Alkaline induces metallothionein gene expression and potentiates cell proliferation in Chinese hamster ovary cells

Lin, Kuei-Ann; Chen, Je-Hsin; Lee, Dong-Fong; Lin, Li‐Yen

doi:10.1002/jcp.20417

Cited by 7 publications

(5 citation statements)

References 52 publications

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“…The CHO-K1 cell line has epithelial morphology and is a subclone of the parental CHO cell line, which was derived from the ovary of an adult Chinese hamster (19). CHO-K1 cells show low-endogenous expression of MT due to MT gene methylation (24). CHO-K1 cells were transfected with the plasmid construct using lipofectin, and stably transfected clones that overexpressed mMT-1 were selected according to their resistance to cadmium.…”

Section: Methodsmentioning

confidence: 99%

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Smith

Wiltshire²,

Furon³

et al. 2008

American Journal of Physiology-Cell Physiology

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Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn(2+), assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd(2+) and Zn(2+) toxicity. In the case of Zn(2+), the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.

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Section: Methodsmentioning

confidence: 99%

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Smith

Wiltshire²,

Furon³

et al. 2008

American Journal of Physiology-Cell Physiology

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“…Protein concentration was determined using a dye-binding assay with chemicals purchased from Bio-Rad Laboratories. The procedures for EMSA have been described previously [20].…”

Section: Preparation Of Whole-cell Extractsmentioning

confidence: 99%

PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity

Lin

Liu

Tam

et al. 2011

Biochemical Journal

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MTF-1 (metal-responsive transcription factor 1) is an essential mammalian protein for embryonic development and modulates the expression of genes involving in zinc homoeostasis and responding to oxidative stress. We report in the present paper that PTEN (phosphatase and tensin homologue deleted on chromosome 10) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced MT (metallothionein) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zinc increased MTF-1-PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of zinc addition and declined afterwards in the cells. The enhanced binding activity occurred mainly in the cytoplasm and reduced after translocating the MTF-1 into the nucleus. Blocking signalling through the PI3K (phosphoinositide 3-kinase) pathway did not alter the zinc-induced MT expression. Analysis of enzymatically inactive PTEN mutants demonstrated that protein but not lipid phosphatase activity of PTEN was involved in the regulation of MTF-1 activity. The same regulatory role of PTEN was also noted in the regulation of ZnT1 (zinc transporter 1), another target gene of MTF-1.

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“…Metallothionein (MT)-1/2 is a group of cysteine-rich and metal binding proteins that play roles in metal homeostasis, detoxification, and oxygen radical scavenging [9-11]. Accumulated data indicate that MTs play protective roles in brain injury models [12-14].…”

Section: Introductionmentioning

confidence: 99%

Hypothermia enhances induction of protective protein metallothionein under ischemia

Park

Lee

Kim

et al. 2013

J Neuroinflammation

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BackgroundHypothermic protection against ischemic stroke has been reported by many studies. Hypothermia is supposed to mitigate the effects of deleterious genes and proteins and promote the activity of protective genes and proteins in the ischemic brain. Metallothionein (MT)-1/2 is thought to be a crucial factor for metal homeostasis, immune function, and apoptosis. This protein was found to exert protective effects in models of brain injury as well. In the present study, we investigated the effect of hypothermia on MT expression and the underlying mechanisms.MethodsCultured bEnd.3 brain endothelial cells were exposed to oxygen glucose deprivation and reperfusion (OGD+R). Reverse transcription PCR and western blot analyses were performed to measure the expression of MT, transcription factors, and methylation regulating factors. Transcription factor binding assays were also performed. Methylation profiles of the promoter area were obtained with pyrosequencing.ResultsHypothermia protected bEnd.3 cells from OGD+R. When the cells were exposed to OGD+R, MT expression was induced. Hypothermia augmented MT levels. While OGD+R-induced MT expression was mainly associated with metal regulatory transcription factor 1 (MTF-1), MT expression promoted by hypothermia was primarily mediated by the signal transducer and activator of transcription 3 (STAT3). Significantly increased STAT3 phosphorylation at Ser727 was observed with hypothermia, and JSI-124, a STAT-3 inhibitor, suppressed MT expression. The DNA demethylating drug 5-aza-2′-deoxycytidine (5-Aza) enhanced MT expression. Some of the CpG sites in the promoter MT=> it should be “the CpG sites in the MT promoter” showed different methylation profiles and some methylation regulating factors had different expressional profiles in the presence of OGD+R and hypothermia.ConclusionsWe demonstrated that hypothermia is a potent inducer of MT gene transcription in brain endothelial cells, and enhanced MT expression might contribute to protection against ischemia. MT gene expression is induced by hypothermia mainly through the STAT3 pathway. DNA methylation may contribute to MT gene regulation under ischemic or hypothermic conditions.

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Alkaline induces metallothionein gene expression and potentiates cell proliferation in Chinese hamster ovary cells

Cited by 7 publications

References 52 publications

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity

Hypothermia enhances induction of protective protein metallothionein under ischemia

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