Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

University of Leicester

During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.

Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes.

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.

Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.

&#x6CDB;&#x7D20;/&#x86CB;&#x767D;&#x9176;&#x4F53;&#x7CFB;&#x7EDF;&#x7684;&#x6709;&#x8FB1;&#x4EBA;&#x683C;&#x7684; polyubiquitinated &#x86CB;&#x767D;&#x8D28;&#x8C03;&#x8282;&#x86CB;&#x767D;&#x8D28;&#x5468;&#x8F6C;&#x3002;&#x5230;&#x76EE;&#x524D;&#x4E3A;&#x6B62;&#xFF0C;&#x6240;&#x6709;&#x7814;&#x7A76;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x4E0E;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x7684;&#x5173;&#x7CFB;&#x90FD;&#x5F3A;&#x8C03;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x5728;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#xFF0C;&#x6839;&#x636E;&#x5176;&#x80FD;&#x529B;&#x964D;&#x4F4E;&#x76D1;&#x7BA1;&#x5206;&#x5B50;&#x53C2;&#x4E0E;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x7684;&#x8C03;&#x63A7;&#x4E2D;&#x53D1;&#x6325;&#x7684;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x3002;&#x6211;&#x4EEC;&#x73B0;&#x5728;&#x6F14;&#x793A;&#x5982;&#x4F55;&#x8BF1;&#x5BFC;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x53EF;&#x89C4;&#x7BA1;&#x7684;&#x6D3B;&#x6027;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x3002;&#x671F;&#x95F4;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x3001; &#x534A;&#x80F1;&#x6C28;&#x9178;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x7ED3;&#x679C;&#x4E2D;&#x7684;&#x4E09;&#x4E2A;&#x7279;&#x5B9A;&#x4E9A;&#x57FA;&#x7684; 19 &#x5C81;&#x76D1;&#x7BA1;&#x590D;&#x6742;&#x7684;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x88C2;&#x89E3;&#xFF1A; S6&#39; (Rpt5) &#x548C; S5a (Rpn10)&#xFF0C;&#x5176;&#x4F5C;&#x7528;&#x662F;&#x8BC6;&#x522B; polyubiquitinated &#x5E95;&#x7269;&#x7684;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x548C; S1 &#xFF08;Rpn2&#xFF09;&#xFF0C;&#x4E0E; S5a &#x548C; S2 &#x7684; (Rpn1) &#x5171;&#x540C;&#x6301;&#x6709;&#x7684;&#x76D6;&#x5B50;&#x548C; 19 &#x5C81;&#x7BA1;&#x7406;&#x590D;&#x6742;&#x7684;&#x57FA;&#x7840;&#x3002;&#x8FD9;&#x79CD;&#x534A;&#x80F1;&#x6C28;&#x9178;&#x86CB;&#x767D;&#x9176;&#x4ECB;&#x5BFC;&#x5206;&#x88C2;&#x72B6;&#x51B5;&#x6291;&#x5236;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x964D;&#x89E3;&#x7684;&#x6CDB;&#x7D20;&#x4F9D;&#x8D56;&#x548C;&#x72EC;&#x7ACB;&#x7684;&#x7EC6;&#x80DE;&#x57FA;&#x8D28;&#xFF0C;&#x5305;&#x62EC;&#x6D46;&#x6DB2;&#x7684;&#x5206;&#x5B50;&#xFF0C;&#x4F8B;&#x5982; Smac&#xFF0C;&#x6240;&#x4EE5;&#x901A;&#x8FC7;&#x63D0;&#x4F9B;&#x4E00;&#x4E2A;&#x524D;&#x9988;&#x6269;&#x589E;&#x5FAA;&#x73AF;&#x4FC3;&#x8FDB;&#x51CB;&#x4EA1;&#x7A0B;&#x5E8F;&#x7684;&#x6267;&#x884C;&#x3002;

&#x534A;&#x80F1;&#x6C28;&#x9178;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x671F;&#x95F4;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x6291;&#x5236;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x529F;&#x80FD;&#x3002;

The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

Caspase activation inhibits proteasome function during apoptosis.

Le syst&#xE8;me ubiquitine / prot&#xE9;asome r&#xE9;gule le renouvellement des prot&#xE9;ines par la d&#xE9;gradation des prot&#xE9;ines polyubiquitinated. &#xC0; ce jour, toutes les &#xE9;tudes sur la relation de l&#x26;#39;apoptose et le prot&#xE9;asome ont soulign&#xE9; le r&#xF4;le cl&#xE9; du prot&#xE9;asome dans la r&#xE9;gulation de l&#x26;#39;apoptose, en vertu de sa capacit&#xE9; &#xE0; d&#xE9;grader les mol&#xE9;cules de r&#xE9;gulation impliqu&#xE9;s dans l&#x26;#39;apoptose. Nous avons maintenant d&#xE9;montrer comment l&#x26;#39;induction de l&#x26;#39;apoptose peut r&#xE9;guler l&#x26;#39;activit&#xE9; du prot&#xE9;asome. Au cours de l&#x26;#39;apoptose, les r&#xE9;sultats activation des caspases dans le clivage de trois sous-unit&#xE9;s sp&#xE9;cifiques du complexe 19S du prot&#xE9;asome r&#xE9;glementaire: S6 &#xBB;(Rpt5) et S5a (Rpn10), dont le r&#xF4;le est de reconna&#xEE;tre substrats polyubiquitinated du prot&#xE9;asome, et S1 (Rpn2), qui, avec S5a et S2 (Rpn1) maintient ensemble le couvercle et la base du complexe 19S r&#xE9;glementaire. Ce clivage de la caspase-m&#xE9;di&#xE9;e inhibe la d&#xE9;gradation du prot&#xE9;asome ubiquitine-d&#xE9;pendante et ind&#xE9;pendants substrats cellulaires, notamment mol&#xE9;cules proapoptotiques comme Smac, de mani&#xE8;re &#xE0; faciliter l&#x26;#39;ex&#xE9;cution du programme apoptotique en fournissant un circuit d&#x26;#39;amplification &#xE0; anticipation.

L&#x26;#39;activation des caspases inhibe la fonction du prot&#xE9;asome cours de l&#x26;#39;apoptose

Caspase-Aktivierung hemmt Proteasom-Funktion w&#xE4;hrend der Apoptose.

Il sistema ubiquitina/proteasoma regola il turnover della proteina degradando le proteine polyubiquitinated. Ad oggi, tutti gli studi sul rapporto tra apoptosi e proteasoma hanno sottolineato il ruolo chiave del proteasoma nella regolazione dell&#39;apoptosi, in virt&ugrave; della sua capacit&agrave; di degradare molecole regolatrici coinvolti nell&#39;apoptosi. Mostriamo ora come induzione di apoptosi pu&ograve; disciplinare l&#39;attivit&agrave; del proteasoma. Durante il apoptosis, attivazione di caspase provoca la scissione delle tre specifiche subunit&agrave; del complesso normativo 19S del proteasoma: S6&#39; (Rpt5) e S5a (Rpn10), cui il ruolo &egrave; di riconoscere polyubiquitinated substrati del proteasoma e S1 (Rpn2), che con S5a e S2 (Rpn1) tiene insieme il coperchio e la base del complesso normativo 19S. Questa scissione caspase-mediata inibisce la degradazione proteosomale di substrati cellulari ubiquitina-dipendente e - indipendenti, tra cui Miselli molecole come Smac, facilitando cos&igrave; l&#39;esecuzione del programma apoptotico fornendo un ciclo di amplificazione feed-forward.

Attivazione di caspase inibisce la funzione del proteasoma durante il apoptosis.

&#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;/&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x7CFB;&#x3092;&#x4F4E;&#x4E0B;&#x3055;&#x305B;&#x308B;&#x30DD;&#x30EA;&#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x5316;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306B;&#x3088;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x4EE3;&#x8B1D;&#x56DE;&#x8EE2;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x73FE;&#x5728;&#x307E;&#x3067;&#x306B;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3068;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x95A2;&#x4FC2;&#x306B;&#x3064;&#x3044;&#x3066;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x8ABF;&#x7BC0;&#x5206;&#x5B50;&#x3092;&#x5206;&#x89E3;&#x3059;&#x308B;&#x80FD;&#x529B;&#x306E;&#x304A;&#x304B;&#x3052;&#x3067;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x5236;&#x5FA1;&#x306B;&#x304A;&#x3051;&#x308B;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x3066;&#x304D;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x4ECA;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x8A98;&#x5C0E;&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x6D3B;&#x6027;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306E;19S&#x8ABF;&#x7BC0;&#x8907;&#x5408;&#x4F53;&#x306E;3&#x3064;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x5207;&#x65AD;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x9593;&#x306B;&#x3001;&#x30AB;&#x30B9;&#x30D1;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x7D50;&#x679C;&#xFF1A;S6 &#x26;quot;&#xFF08;Rpt5&#xFF09;&#x3068;&#x5F79;&#x5272;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x30DD;&#x30EA;&#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x5316;&#x57FA;&#x8CEA;&#x3092;&#x8A8D;&#x8B58;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3042;&#x308B;S5A&#xFF08;Rpn10&#xFF09;&#x3068;&#x3001;S1&#xFF08;Rpn2&#xFF09; S5A&#x3068;S2&#x3068;&#x306A;&#x308B;&#xFF08;Rpn1&#xFF09;&#x304C;&#x4E00;&#x7DD2;&#x306B;19S&#x8ABF;&#x7BC0;&#x8907;&#x5408;&#x4F53;&#x306E;&#x84CB;&#x3068;&#x30D9;&#x30FC;&#x30B9;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30AB;&#x30B9;&#x30D1;&#x30FC;&#x30BC;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x5207;&#x65AD;&#x306E;&#x3067;&#x30D5;&#x30A3;&#x30FC;&#x30C9;&#x30D5;&#x30A9;&#x30EF;&#x30FC;&#x30C9;&#x5897;&#x5E45;&#x30EB;&#x30FC;&#x30D7;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3001;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x306E;&#x5B9F;&#x884C;&#x3092;&#x5BB9;&#x6613;&#x306B;&#x3059;&#x308B;&#x306A;&#x3069;&#x3001;Smac&#x306E;&#x3088;&#x3046;&#x306A;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x4FC3;&#x9032;&#x5206;&#x5B50;&#x3092;&#x542B;&#x3080;&#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x304A;&#x3088;&#x3073;&#x975E;&#x4F9D;&#x5B58;&#x6027;&#x7D30;&#x80DE;&#x57FA;&#x8CEA;&#x306E;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306B;&#x3088;&#x308B;&#x5206;&#x89E3;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x3002;

&#x30AB;&#x30B9;&#x30D1;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x306F;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x969B;&#x306B;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x6A5F;&#x80FD;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;

&#xC720;&#xBE44;&#xD034;&#xD2F4;/&#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880; &#xC2DC;&#xC2A4;&#xD15C; polyubiquitinated &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xC800;&#xD558; &#xD558; &#xC5EC; &#xB2E8;&#xBC31;&#xC9C8; &#xD68C;&#xC804;&#xC728;&#xC744; &#xC81C;&#xC5B4;. Apoptosis&#xB294; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;&#xC758; &#xAD00;&#xACC4;&#xC5D0; &#xB300; &#xD55C; &#xBAA8;&#xB4E0; &#xC5F0;&#xAD6C;&#xB294; apoptosis, apoptosis&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xADDC;&#xC81C; &#xBD84;&#xC790; &#xC800;&#xD558; &#xB420; &#xC218; &#xC788;&#xB2E4;&#xB294; &#xBBF8; &#xB355;&#xC758; &#xADDC;&#xCE59;&#xC5D0; &#xC788;&#xB294; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;&#xC758; &#xD575;&#xC2EC;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xAC15;&#xC870; &#xD558; &#xACE0; &#xC788;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC9C0;&#xAE08; apoptosis &#xC720;&#xB3C4; &#xC218;&#xB294; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;&#xC758; &#xD65C;&#xB3D9;&#xC744; &#xC870;&#xC808; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. Caspase&#xC758; &#xD65C;&#xC131;&#xD654;&#xB294; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;&#xC758; 19S &#xADDC;&#xC81C; &#xB2E8;&#xC9C0; &#xC138; &#xAC00;&#xC9C0; &#xD2B9;&#xC815; subunits &#xBD84;&#xC5F4; &#xADC0;&#xCC29;&#xB418; apoptosis, &#xC911;: S6&#39; (Rpt5) &#xBC0F; S5a (Rpn10) &#xC5ED;&#xD560; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;, s 1&#xC758; polyubiquitinated &#xAE30;&#xD310; &#xC778;&#xC2DD; &#xD558;&#xB294; &#xAC83; (Rpn2) s5a&#xC640; s 2&#xB294; &#xB69C;&#xAED1;&#xACFC; &#xADDC;&#xC81C; 19S &#xBCF5;&#xD569;&#xCCB4;&#xC758; &#xAE30;&#xC9C0; (Rpn1) &#xD568;&#xAED8; &#xBCF4;&#xC720; &#xD558;. Caspase &#xC911;&#xC7AC; &#xBD84;&#xC5F4;&#xC774; &#xC720;&#xBE44;&#xD034;&#xD2F4; &#xC885;&#xC18D; &#xBC0F; &#xB3C5;&#xB9BD; &#xC140;&#xB8F0;&#xB7EC; &#xAE30;&#xD310;, proapoptotic &#xBD84;&#xC790; Smac, &#xADF8;&#xB798;&#xC11C; &#xD53C;&#xB4DC; &#xD3EC;&#xC6CC;&#xB4DC; &#xC99D;&#xD3ED; &#xB8E8;&#xD504;&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xC5EC; apoptotic &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC758; &#xC2E4;&#xD589;&#xC744; &#xCD09;&#xC9C4; &#xB4F1;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; proteasomal &#xC800;&#xD558;&#xB97C; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;.

Caspase &#xD65C;&#xC131;&#xD654; apoptosis &#xB3D9;&#xC548; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880; &#xAE30;&#xB2A5;&#xC744; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;.

El sistema de ubiquitina / proteasoma regula el volumen de negocios de prote&#xED;nas por degradaci&#xF3;n de las prote&#xED;nas polyubiquitinated. Hasta la fecha, todos los estudios sobre la relaci&#xF3;n de la apoptosis y el proteasoma han puesto de relieve el papel clave del proteasoma en la regulaci&#xF3;n de la apoptosis, en virtud de su capacidad para degradar las mol&#xE9;culas reguladoras implicadas en la apoptosis. Ahora demuestran c&#xF3;mo la inducci&#xF3;n de la apoptosis puede regular la actividad del proteasoma. Durante la apoptosis, las caspasas resultados de activaci&#xF3;n en la divisi&#xF3;n de tres subunidades espec&#xED;ficas del complejo 19S reguladora del proteosoma: S6 &#x26;#39;(Rpt5) y S5 (Rpn10), cuya funci&#xF3;n es reconocer sustratos polyubiquitinated del proteasoma, y &#x200B;&#x200B;S1 (Rpn2), que con S5a y S2 (Rpn1) mantiene unida la tapa y la base del complejo regulador 19S. Esta escisi&#xF3;n mediada por la caspasa inhibe la degradaci&#xF3;n proteasomal de ubiquitina-dependientes e independientes sustratos celulares, incluyendo mol&#xE9;culas proapopt&#xF3;ticos tales como SMAC, de modo que faciliten la ejecuci&#xF3;n del programa de apoptosis, proporcionando un bucle de amplificaci&#xF3;n de alimentaci&#xF3;n hacia adelante.

Inhibe la activaci&#xF3;n de la caspasa la funci&#xF3;n del proteasoma Durante la apoptosis

Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

Death receptor-induced apoptosis reveals a novel interplay between the chromosomal passenger complex and CENP-C during interphase.

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

Inhibitor of apoptosis (IAP) proteins are widely expressed throughout nature and suppress cell death under a variety of circumstances. X-linked IAP, the prototypical IAP in mammals, inhibits apoptosis largely through direct inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once thought to also inhibit caspases, but more recent studies have suggested otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the proteolytic activities of effector caspases on fluorogenic or endogenous substrates. However, cIAP1 does bind to caspase-3 and -7 and does so, remarkably, at distinct steps prior to or following the removal of their prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif, AKPD, on the N terminus of the large subunit of fully mature caspase-7, whereas cIAP1 bound to partially processed caspase-3 in a manner that required its prodomain and cleavage between its large and small subunits but did not involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier protein/ubiquitin-conjugating enzymes), and in the case of caspase-3, differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric caspase-3 (bearing the AKPD motif) were degraded in vivo in a proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least in part, by facilitating the ubiquitination and turnover of active effector caspases in cells.

The E3 ubiquitin ligase cIAP1 binds and ubiquitinates caspase-3 and -7 via unique mechanisms at distinct steps in their processing.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.

Metabolism is a prerequisite for the development of benzene-mediated myelotoxicity. Benzene is initially metabolized via cytochromes P450 (primarily CYP2E1 in liver) to benzene-oxide, which subsequently gives rise to a number of secondary products. Benzene-oxide equilibrates spontaneously with the corresponding oxepine valence tautomer, which can ring open to yield a reactive alpha,beta-unsaturated aldehyde, trans-trans-muconaldehyde (MCA). Further reduction or oxidation of MCA gives rise to either 6-hydroxy-trans-trans-2,4-hexadienal or 6-hydroxy-trans-trans-2,4-hexadienoic acid. Both MCA and the hexadienal metabolite are myelotoxic in animal models. Alternatively, benzene-oxide can undergo conjugation with glutathione (GSH), resulting in the eventual formation and urinary excretion of S-phenylmercapturic acid. Benzene-oxide is also a substrate for epoxide hydrolase, which catalyzes the formation of benzene dihydrodiol, itself a substrate for dihydrodiol dehydrogenase, producing catechol. Finally, benzene-oxide spontaneously rearranges to phenol, which subsequently undergoes either conjugation (glucuronic acid or sulfate) or oxidation. The latter reaction, catalyzed by cytochromes P450, gives rise to hydroquinone (HQ) and 1,2,4-benzene triol. Co-administration of phenol and HQ reproduces the myelotoxic effects of benzene in animal models. The two diphenolic metabolites of benzene, catechol and HQ undergo further oxidation to the corresponding ortho-(1,2-), or para-(1,4-)benzoquinones (BQ), respectively. Trapping of 1,4-BQ with GSH gives rise to a variety of HQ-GSH conjugates, several of which are hematotoxic when administered to rats. Thus, benzene-oxide gives rise to a cascade of metabolites that exhibit biological reactivity, and that provide a plausible metabolic basis for benzene-mediated myelotoxicity. Benzene-oxide itself is remarkably stable, and certainly capable of translocating from its primary site of formation in the liver to the bone marrow. However, therein lies the challenge, for although there exists a plethora of information on the metabolism of benzene, and the fate of benzene-oxide, there is a paucity of data on the presence, concentration, and persistence of benzene metabolites in bone marrow. The major metabolites in bone marrow of mice exposed to 50 ppm [(3)H]benzene are muconic acid, and glucuronide and/or sulfate conjugates of phenol, HQ, and catechol. Studies with [(14)C/(13)C]benzene revealed the presence in bone marrow of protein adducts of benzene-oxide, 1,4-BQ, and 1,4-BQ, the relative abundance of which was both dose and species dependent. In particular, histones are bone marrow targets of [(14)C]benzene, although the identity of the reactive metabolite(s) giving rise to these adducts remain unknown. Finally, hematotoxic HQ-GSH conjugates are present in the bone marrow of rats receiving the HQ/phenol combination. In summary, although the fate of benzene-oxide is known in remarkable detail, coupling this information to the site, and mechanism of action, remains to be established.

The fate of benzene-oxide.

Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells.

Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells.

Owing to the high levels of antiapoptotic B-cell lymphoma 2 (BCL-2) family members observed in several cancers, there has been a major effort to develop inhibitors of the BCL2-family as chemotherapeutic agents. Of the different members in the BCL-2 family, myeloid cell leukemia sequence 1 (MCL-1) is commonly amplified in human tumors and is associated with their relapse and chemoresistance. As a result, specific inhibitors of MCL-1 are being designed to treat resistant tumors. However, there is increasing evidence for other nonapoptotic roles of the BCL-2 family, ranging from ionic homeostasis and autophagy to the regulation of fission-fusion dynamics in subcellular organelles, including the endoplasmic reticulum and mitochondria. In this study, we characterize the specificity of two novel putative MCL-1 inhibitors, BI97C1 (Sabutoclax) and BI112D1, in inducing apoptosis in a BAX/BAK-dependent manner and in an MCL-1-dependent system. In addition to their being proapoptotic, these inhibitors also cause enhanced mitochondrial fragmentation that accompanies a time-dependent loss of optic atrophy 1 (OPA1), suggesting an impairment of mitochondrial fusion. This mitochondrial fragmentation occurs independently of dynamin-related protein 1 (DRP1)-mediated fission activity and, unlike most apoptotic stimuli, occurs upstream of and/or independent of BAX, BAK, and other BH3-only proteins. Furthermore, this mitochondrial fragmentation occurred rapidly and preceded other hallmarks of apoptosis, including the loss in mitochondrial membrane potential and the release of cytochrome c. Although such mitochondrial fragmentation did not deplete total cellular adenosine triphosphate (ATP) or alter other mitochondrial complexes, there was significant accumulation of reactive oxygen species.

Michael Butterworth

Leicester Cancer Research Center

University of Leicester

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Michael Butterworth

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University of Leicester

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Leicester Cancer Research Center