Search

Abstract Background In vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines. Methods We established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein. Results As a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1. Conclusions Our study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

Abstract Background To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. Methods We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. Results With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. Conclusions With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

Abstract Background The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. Results Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. Conclusions The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.