3 (sindattilia di tutte le dita).

In alcuni casi si ha ritardo mentale.

Questa sindrome è dovuta a mutazioni nel gene del Recettore dei Fattori di Crescita di tipo 2 (FGFR2), mappato in 10q25.3-q26.

Sono state individuate nell’esone 7 (IIIa) due mutazioni puntiformi che provocano sostituzione aminoacidica.

La prima, S252W si riscontra con una frequenza del 71% circa, la seconda, P253R con una frequenza del 26% circa. Diversi studi hanno evidenziato che non ci sono importanti differenze tra le due mutazioni per quanto riguarda i dismorfismi cranio-facciali, mentre le forme più gravi di sindattilia correlano usualmente con la mutazione P253R. Nei pazienti con ACS I la mutazione causante la patologia è stata trovata nel 99% dei casi ed è stato dimostrato che, nei casi sporadici, questa è sempre di origine paterna e che correla con l’età avanzata del padre.

Scheda tratta da RETEGEM
 (Rete di Genetica Medica)

Death of a Cell by Suicide

In the life sciences, death has become a hot topic

By William Allstetter

Columbia University Medical Center
www.cumc.columbia.edu  
download 27 agosto 2006

Most people have seen a cell divide. Short movies showing the sequential phases of mitosis are a mainstay of science education. But few have seen a cell die. That may be because, until recently, few people paid much attention to cell death.

Most scientists considered it the final, relatively dull endgame of a cell's existence. They believed that once toxic insult or injury destroys a cell's ability to carry on metabolism and maintain homeostasis, there is nothing to be done. The cell and its organelles swell, their membranes break apart, and the cell's contents spill into the intercellular space. Immune system cells and molecules sweep in to clean up the mess, causing inflammation and additional damage in the process. Necrosis, as it is called, was described long ago and offered little of interest to researchers.

In the 1990s, however, it has become abundantly clear that cell death is considerably more complex and interesting. Cells don. t always wait for deadly forces to do them in. They commit suicide, or, in a more scientific term, apoptosis.

In fact, each cell in our bodies contains a complex machinery of death. A host of genes, receptors, and enzymes carefully controls when and under what conditions a cell will kill itself. Some cells initiate apoptosis after chemical messengers bind to their "death receptors." In others, suicide is the default, preventable only by a constant stream of reassuring messages from other cells. Cells also respond to internal signals, condemning to death any cell that poses a threat to the larger organism.

"Apoptosis is a normal developmental and safety process," says Dr. Michael Shelanski, Delafield Professor and Chairman of Pathology. "It gets sick cells out of the way and makes sure we develop properly."

The hand of a developing fetus begins as a flat paddle. Apoptosis sculpts it into individual fingers. A developing brain makes more than twice the neurons it will eventually employ; ones that fail to make the right connections are eliminated. When genetic damage occurs, internal sentries, such as p53, halt cell division until repairs can be made. If the damage is beyond repair, suicide is invoked. Cells in the gut, skin, and elsewhere commit suicide every day as part of the normal maintenance of tissue.

But when the death machinery goes awry, disease can result. In neurodegenerative disorders, autoimmune disorders, and stroke, cells die prematurely. In cancer, cells fail to die when they should.

Today, apoptosis is one of the hottest topics in biomedical research. Basic scientists have a whole new phenomenon to examine and describe. Clinical scientists are hoping to cure several diseases by preventing or inducing apoptosis. Thousands of papers on apoptosis are published every year, and entire conferences are organized around individual elements of the process.

"Something about it intuitively appeals," says Dr. Beth Levine, assistant professor of medicine. "It has implications for so many aspects of biology."

Some complain that apoptosis has become the latest research bandwagon, with scientists seeing it where it doesn't exist and scrambling to include it in their grant applications. A handful of P&S researchers, however, were ahead of the curve, investigating apoptosis before it became the latest biological buzzword. They helped reveal basic elements of apoptosis in all cells. They have discovered how and where it occurs in both healthy and diseased tissue. They have even demonstrated its role in interspecies competition. Several are now developing therapies based on apoptosis.

Apoptosis was first described in a 1972 paper by John Kerr, Andrew Wyllie, and Alastair Currie, three researchers at the University of Aberdeen. The researchers also coined the term, which comes from the Greek word that describes the falling of leaves from a tree or petals from a flower. In contrast to the messy process of necrosis, apoptosis is quick and neat. Instead of swelling, a cell undergoing apoptosis shrivels and separates from its neighbors. Its DNA and organelles condense. The cell then "blebs," dividing into several small vesicles, which are consumed by neighboring cells. Apoptosis can occur in as little as 20 minutes and leaves no trace behind, which may explain why biologists failed for so long to see it.

Although the paper by Kerr, Wyllie, and Currie is now considered a classic, their concept of apoptosis languished, largely unnoticed, for many years. Then, in the mid-1980s Robert Horvitz at the Massachusetts Institute of Technology began investigating cell death in the nematode worm, C. elegans. He learned that precisely 131 of the worm's 1,092 cells disappear during development. In 1986 he published a paper identifying two genes, ced-3 and ced-4, involved in their disappearance. Then, in 1992, he found a third apoptosis gene, ced-9, which was nearly identical to bcl-2, a human gene implicated in lymphatic cancer. At that point, many biologists sat up and took notice of apoptosis. Clearly, if a gene controlling apoptosis in a worm had persisted through the eons of evolution to humans, then apoptosis must be a universally important biological function.

Columbia's Dr. Taka-Aki Sato and Dr. Thomas Franke have helped biologists understand pathways that transmit apoptotic signals from a receptor on the cell surface usually to the mitochondrion. The mitochondrion releases cytochrome c, which leads to the activation of caspases, the enzymatic executioners. The caspases, about a dozen of which have so far been identified, chew up the cell's DNA, destroy molecules that attach a cell to its neighbors, and dismantle structural proteins within the cell.

Taka-Aki Sato

Dr.Taka-Aki Sato, associate professor of molecular oncology (in otolaryngology/head & neck surgery and pathology), did much of his early work on the bcl-2 family of genes. The bcl-2 protein plays a central role in apoptosis. It sits on the outer membrane of the mitochondrion and inhibits apoptosis. Several closely related proteins, such as BAD, Bax, and bcl-xl, interact with bcl-2 and each other to either promote or inhibit apoptosis. Dr. Sato, in collaboration with Dr. John Reed at the Burnham Institute, identified, among other genes, BAG-1, a bcl-2 family member that inhibits apoptosis.

Several years ago Dr. Sato made the surprising discovery that yeast can serve as a powerful model for studying apoptosis. A single-celled organism would not be likely to have a suicide program, and yeast does not. But when two genes, antiapoptotic bcl-2 and its proapoptotic cousin Bax, are transferred into yeast, they establish a primitive form of the death machinery.

This allowed Dr. Sato to use a clever system, known as the yeast two-hybrid system, to find several other genes involved in apoptosis. When Dr. Sato has one protein in a pathway and wants to know what it interacts with, he attaches half the amino acids of a transcription factor to the protein he already knows. He then attaches the other half of the transcription factor to proteins that might interact with the original protein. If two proteins bind in their normal interaction, the two halves of the transcription also join together. The unified transcription factor turns on a marker gene, beta-galactosidase, which turns the yeast blue.

"Using a yeast system is a very powerful way to pull out proteins," says Dr. Sato.

P75NTR is a neural receptor whose stimulation can either induce apoptosis or prevent it, depending upon the tissue in which it is expressed. Dr. Sato recently discovered the gene for the first protein known to interact with P75NTR. The gene, which Dr. Sato named NADE, promotes apoptosis.

Dr. Sato hopes eventually to induce apoptosis in cancer cells. Early in his career, he worked for a company trying to develop Interluekin-2 as a cure for cancer. Interleukin-2 is a growth hormone-like substance that binds to receptors on cell surfaces. But interleukin-2's actions were non-specific, causing both positive and negative effects, depending upon the type of cell. Dr. Sato believed that the various apoptosis pathways offered more specific and promising targets for therapy.

"I thought apoptosis offered the fastest way to make a new drug against cancer," says Dr. Sato.

He has been able to induce apoptosis in colon cancer cells by injecting the cells with a tiny molecule that binds to FAP-1, a protein he discovered in the Fas signaling pathway. By binding to FAP-1, the tripeptide molecule kills colon-cancer cells by turning on their apoptotic machinery.

Thomas Franke and the Akt signaling pathway

Dr. Thomas Franke, assistant professor of pharmacology, has focused most of his work in apoptosis on the Akt pathway, so named for one of its central characters. Originally discovered as a viral oncogene, Dr. Franke cloned and characterized the cellular Akt gene in mice in 1993. Although he knew that the human homologue is overexpressed in some forms of ovarian cancer, it was considered an "orphan" because no one knew what function the Akt protein served or what molecules it interacts with. In 1995, Dr. Franke linked Akt to a growth-factor receptor, PDGF, and an important enzyme, PI3-kinase, which regulates numerous physiological processes. In 1997, Dr. Franke outlined the explicit biochemical steps leading from growth-factor receptors through PI3-kinase to the activation of Akt protein and showed that the pathway controlled cell survival in neurons and hematopoietic cells.

"The . orphan. kinase has now moved to center stage as a crucial regulator of life and death decisions emanating from the cell membrane," wrote Dr. Brian A. Hemmings in a commentary accompanying two papers in Science.

Since joining the P&S faculty in August 1997, Dr. Franke has helped identify many of the downstream targets of Akt, of which about a dozen have been identified. Last November he helped show that Akt inactivates caspase 9, the "effector" caspase that activates other caspases in the final march to death. In March he helped show that Akt promotes the production of nitric oxide in blood vessels. And in April he contributed to a paper showing that calcium influx induces apoptosis by triggering phosphorylation of BAD, a downstream target of Akt.

"It has been wonderful to see how this pathway has fallen into place," says Dr. Franke.

Dr. Franke is now turning his attention to the role of Akt in cancer and heart disease. He is studying how the expression of caspase 9 is altered in patients with ovarian cancer. He is also beginning a study of the role Akt plays in prevention of apoptosis in cardiac myocytes.

"You want your research to have human implications," says Dr. Franke. "Columbia is a very good environment for connecting basic research to clinical research."

The signaling pathways elucidated by Drs. Sato and Franke trigger apoptosis in many cells. But the biological role that apoptosis plays and the signals that trigger it vary from tissue to tissue. Several P&S researchers have studied the biology of apoptosis in varied tissues ranging from lymph nodes to neurons and the prostate.

Seth Lederman and B-cell selection

Dr. Seth Lederman, associate professor of medicine, showed how apoptosis helps select B cells that make infection-fighting antibodies. Each B cell is capable of making a unique antibody. As with many other cell types, apoptosis is the default program for B cells; they commit suicide before maturing unless they receive a signal that saves them. Helper T cells deliver the signal that saves B cells from apoptosis. Dr. Lederman described the molecules on the surfaces of B cells and T cells, known as CD40 and CD40-ligand, respectively, that interact to save B cells.

"Our studies showed how the antibody response develops through apoptosis and the rescue of selected cells," says Dr. Lederman.

The selection of B cells occurs in the germinal centers of lymph nodes. In those centers dendritic cells release a limited number of antigens, which are fragments of the invading organism. B cells compete to bind with the antigens. The B cells whose antibodies bind most strongly to the antigen, consume it, and present it on their surface. Helper

T cells bind to the B cell presenting those antigens and rescue it from death. The saved B cell then begins to proliferate.

But the antibody genes in B cells mutate frequently. As a result, many of the daughter cells produce antibodies slightly different from the parent B cell. The daughter cells once again compete to bind the antigen. The one making the antibody with the best fit, slightly better than the parent cell, is saved by the T cell. The B cells go through several rounds of this accelerated evolutionary process in which the "fittest" B cells survive to produce another generation of B cells. It eventually produces a B cell whose antibodies bind tightly to the antigen and neutralize the foreign body. At that point, it is released from the germinal center and begins producing massive numbers of antibodies.

"The process of selecting B cells and refining the structure of their antibody molecules is iterative," says Dr. Lederman. "Like other forms of evolution, it tinkers with the antibody until the structure is best."

Dr. Lederman believes apoptosis has transformed the field of physiology. Physiologists used to define homeostasis largely in terms of the plasma levels of various molecules, such as electrolytes or hormones. Now they take a more cellular approach. "Apoptosis has focused physiologists. attention on homeostatic mechanisms that balance cell division and death."

Ralph Buttyan: apoptosis in prostate cancer

Dr. Ralph Buttyan, associate professor of pathology (in urology), was probably the first person at P&S to study apoptosis, although he didn. t recognize its signature when he first observed it in 1984. Apoptosis was still an obscure subject at that time, but it was well known that prostate cells require the male steroid hormones known as androgens to survive. That is why various forms of castration therapy are used to treat advanced prostate cancers.

When Dr. Buttyan castrated rats with prostate cancer, most of the prostate cells did dutifully die. But Dr. Buttyan made the enigmatic observation that the prostate cells were becoming more active just before their demise, synthesizing new gene products that are often made by cells in the process of division.

"This was such an unexpected observation," says Dr. Buttyan 15 years later. "That's why I decided to pursue it."

A short time later, Dr. Buttyan came across the literature of Kerr and his colleagues describing apoptosis, which helped him understand what was occurring in those prostate cells and led to the publication of his findings in 1988. Several of the gene products he saw in the dying prostate cells, such as c-myc, c-fos, and p53, have since been proved to participate in apoptotic signaling and regulation. As Dr. Buttyan and others have since discovered, apoptosis is closely linked to mitosis.

"Proliferation is a good time to weed out bad cells," says Dr. Buttyan. "It presents the perfect opportunity for a cell to recognize internal genetic problems so that they can be eliminated before they are passed on to progenitors."

Dr. Buttyan has continued to study the role of apoptosis in the prostate, especially in prostate cancers. His recent studies have shown that castration induces apoptosis in endothelial cells lining the blood vessels of the prostate. When those cells die, the blood supply to the cancerous tissue is disrupted, depriving other cells in the prostate of oxygen. While oxygen deprivation eventually causes such damage that cells undergo necrosis, it appears that the prostate cells sense this impending disaster and trigger their own suicidal program, thus removing those cells in a more controlled and cleaner process.

"I now tell urologists, . In your use of castration therapy for prostate cancer, you have been using antiangiogenic ther-apy for 50 years,. " says Dr. Buttyan. Antiangiogenic therapy, in which tumors are starved of their blood supply, has received attention recently as a promising anticancer therapy.

Some prostate cancer cells inevitably survive castration. Dr. Buttyan has shown that these cells express high levels of the antiapoptotic protein bcl-2, making them resistant not only to castration but also to other important cancer therapies. Dr. Buttyan and associate research scientist Thambi Dorai have developed an antisense ribozyme that degrades bcl-2 messenger RNA in cells and destroys the ability of prostate cancer cells to make bcl-2 protein. They are now testing the approach in animal models and believe that it holds promise as a treatment for prostate cancer cells that resist other forms of therapy.

Dr. Bernard Weinstein, the Frode Jensen Professor of Medicine, says the concept of apoptosis has completely transformed the study of cancer. Until recently, cancer was viewed as a disease that results from the uncontrolled proliferation of cells. But today, it has become clear that the failure of cells to die at the appropriate time is just as important in the development of cancer.

"Apoptosis is a dominant theme in cancer research," says Dr. Weinstein. "The concept has provided insight into why drugs work or don. t work and has opened up whole new lines of research. Investigators are looking for new forms of cancer therapy by screening for drugs that cause apoptosis."

Lloyd Greene and neuronal survival

Dr. Lloyd Greene, professor of pathology (in the Center for Neurobiology and Behavior), has for many years studied the growth and differentiation of neurons. His primary tool has been a cell line, PC12, that he and Dr. Arthur Tischler developed in 1975. (Dr. Greene jokes that some of the PC12 cells in his laboratory are older than some of the people working there.) PC12 cells, originally derived from tumors in the adrenal glands of rats, develop into cells resembling sympathetic neurons when exposed to nerve growth factor (NGF). They are one of the few models of neuronal cells and are widely used in neuronal research.

In the early 1990s Dr. Greene became interested in how NGF promotes cell survival. One of his students, Anna Batistatou, suggested that depriving neurons of NGF might trigger apoptosis. Although researchers at a recent conference reached a consensus that apoptosis probably does not occur in neurons, Dr. Greene agreed to look for it anyway. Dr. Green and Ms. Batistatou became one of the first groups to show that neurons do indeed undergo apoptosis when deprived of growth factors.

In subsequent years, Dr. Greene, working with Drs. Carol Troy, Leonidas Stefanis, and Michael Shelanski, has described many features of neuronal apoptosis. The researchers learned that removal of NGF initiates changes in the expression and activity of molecules associated with the cell cycle. Mature neurons don't normally divide.

"When you turn on the cell cycle in neurons, then they are in trouble and can die," says Dr. Greene.

But Dr. Greene and his colleagues have uncovered evidence that the signal turning on the cell-cycle genes cannot initiate apoptosis by itself. It appears that a second signaling pathway, one normally associated with stress, also must be stimulated for neurons to undergo apoptosis. Dr. Greene believes neurons may need two independent signals to begin apoptosis because they are so important to an organism's survival and quite difficult to replace.

"You don. t want neurons to make a mistake and die if they don. t have to," says Dr. Greene. His "two-key" hypothesis equates neuronal apoptosis to nuclear missiles; both require two independent signals for their launch.

Dr. Greene has turned his attention to the mitochondrion and the changes it undergoes before releasing cytochrome c. He is using a powerful technique developed by cancer researcher Bert Vogelstein to determine not only genes newly expressed during apoptosis, but also changing levels of gene expression. This technique, called SAGE, requires high-throughput sequencing of genes. To that end, Drs. Greene and Shelanski have purchased an AB prism 3700, the fastest gene sequencer available and the first one in New York City.

Dr. Greene says he is most surprised by the fact that the three genes in C. elegans originally discovered by Robert Horvitz describe the basic process of apoptosis in higher animals. One gene codes for the enzyme that cleaves the cell's proteins. One codes for a protein that activates that enzyme, and another codes for a protein that inhibits apoptosis.

"In mammalian cells, it has become much more baroque," says Dr. Greene. "At the beginning I thought it would be a switch. You turn it on and the cell dies. It's not that easy. The more I think and read about this the more complex it gets. It's getting embarrassingly complex."

Robert Burke and Parkinson's disease

Neurons undergo apoptosis in several neurodegenerative diseases, including Alzheimer' s disease, Huntington's disease, and amyotrophic lateral sclerosis. But the exact role of apoptosis is unclear.

"Is it part of the disease? Can you stop it and make cells survive? Or is the neuron so damaged that it must be removed?" asks Dr. Shelanski.

Dr. Robert Burke, professor of neurology (in pathology), has focused on unraveling the role of apoptosis in Parkinson's disease. In that movement disorder, dopamine-emitting neurons in the substantia nigra region of the brain die prematurely. In the early 1990s Dr. Burke discovered that the dopamine neurons undergo apoptosis during development if the target neurons are destroyed. The target neurons are believed to provide growth factors to prevent the default apoptosis. Dr. Burke has also shown that dopamine neurons undergo apoptosis in a rat model of Parkinson's disease.

"There's no question that apoptosis occurs in dopamine neurons," says Dr. Burke.

The key will be showing definitively that the dopamine neurons in the substantia nigra undergo apoptosis in the brains of Parkinson's patients. But it can be very hard to spot since apoptosis occurs rapidly and leaves no trace. Dr. Burke has discovered that those neurons undergoing apoptosis in rats express the caspase 3 gene. He plans to search for expression of that gene in the brains of Parkinson's patients who have died.

Beth Levine and apoptosis in viruses

Apoptosis plays a role in more than just the proper development and protection of a multicellular organism. It is a weapon in the conflict between species. When a cell becomes infected by a virus, it often undergoes apoptosis.

"The cell commits this altruistic suicide for the good of the organism," says Dr. Beth Levine, assistant professor of medicine. "Apoptosis may have evolved as a defense mechanism."

But in the arms race of interspecies conflict, organisms often evolve new ways to overcome the defenses. Viruses want a cell to survive long enough for the viruses inside to reproduce many times. Then the cell lyses, releasing the new viruses to infect other cells.

"Viruses want to keep a cell alive. They have evolved a number of defenses to block apoptosis," says Dr. Levine. One of the first people to study apoptosis in connection with viruses, Dr. Levine explains that many viruses have picked up antiapoptotic genes from the cells they infect. For example, in 1997 Dr. Sato and Dr. Yuan Chang, associate professor of pathology, showed that the Kaposi's sarcoma-associated herpesvirus expresses a bcl-2 gene that prevents infected cells from committing suicide.

Most of Dr. Levine's work has focused on the Sindbis virus, one of a family of RNA viruses that can cause acute encephalitis. The Sindbis virus is an evolutionarily simple virus with such a tiny genome that it has no gene to prevent apoptosis. But it has developed its own strategy for avoiding removal by apoptosis.

"Sindbis virus preferentially infects neurons, which are more resistant to virus-induced apoptosis," says Dr. Levine.

Most virally infected cells present a signal on their surface that tells killer T cells to destroy them. But neurons do not. As a result the immune system can. t kill the infected neurons, allowing the virus to persist in those cells. Dr. Levine showed that the Sindbis virus, previously believed to cause only acute infections, persists indefinitely in neurons. She believes other viruses also may establish persistent infections within neurons. "Our neurons are probably loaded with viruses," says Dr. Levine.

Dr. Levine also showed that bcl-2 protects neurons from the apoptosis that occurs when Sindbis infects other cell types. Bcl-2 also slows the replication of the Sindbis virus inside neurons. Recently Dr. Levine discovered a new gene, beclin 1, whose protein product interacts with bcl-2 to protect neurons against apoptosis. Counterintuitively, it also appears to play a role in tumor suppression and is mutated in several breast-cancer cell lines. Her most recent work has identified beclin 1 as the first mammalian gene known to control the process of autophagy.

"Autophagy is the process by which cells degrade their own cytoplasmic contents," says Dr. Levine. "That process is defective in tumor cells, which raises the interesting possibility that there are genetic links between the regulation of apoptosis, tumor suppression, and autophagy."

Future directions and therapy

Many more people at P&S are studying apoptosis in a variety of contexts. Among them are Dr. Jean Gautier, assistant professor of genetics and development (in dermatology), who has described apoptosis in the earliest stages of development in the frog, Xenopus. Dr. Ramon Parsons, assistant professor of pathology, has shown the tumor suppressor he co-discovered, PTEN, is an antiapoptic element in the Akt signaling pathway. Dr. Steven Moss, assistant professor of medicine, has shown that Helicobacter pylori, the bacterium associated with ulcers and other gastrointestinal disorders, induces apoptosis in the gut. Dr. Abraham Spector, the Malcolm P. Aldrich Research Professor of Ophthalmology, has shown that apoptosis contributes to the development of cataracts in the eyes. P&S researchers also have seen evidence of apoptosis in stroke, heart attacks, and diseases of the kidney.

It is also becoming clear that several anticancer therapies work by promoting apoptosis. DNA damage caused by radiation causes apoptosis. Several established chemotherapies, such as taxol, also have been shown to induce apoptosis.

New therapies developed as a result of new knowledge about apoptosis are mostly in the preclinical stages. Several researchers have shown promising results in cell-culture and animal studies. The drug sulindac sulfone, a derivative of the non-steroidal anti-inflammatory drug sulindac, is being tested in clinical trials as a therapy against prostate cancer. Dr. Weinstein first showed that the drug and related compounds induce apoptosis in human prostate cancer cells in culture. This occurs even if the cancer cells lack the important tumor suppressor gene p53, have increased expression of bcl-2, or have become androgen-independent. Based on those findings, Dr. Erik Goluboff, assistant professor of urology, has begun a Phase III trial of sulindac sulfone on 90 patients whose prostates have been removed but whose rising levels of prostate-specific antigen indicate that the cancer has not been vanquished.

Apoptosis has brought a certain balance to biology. Scientists are now learning as much about the end of a cell's life as they already know about its beginning. And in its complexity they see opportunities for therapy. They realize that a better understanding of death may one day save lives.