Characterization of Jumping translocation breakpoint (JTB) gene product isolated as a TGF-β1-inducible clone involved in regulation of mitochondrial function, cell growth and cell death

Abstract

Jumping translocation breakpoint (JTB) is a gene located on human chromosome 1 at q21 that suffers an unbalanced translocation in various types of cancers, and potentially encodes a transmembrane protein of unknown function. The results of cancer profiling indicated that its expression was suppressed in many cancers from different organs, implying a role in the neoplastic transformation of cells. Recently, we isolated JTB as a TGF-b1-inducible clone by differential screening. In this study, we characterized its product and biological functions. We found that it was processed at the N-terminus and located mostly in mitochondria. When expressed in cells, JTB-induced clustering of mitochondria around the nuclear periphery and swelling of each mitochondrion. In those mitochondria, membrane potential, as monitored with a JC-1 probe, was significantly reduced. Coinciding with these changes in mitochondria, JTB retarded the growth of the cells and conferred resistance to TGF-b1-induced apoptosis. These activities were dependent on the N-terminal processing and induced by wild-type JTB but not by a mutant resistant to cleavage. These findings raised the possibility that aberration of JTB in structure or expression induced neoplastic changes in cells through dysfunction of mitochondria leading to deregulated cell growth and/or death.

Transcriptional induction of MMP-10 by TGF-β, mediated by activation of MEF2A and downregulation of class IIa HDACs

“…NMuMG mouse mammary epithelial cells were maintained as described previously (Kanome et al, 2007). MCF10A human mammary epithelial cells were cultured in Dulbecco's modified Eagle's medium/ F-12 1:1 mixture (Invitrogen, San Diego, CA, USA) supplemented with 5% horse serum (Invitrogen), 20 ng/ml epidermal growth factor (Wako Pure Chemical Industries, Osaka, Japan), 10 mg/ml insulin (Wako), 0.5 mg/ml hydrocortisone (Sigma, St Louis, MO, USA), 100 U/ml penicillin and 50 mg/ml streptomycin.…”

Competitive Nuclear Export of Cyclin D1 and Hic-5 Regulates Anchorage-Dependence of Cell Growth and Survival

“…The retroviral expression vectors and the procedure for infection have previously been described previously (Kanome et al, 2007). The efficiency of the infection, as monitored by enhanced green fluorescent protein (EGFP) expression, was above 80%.…”

“…First, 5 ϫ 10 5 cells were placed in suspension for 48 h, collected, and stained with APOPercentage dye according to the manufacturer's instructions and as previously described (Kanome et al, 2007). The values were normalized to the total cell number.…”

“…To generate the HA-tagged cyclin D1 mutants, T286A and CRM(Ϫ) (V290A, V293A, I295A), point mutations were introduced into HA-tagged cyclin D1 using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) with a mutated primer according to the manufacturer's instructions (Kanome et al, 2007). A coding region of v-Ki-ras/pBR322 was PCR-amplified and inserted into pcDNA3 for the Flag-tagged v-Ki-ras expression vector.…”

“…To generate the HA-tagged cyclin D1 mutants, T286A and CRM(Ϫ) (V290A, V293A, I295A), point mutations were introduced into HA-tagged cyclin D1 using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) with a mutated primer according to the manufacturer's instructions (Kanome et al, 2007). A coding region of v-Ki-ras/pBR322 was PCR-amplified and inserted into pcDNA3 for the Flag-tagged v-Ki-ras expression vector.The expression vectors were introduced into the cells by the conventional calcium phosphate precipitation method, and the cells were processed for analysis 24 h after transfection.The retroviral expression vectors and the procedure for infection have previously been described previously (Kanome et al, 2007). The efficiency of the infection, as monitored by enhanced green fluorescent protein (EGFP) expression, was above 80%.…”