Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

Tabuchi, Yoshiaki; Toyama, Yoshiro; Toshimori, Kiyotaka; Komiyama, Masatoshi; Mori, Chisato; Kondo, Takashi

doi:10.1016/j.bbrc.2005.02.066

Cited by 10 publications

(15 citation statements)

References 59 publications

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“…The epididymal epithelial cells were isolated according to the methods as previously reported [28],[29]. In brief, forty day old mice were killed by cervical dislocation, and the epididymes were microdissected in sterile conditions, freed from adhering fat and adventitia, and cut into small pieces (2∼3 mm).…”

Section: Methodsmentioning

confidence: 99%

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

Shen

et al. 2014

PLoS ONE

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In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.

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Section: Methodsmentioning

confidence: 99%

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

Shen

et al. 2014

PLoS ONE

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“…Thus, it seems plausible that similar interactions occur in other reproductive tissues, including the epididymis; however, this remains to be examined. The characterization of recently established epididymal cell lines should facilitate further investigation into the interactions between androgen and growth factor signaling pathways in the epididymis (Kirchhoff et al 2004, Tabuchi et al 2005.…”

Section: Growth Factor Systems In the Epididymismentioning

confidence: 99%

Region-specific expression of androgen and growth factor pathway genes in the rat epididymis and the effects of dual 5α-reductase inhibition

Henderson¹,

Cooke²,

Robaire³

2006

Journal of Endocrinology

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Dihydrotestosterone (DHT) is the primary androgen acting in the epididymis, the site of sperm maturation. Previously, we showed that the treatment of male rats with PNU157706, an inhibitor that acts on both isoforms of 5a-reductase to prevent DHT formation, has effects on the expression of genes implicated in processes that create the optimal luminal microenvironment required for sperm maturation, and on sperm maturation itself. However, signaling pathways involved in regulating or mediating DHT actions in the epididymis remain largely unknown. The goals of this study were to determine the expression profiles of potential signaling systems in the epididymis and assess their DHTdependence using two different dual 5a-reductase inhibitors. Rats were untreated or gavaged with vehicle, 10 mg/kg per day PNU157706 or 32 mg/kg per day FK143 for 28 days and epididymal gene expression was analyzed. Gene array analysis revealed analogous effects of FK143 on overall epididymal gene expression when compared with previous PNU157706 studies. Quantitative RT-PCR analysis of the expression of the 5a-reductase isozymes, androgen receptor, and members of the IGF, FGF, TGF, and VEGF families revealed novel region-specific expression profiles in the epididymis that were differentially affected by 5a-reductase inhibition; the two inhibitors had parallel effects. Specifically, in proximal regions, 5a-reductase 1, androgen receptor, and TGF-b1 expression increased after treatment, while in distal regions expression of IGF-I, IGFBP-5, IGFBP-6, and FGF-10 decreased. These results provide insight into epididymal signaling mechanisms and indicate potential candidates acting either upstream or downstream of DHT to regulate and/or mediate its actions in the epididymis.

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“…] cells immortalized by the tsSV40 large T-antigen is associated with growth arrest, apoptosis, and differentiation, and leads to the activation of p53 and the induction of p21 waf1 [Guenal and Mignotte, 1995]. As expected, the RCG-12 cells showed temperature-sensitive growth characteristics, as do other tsSV40 large T-antigen-expressed cell lines such as GSM06 [Sugiyama et al, 1993], TTE3 [Tabuchi et al, 2005a], and MEPC5 [Tabuchi et al, 2005b]. Although high expression level of large T-antigen was observed at 338C in the RCG-12 cells, the level was gradually decreased at 398C.…”

Section: Discussionmentioning

confidence: 65%

“…Stable transfectants were selected in DMEM containing 10% FBS and 0.5 mg/mL G418 (Nacalai Tesque, Tokyo, Japan), and were cloned by a single cell culture method. The cloned cells were maintained in DMEM containing 10% FBS and 0.5 mg/mL G418 in a culture dish at a permissive temperature of 338C [Tabuchi et al, 2005b].…”

Section: Establishment Of Immortalized Cell Linesmentioning

confidence: 99%

“…Thus, cell lines harboring the tsSV40 large T-antigen grow continuously at permissive temperatures, whereas inactivation of large T-antigen at non-permissive temperature is associated with growth arrest, apoptosis, and differentiation [Guenal and Mignotte, 1995]. To date, many kinds of conditionally immortalized cell lines have been developed by using the tsSV40 Large T-antigen, e.g., hepatocyte cells [Yanai et al, 1991], gastric surface mucus cells [Sugiyama et al, 1993], and epididymis caput epithelial cells [Tabuchi et al, 2005b]. Immortalized astrocyte cell lines have also been established by the tsSV40 large T-antigen gene [Groves et al, 1993;Whittemore et al, 1994;Kitamura et al, 1997;Tetsuka et al, 2001].…”

mentioning

confidence: 99%

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Identification of genetic networks involved in the cell growth arrest and differentiation of a rat astrocyte cell line RCG‐12

Takasaki

Takarada

Yasuda

et al. 2007

J of Cellular Biochemistry

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The purpose of the present study is to establish and characterize a conditionally immortalized astrocyte cell line and to clarify the genetic networks responsible for the cell growth arrest and differentiation. A conditionally immortalized astrocyte cell line, RCG-12, was established by infecting primary cultured rat cortical glia cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and cells grew continuously. On the other hand, the down-regulation of T-antigen at a non-permissive temperature of 39 degrees C led to growth arrest and differentiation. The cells expressed astrocyte-expressed genes such as glial fibrillary acidic protein. Interestingly, the differentiated condition induced by the non-permissive temperature significantly elevated the expression levels of several astrocyte-expressed genes. To identify the detailed mechanisms by which non-permissive temperature-induced cell growth arrest and differentiation, we performed high-density oligonucleotide microarray analysis and found that 556 out of 15,923 probe sets were differentially expressed 2.0-fold. A computational gene network analysis revealed that a genetic network containing up-regulated genes such as RB, NOTCH1, and CDKN1A was associated with the cellular growth and proliferation, and that a genetic network containing down-regulated genes such as MYC, CCNB1, and IGF1 was associated with the cell cycle. The established cell line RCG-12 retains some characteristics of astrocytes and should provide an excellent model for studies of astrocyte biology. The present results will also provide a basis for understanding the detailed molecular mechanisms of the growth arrest and differentiation of astrocytes.

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Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

Cited by 10 publications

References 59 publications

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

Region-specific expression of androgen and growth factor pathway genes in the rat epididymis and the effects of dual 5α-reductase inhibition

Identification of genetic networks involved in the cell growth arrest and differentiation of a rat astrocyte cell line RCG‐12

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