RESUMO
Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Caspases/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Microdomínios da Membrana/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Alemtuzumab , Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais Humanizados , Anticorpos Antineoplásicos/efeitos dos fármacos , Antígenos CD/biossíntese , Antígenos CD/metabolismo , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/metabolismo , Antígeno CD52 , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Gangliosídeo G(M1)/biossíntese , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Humanos , Técnicas In Vitro , Microdomínios da Membrana/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.
Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Inibidores de Histona Desacetilases , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Linfocítica Crônica de Células B/patologia , Piridinas/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
The authors report two cases, a 44-year-old woman and a 6-year-old girl who had mental status changes and hyponatremia. Serum sodium levels in both of these cases were corrected quickly with further decline in their mental status, and the patients became quadriparetic. Magnetic resonance imaging (MRI) studies performed then did not reveal any abnormalities, whereas a repeat imaging study performed 10-14 days after the shift in serum sodium revealed evidence for central pontine myelinolysis and extrapontine demyelination. The clinical manifestations and distribution of lesions seen on the imaging studies demonstrated that the above presentation of neurologic illness is the result of hyponatremia and its correction. The authors conclude that imaging studies performed early during the illness may be unremarkable, but still a diagnosis of central pontine myelinolysis should be suspected and, most importantly, a repeat imaging study might be required in 10-14 days to establish the diagnosis of central pontine myelinolysis.