RESUMEN
In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.
Asunto(s)
Resistencia a Antineoplásicos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Complejos Multiproteicos/metabolismo , Células 3T3 , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Compuestos de Bifenilo/farmacología , Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Muerte Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Ratones , Mutación/genética , FN-kappa B/metabolismo , Nitrofenoles/farmacología , Piperazinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Ubiquitina-Proteína LigasasRESUMEN
In an attempt to improve TRAIL's (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor-targeting properties. We studied tumor targeting and apoptosis induction of scFv-scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv-scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL's antitumoral action and to minimize potential unwanted actions on normal tissues.Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Animales , Antineoplásicos/sangre , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Epítopos , Semivida , Humanos , Ratones , Ratones Desnudos , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos de Cadena Única/sangre , Anticuerpos de Cadena Única/farmacocinética , Anticuerpos de Cadena Única/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Ligando Inductor de Apoptosis Relacionado con TNF/farmacocinética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE(10) decreasing S and one at SAFD(135) decreasing G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Glutámico , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células COS , Caspasa 7 , Chlorocebus aethiops , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/química , Dimerización , Humanos , Inmunoglobulina M/farmacología , Células Jurkat , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Mapeo Peptídico , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transfección , Receptor fas/fisiologíaRESUMEN
Mad1 is a member of the Myc/Max/Mad network of transcriptional regulators that play a central role in the control of cellular behavior. Mad proteins are thought to antagonize Myc functions at least in part by repressing gene transcription. To systematically examine the function of Mad1 in growth control and during apoptosis, we have generated U2OS cell clones that express Mad1 under a tetracyline-regulatable promoter (UTA-Mad1). Mad1 was induced rapidly and efficiently, localized to the nucleus, and bound to DNA as a heterodimer with Max. The induction of Mad1 reduced cellular growth and, more profoundly, inhibited colony formation of UTA-Mad1 cells. Conditioned medium neutralized this inhibitory effect implying that Mad1 function is regulated by extracellular signals. In addition Mad1 interfered with Fas-, TRAIL-, and UV-induced apoptosis, which coincided with a reduced activation of caspase-8 during Fas-mediated apoptosis in response to Mad1 expression. Furthermore, microinjection of Mad1-expressing plasmids into fibroblasts inhibited apoptosis induced by the oncoproteins c-Myc and E1A. Thus, Mad1 not only interferes with cellular proliferation but also with apoptosis, which defines a novel aspect of Mad1 function.