ABSTRACT
Molecular mechanisms responsible for lymphoma resistance to apoptosis often involve the bcl-2 pathway. In this study, we investigated the cell signaling pathways activated in bcl-2-overexpressing human mantle cell lymphoma cell lines (JVM-2 and Z-138) that have been treated with oblimersen, a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Z-138 cells expressed higher levels of bcl-2 and were more sensitive to the effects of bcl-2 silencing, mediated by oblimersen or bcl-2 small interfering RNA, in vitro. Tumors derived following injection of Z-138 cells were sensitive to oblimersen as judged by decreases in tumor growth rate and decreases in cell proliferation (as measured by Ki-67). Immunohistochemistry and Western blot analysis of oblimersen-treated Z-138 tumors revealed a dose-dependent decrease in bcl-2 levels and an associated increase in the proapoptotic proteins caspase-3 and caspase-9. Silencing bcl-2 in Z-138 xenografts revealed an associated dose-dependent suppression of bax, a decrease in nuclear factor-kappaB and phospho-nuclear factor-kappaB, and transient loss of p53 levels. Coimmunoprecipitation studies suggest that the latter observation is mediated by an association between bcl-2 and phospho-mdm2. Bcl-2 silencing also led to p27 down-regulation and coimmunoprecipitation studies point to a role for bcl-2 in regulation of p27 localization/degradation. Bcl-2 silencing was also correlated with loss of cyclin D1a protein levels but not cyclin D1b levels. Coimmunoprecipitation studies indicate that bcl-2 may mediate its effects on cyclin D1a via interaction with p38 mitogen-activated protein kinase as well as a previously unreported interaction between bcl-2 and cyclin D1a.
Subject(s)
DNA-Binding Proteins/genetics , Disease Models, Animal , Gene Silencing , Lymphoma, Mantle-Cell/genetics , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Apoptosis/physiology , Blotting, Western , Cell Proliferation , Cyclin D , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclins/genetics , Cyclins/metabolism , DNA-Binding Proteins/physiology , Humans , Immunoenzyme Techniques , Immunoprecipitation , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/prevention & control , Male , Mice , Mice, Knockout , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Phosphorylation , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thionucleotides/pharmacology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
The objectives of this study were foremost to further characterize pre-existing cell lines containing the t(11;14)(q13;q32) translocation. This translocation along with cyclin D1 overexpression is characteristic of Mantle Cell Lymphoma (MCL), an aggressive B cell neoplasm. Considerable variation in the abundance of cyclin D1 expression was observed. mRNA levels were examined by RT-PCR as differences in cyclin D1 mRNA abundance have been shown to synergize with INK4A/Arf deletions to dictate proliferation rate and survival in MCL patient samples. In this study, the cell lines, Z-138 and HBL-2, which exhibited the fastest growth rates and the shortest survival times in Rag2-M mice, had high expression of either one or both cyclin D1 mRNA isoforms and had negligible expression of p16. On the other hand, NCEB-1 and JVM-2 had low expression of both mRNA isoforms, retained p16 expression, and had slower growth rates and exhibited longer survival times in Rag2-M mice. Furthermore, JVM-2, which was found to have the lowest expression of cyclin D1, was the only cell line that expressed cyclin D2. The results of the characterization of Z-138, HBL-2, NCEB-1 and JVM-2 reveal that this group of cell lines represents both classic and variant features of MCL.
