ApoptosisApoptosis:: Architecture of ChromosomalArchitecture of Chromosomal Territories in Apoptotic CellsTerritories in Apoptotic Cells E. Bártová •Institute of Biophysics Academy of Sciences of the Czech Republic Cellular death-by-suicide is part of normal development, and is termed apoptosis or programmed cell death (PCD). Cysteine Aspartate Specific ProteASEs – caspases – are active in apoptosis, as are p53, a tumor suppressor gene, and FAS gene, which is member 6 of the tumor necrosis factor receptor superfamily (TNF). In contrast to apoptosis, necrosis is cell death that results from cytotoxic, injurious stresses that are too severe for correction by the cellular stress response. ApoptosisApoptosis isis a parta part ofof normalnormal cellcell turnoverturnover andand tissuetissue homeostasishomeostasis „„HistoryHistory““ ofof molecularmolecular biologybiology ofof cellcell deathdeath Horvitz (1992Horvitz (1992--3)3) identification of „cell death genes“ inidentification of „cell death genes“ in CaenorhabditisCaenorhabditis eleganselegans {{cedced--33 (ICE),(ICE), cedced--44 (0),(0), cedced--99 ((bclbcl--22)})} ((CerrettiCerretti 1992, Thornberry 1992)1992, Thornberry 1992) uncovering of the homologyuncovering of the homology betweenbetween cedced--33 gene product and ICEgene product and ICE (interleukin(interleukin--11ββ convertingconverting enzymeenzyme))]] proteaseprotease 1990 Discovery of new family of mammalian cysteine proteases - CASPASES KerrKerr et alet al., 1972:., 1972: IdentificationIdentification ofof thethe cellcell deathdeath APOPTOSISAPOPTOSIS KerrKerr,, WylieWylie andand CurrieCurrie Apoptosis: a basic biological pehenomenon with wide-ranging implications in tissue kinestics. Br. J.Cancer 1972;26:239-257 ApoptosisApoptosis isis involvedinvolved in ain a widewide rangerange ofof physiologicalphysiological andand pathologicalpathological processesprocesses.. >> DevelopmentDevelopment ((embryonicembryonic,, neuronalneuronal developmentdevelopment)) >> InflammationInflammation andand involutioninvolution ofof tissuestissues > In> In thethe immuneimmune systemsystem ((ApoptosisApoptosis isis employedemployed as aas a methodmethod ofof cytotoxiccytotoxic TT--cellcell mediatedmediated killingkilling ofof infectedinfected cellscells)) > In> In ageingageing ApoptosisApoptosis playsplays aa pivotalpivotal role inrole in thethe pathophysiologypathophysiology ofof ageingageing'.'. TheThe freefree radicalradical theorytheory ofof ageingageing linkslinks senescencesenescence toto damagedamage inflictedinflicted byby superoxidesuperoxide--derivedderived radicalsradicals andand otherother oxidantsoxidants generatedgenerated primarilyprimarily inin mitochondrialmitochondrial respirationrespiration.. TheThe mitochondrialmitochondrial theorytheory ofof ageingageing,, proposesproposes thatthat ageingageing isis thethe resultresult ofof accumulatedaccumulated freefree radicalradical damagedamage toto mitochondrialmitochondrial DNA (DNA (mtDNAmtDNA).). TheThe accumulationaccumulation ofof errorserrors inin mtDNAmtDNA leadsleads toto errorserrors inin thethe polypeptidespolypeptides encodedencoded byby mtDNAmtDNA,, i.e.,i.e., thethe fourfour mitochondrialmitochondrial enzymaticenzymatic complexescomplexes.. DefectiveDefective complexescomplexes produceproduce moremore freefree radicalsradicals leadingleading to ato a viciousvicious cyclecycle ofof increasingincreasing mtDNAmtDNA damagedamage,, radicalradical generationgeneration,, andand possiblypossibly apoptosisapoptosis ApoptosisApoptosis inin contrastcontrast toto necroticnecrotic cellcell deathdeath RocheRoche: Cell: Cell DeathDeath -- ApoptosisApoptosis andand NecrosisNecrosis UvolněníUvolnění lysosomálníchlysosomálních enzymůenzymů NuclearNuclear morfologymorfology in HLin HL--60 cells60 cells (P. Mlejnek 2001)(P. Mlejnek 2001) I) II) III) I) Control II) Apoptosis III) Necrosis Cell death classification by Clarke CM – cyt. membrane J – nuclei M – mitochondrion ER – endopl. reticulum GA – Golgy complex L – lysosomes Apoptosis Autophagy Nelysosomal disintegration Cell death classification by ClarkCell death classification by Clarkee AApoptopoptosissis AutophagyAutophagy NonlysosomalNonlysosomal disintegrationdisintegration - heterophagy, final cell destruction is done by lysosomes of other cells - final cell destruction is done by its own lysosomes - cell destruction is mediated by unknown nonlysosomal proteases AnoikisAnoikis is a form of programmed cell death which is induced by anchorageis a form of programmed cell death which is induced by anchorage--dependent cells detachingdependent cells detaching from the surroundingfrom the surrounding extracellularextracellular matrix (ECM)[1]. Usually cells stay close to the tissue to whicmatrix (ECM)[1]. Usually cells stay close to the tissue to which theyh they belong since the communication between proximal cells as well asbelong since the communication between proximal cells as well as between cells and ECM providebetween cells and ECM provide essential signals for growth or survival. When cells are detacheessential signals for growth or survival. When cells are detached from the ECM, i.e. there is a loss ofd from the ECM, i.e. there is a loss of normal cellnormal cell--matrix interactions, they may undergomatrix interactions, they may undergo anoikisanoikis. However,. However, metastaticmetastatic tumor cells may escapetumor cells may escape fromfrom anoikisanoikis and invade other organs.and invade other organs. MorphologicalMorphological featuresfeatures ofof apoptosisapoptosis ScanningScanning electronelectron micrographmicrograph TransmissionTransmission electronelectron micrographmicrograph CC--KnudsonKnudson@@uniowa.eduuniowa.edu Apoptotic DNA degradation is followedApoptotic DNA degradation is followed byby phogocytosisphogocytosis of apoptotic bodiesof apoptotic bodies RichRich etet alal.,., NatureNature 20002000 HengartnerHengartner M.O.,M.O., NatureNature 20002000 TwoTwo majormajor apoptoticapoptotic pathwayspathways inin mammalianmammalian cellscells DeathDeath--receptorreceptor pathwaypathway:: DeathDeath receptorreceptor superfamilysuperfamily:: CD95 receptorCD95 receptor andand tumourtumour necrosisnecrosis factorfactor receptor. CD95receptor. CD95 ligandligand bindsbinds to CD95 receptorto CD95 receptor -- toto formform death inducing signalingdeath inducing signaling complexcomplex.. ThisThis complexcomplex recruitsrecruits viavia thethe adaptoradaptor moleculemolecule FADDFADD ((FasFas--associatedassociated deathdeath domaindomain protein).protein). ProcaspaseProcaspase 88 bindsbinds toto this complex in orderthis complex in order toto activateactivate CaspaseCaspase--88 andand subsequentysubsequenty activation ofactivation of CaspaseCaspase--33 is inducedis induced.. Activation ofActivation of procaspaseprocaspase--88 can becan be blockedblocked throughthrough degenerate caspasedegenerate caspase homoloquehomoloque cc--FLIP.FLIP. HengartnerHengartner M.O.,M.O., NatureNature 20002000 TwoTwo majormajor apoptoticapoptotic pathwayspathways inin mammalianmammalian cellscells The mitochondrial pathwayThe mitochondrial pathway -- activated afteractivated after DNADNA damagedamage -- proapoptoticproapoptotic membersmembers ofof BclBcl--22 familyfamily,, locatedlocated onon thethe surfacesurface ofof mitochondriamitochondria,, areare activatedactivated -- CytochromeCytochrome cc is releasedis released from mitochondriafrom mitochondria andand forms complex withforms complex with ApafApaf--11 andand ProcaspaseProcaspase 9.9. -- TheThe complexcomplex isis calledcalled APOPTOSOME.APOPTOSOME. BothBoth apoptoticapoptotic pathwayspathways convergeconverge onon thethe levellevel ofof CaspaseCaspase--33 activationactivation CaspaseCaspase--33 activationactivation isis antagonizedantagonized by IAPby IAP releasedreleased fromfrom mitochondriamitochondria Sigma (Sigma (ApoptosisApoptosis andand LifeLife Science)Science) www.www.cellsignalcellsignal..comcom DNADNA repairrepair DNA damage stimulates apoptosis. For example p53 is a tumour suppressor gene. MDM2 inhibits the activity of p53 participating in the ubiquitination of p53. p53 is activated when MDM2 is inhibited by signalling from factors such as DNA damage. p53 is a transcription factor. Active p53 induces the transcription of many genes, including Bax, which promotes apoptosis by stimulating the release of cytochrome c and the formation of apoptosomes. PARP-1 is a nuclear enzyme involved in DNA repair. When overactive, it can cause apoptosis or necrosis. PARP-1 is activated by single stranded DNA. Active PARP-1 cleaves NAD+ as shown in figure. Cleavage of NAD+ by PARP-1. PARP-1 catalyses the addition of an ADP-ribose polymer of 50-200 residues to nuclear proteins such as histones, which stimulates DNA repair enzymes. However, overactive PARP-1 causes depletion of NAD+, and consequently the depletion of ATP. • ATP depletion leads to ion pump failure. The cell swells and bursts due to osmotic pressure. This is necrosis. • Alternatively, the depletion of NAD+ from mitochondria appears to induce AIF translocation from the mitochondria to the cytoplasm. This leads to apoptosis. • There may be a PARP-1 activity threshold, which determines whether the cell engages in DNA repair, apoptosis or necrosis. Apoptosis is ATP dependent. Apoptosis involves chromatin fragmentation, which would be predicted to cause PARP-1 overactivity and drive the cell into necrosis. DNADNA fragmentationfragmentation duringduring apoptosisapoptosis 1.1. High molecular weight DNAHigh molecular weight DNA fragmentation (50fragmentation (50--300300 kbpkbp)) 2.2. OligonucleosomalOligonucleosomal DNADNA fragmentation (180fragmentation (180--200200 bpbp)) 3.3. SingleSingle-- strand cleavagestrand cleavage BortnerBortner C.D.C.D. etet alal., 1995., 1995 APOPTOSIS DETECTION DNA fragmentation test LargeLarge and oligonucleosomaland oligonucleosomal DNADNA fragmentation infragmentation in apoptoticapoptotic cellscells (M. Fojtová, BFÚ Brno)(M. Fojtová, BFÚ Brno) FieldField inversioninversion electrophoresiselectrophoresis (FIGE)(FIGE) DNADNA fragmentationfragmentation testtest PolyPoly(ADP(ADP--ribosylribosyl))ationation andand apoptosisapoptosis AntiAnti--PARP p85PARP p85 fragmentfragment pAbpAb WesternWestern blotsblots andand detectiondetection ofof apoptosisapoptosis Lamin BLamin B PARPPARP cleavagecleavage 1. Etoposide 2. Cis-platin 3. Vincristine 4. Gamma-irradiation 5. Serum deprivation Apoptosis was detected in human erythroleukemia cell line K-562 and human retinoblastoma cell line Y79 ControlControl SerumSerum deprivationdeprivation EtoposideEtoposide AnnexinAnnexin VV / PI/ PI Hoechst33342Hoechst33342 / PI/ PI ControlControlEtoposideEtoposide AnexinAnexin VV bindsbinds toto phosphatidylserinesphosphatidylserines thatthat areare traslocatedtraslocated fromfrom thethe innerinner sideside odod thethe plasmaplasma membranemembrane toto thethe cellcell surfacesurface soonsoon afterafter thethe inductioninduction ofof apoptosisapoptosis CaspSCREENCaspSCREEN ((tmtm)) BioVisionBioVision kitkit TMRETMRE EtoposideEtoposide ControlControl TheThe resultsresults ofof TUNEL testTUNEL test Nuclear organisationNuclear organisation of chromosomalof chromosomal territoriesterritories ((CremerCremer T.T. andand CremerCremer C., 2001)C., 2001) Territory of chromosomeTerritory of chromosome 1111 andand 1717 Territory of chromosomeTerritory of chromosome 33 ArcitectureArcitecture of chromosomal territoriesof chromosomal territories duringduring apoptosisapoptosis ApoptosisApoptosis andand HSA 21 in KHSA 21 in K--562562 cellscells ApoptosisApoptosis andand chromosomalchromosomal territoryterritory andand centromericcentromeric regionregion ofof HSA 11 in KHSA 11 in K-- 562562 leukemicleukemic cellscells RetinoblastomaRetinoblastoma Y79Y79 cellscells andand HSRHSR MYCN (2p24)MYCN (2p24) TUNEL and PI staining of fixed cells ApoptosisApoptosis inin patientpatient sufferingsuffering fromfrom retinoblastomaretinoblastoma TUNELTUNEL andand DAPIDAPI stainingstaining A.A. LiepinsLiepins/SPL/SPL ConclusionsConclusions ** Differences in DNA fragmentationDifferences in DNA fragmentation ** Differences in the number of nuclear apoptotic bodiesDifferences in the number of nuclear apoptotic bodies * Chromosomal territories cleaved into high molecular* Chromosomal territories cleaved into high molecular DNA fragments were variably disassembled intoDNA fragments were variably disassembled into apoptotic bodies whose induction is the main effort ofapoptotic bodies whose induction is the main effort of anticancer therapy.anticancer therapy. * Apoptotic nuclear segmentation can be observed at* Apoptotic nuclear segmentation can be observed at centromericcentromeric regions.regions. * Disassembly of chromosomal territories was also* Disassembly of chromosomal territories was also found in prefound in pre--apoptotic (TUNEL positive) nuclei.apoptotic (TUNEL positive) nuclei. * Apoptosis can be observed not only after experimental* Apoptosis can be observed not only after experimental and/or clinicaland/or clinical treatment but also spontaneouslytreatment but also spontaneously..