RESUMEN
Malignant transformation of melanocytes to melanoma cells closely parallels activation of MIA expression and involves a promoter region that we referred to previously as a HCR (highly conserved region). The HCR element interacts with the melanoma-associated transcription factor and confers strong activation of the promoter. Furthermore, mutation and deletion studies described in this study revealed that the permissive site for cell-specific promoter activity was located directly 5' to the HCR region. Changes in the DNA sequence 5' adjacent to the melanoma-associated transcription factor binding site led to an MIA promoter activity in benign melanocytes and nonmelanocytic cells that usually do not express MIA. Detailed analysis revealed binding of T-cell factor family transcription factors to the repressor element. Because this family is known to interact with COOH-terminal binding protein, we explored the role of COOH-terminal binding protein 1(CtBP1) in silencing MIA gene expression. By reporter gene analysis, we determined a strict negative regulation of MIA promoter activity in melanoma cells by CtBP1. Furthermore, we observed strong expression of CtBP1 in primary melanocytes but a loss of wild-type CtBP1 expression in malignant melanoma in vitro and in vivo. Therefore, we speculate that CtBP1 has an important negative role in MIA regulation, and loss of CtBP1 is implicated in melanoma progression.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Melanoma/metabolismo , Proteínas de Neoplasias/biosíntesis , Fosfoproteínas/biosíntesis , Oxidorreductasas de Alcohol , Animales , Secuencia de Bases , Secuencia Conservada , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Proteínas de la Matriz Extracelular , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Ratones , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Transfección , Células Tumorales CultivadasRESUMEN
We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.