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Additional file 7:Figure S3. Down-regulation of asymmetric arginine methylation in MS023-treated CRC cells. DLD1 and HCT116 cells were treated with increasing concentrations of MS023 for 48 h as indicated above. Equal amounts of total protein (20 μg) were loaded to polyacrylamide gel followed by Western blot using anti-ADMA antibodies.

Additional file 8:Figure S4. Representative flow cytometry plots of control and MS023-treated CRC cells. DLD1 (A) and HCT116 cells (B) were treated with or without MS023 (200 μM) for 48 h. Detached and adherent cells were collected and stained with Annexin V and propidium iodide as described in Methods.

Additional file 6:Figure S2. Venn diagram showing overlaps of MMA and ADMA sites between CRC tissues and HCT116 cells. Overlap between arginine methylation sites identified by (A) MMA and (B) ADMA antibodies using protein extracts from CRC tissues and HCT116 cells.

Additional file 5:Figure S1. Venn diagram showing overlaps of monomethylated and asymmetric dimethylated proteins between CRC tissues and HCT116 cells. Overlap between proteins identified by (A) MMA and (B) ADMA antibodies using protein extracts from CRC tissues and HCT116 cells. The Venn diagram was generated using online program VENNY (http://bioinfogp.cnb.csic.es/tools/venny/ index.html).

Protein arginine methylation is involved in cellular differentiation and proliferation. Recently, aberrant expression of protein arginine methyltransferases, which are responsible for the methylation reaction, has been reported in various types of cancer. However, there is no clear evidence regarding the prognostic value of abnormal PRMT6 expression in colorectal cancer or the effect of PRMT6 regulation on CRC cells. We investigated the expression patterns of PRMT6 in patients with stage II and III CRC. We detected nuclear expression of PRMT6 in 23.7% of carcinoma samples by immunohistochemistry. Among the clinicopathological parameters, the ratio of poorly differentiated cancer cells was approximately two-fold higher in patients with PRMT6-positive disease than in those with PRMT6-negative disease (p = 0.002). Patients with PRMT6-positive CRC had a shorter disease-free survival than those with PRMT6-negative CRC in both univariate and multivariate analyses (p = 0.018 and p = 0.035, respectively). siRNA-mediated inhibition of PRMT6 expression in CRC cells induced p21WAF1/CIP1 overexpression and suppressed cell growth and colony-forming ability. Concomitantly, apoptosis was induced in PRMT6-suppressed CRC cells. These data suggest that PRMT6 can serve as a biomarker for unfavorable prognosis and as a therapeutic target in CRC.

Protein arginine methyltransferases (PRMTs) have been proposed as a transcriptional coactivators in the induction of CYP1A1. In this study, we investigated the change of AhR and CYP1A1 expression by inhibition of CARM1 (also known as PRMT4) to show the involvement of protein arginine methylation in CYP1A1 gene expression. Also, we tested the hypothesis that CARM1 recruitment to the CYP1A1 promoter and subsequent histone arginine methylation are required for the activation of transcription. We found that treatment of HepG2 cells with silencing RNA targeting CARM1 or an arginine methyltransferase inhibitor (AMI-5) significantly decreased the CYP1A1 gene expression level. Furthermore, treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which increases histone arginine methylation in histone H3R17, also caused increases in CARM1 and H3R17 signals in chromatin immunoprecipitation assays. These results indicate that CARM1 is recruited to the CYP1A1 promoter region as a coactivator, and histone arginine methylation makes XRE (xenobiotic responsive element) regions more accessible for transcription.

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high-resolution mass spectrometry, a total of 147 symmetric dimethyl-arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH-type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.

The relationship between protein arginine methyltransferases (PRMTs) and insulin synthesis in β cells is not yet well understood. In the present study, we showed that PRMT4 expression was increased in INS-1 and HIT-T15 pancreatic β cells under high-glucose conditions. In addition, asymmetric dimethylation of Arg17 in histone H3 was significantly increased in both cell lines in the presence of glucose. The inhibition or knockdown of PRMT4 suppressed glucose-induced insulin gene expression in INS-1 cells by 81.6 and 79% respectively. Additionally, the overexpression of mutant PRMT4 also significantly repressed insulin gene expression. Consistently, insulin secretion induced in response to high levels of glucose was decreased by both PRMT4 inhibition and knockdown. Moreover, the inhibition of PRMT4 blocked high-glucose-induced insulin gene expression and insulin secretion in primary pancreatic islets. These results indicate that PRMT4 might be a key regulator of high-glucose-induced insulin secretion from pancreatic β cells via H3R17 methylation.

It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.

Protein arginine methyltransferases (PRMTs) generate asymmetric and symmetric dimethyl-arginines by catalyzing the transfer of methyl groups from S: -adenosyl-L-methionine to arginines in target proteins. Previously, we observed that the expression and activity of PRMTs were significantly down-regulated in replicatively senescent fibroblasts compared to young fibroblasts. In this study, we determined the level of three PRMT family members (PRMT1, PRMT4, and PRMT5) and the arginine methylation status in eight tissues from 6- and 24-month-old rats. We observed tissue-specific down-regulation of individual PRMT members in testis, thymus, kidney, lung, and heart from 24-month-old as compared to 6-month-old rats. Specifically, we observed reduced levels of PRMT1 in thymus and lung, reduced levels of PRMT4 in testis, thymus, and hearts, and reduced levels of PRMT5 in all five tissues. PRMT enzyme activity on histones generally correlated with PRMT expression. Furthermore, we observed a reduction in asymmetric and symmetric dimethylation on proteins in aged thymus and lung, and a reduction in symmetric dimethylation in aged testes relative to the testes harvested from young rats. These results suggest that individual PRMT proteins have tissue-specific functions and are regulated in a tissue-specific and age-dependent manner.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known carcinogen, however, the biological mechanism of carcinogenesis by TCDD has not been established. Recently, protein arginine methyltransferases (PRMTs) have been identified as secondary transcription co-activators and are proposed to be co-activators of aryl hydrocarbon receptors binding to xenobiotic response elements. Both PRMT1 and PRMT4 were also reported to be involved with carcinogenesis. The aim of this study was to identify cancer-related genes that are regulated by TCDD exposure and the effect of arginine methylation on TCDD toxicity by transfecting human hepatocarcinoma cells with PRMT1 and PRMT4 siRNA. By microarray analysis, 1,461 genes were up-regulated and 1,591 genes were down-regulated by TCDD exposure. Among the 16 up-regulated genes which had functions related to cancer or metastasis, 13 genes were confirmed by quantitative real time RT-PCR: ABCG2, NRP1, SOX5, BIRC3, CD109, CYP1A1, ERBB2, MTA1, FURIN, F3, PIK3R3, NPTN and NTN4. Co-inhibition of PRMT1 and PRMT4 resulted in decreased expression of eight of these genes, MTA1, ERBB2, SOX5, CD109, FURIN, NRP1, PIK3R3 and ABCG2, all of which have been reportedly involved in breast, ovary, prostate and lung cancers, and metastasis.

The high frequency of intrinsic resistance to TNF-related apoptosisinducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.

Enzymatic methylation of endogenous proteins in clonal pancreatic β-cell, HIT-T15, was investigated. When cell extract incubated with S -adenosyl- l -[ methyl - 3 H]methionine was subjected to SDS–PAGE followed by fluorography, endogenous 20-kDa protein was highly [ methyl - 3 H]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5 mM glucose, insulin secretion increased by 2.5-fold, while the 20-kDa methylation to about 3.2-fold. In the case of forskolin, another secretagogue, at 0.1 mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3 mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [ methyl - 3 H]-labeled amino acids were identified by thin layer chromatography as N G -methylated arginines. While arginyl residues in Gly– Arg –Gly sequence are known to be posttranslationally methylated, a synthetic nonapeptide, GG R G R G R GG, competed with the 20-kDa methylation; at 1 and 10 μM nonapeptides, 62% and 78% of 20-kDa methylation were inhibited, respectively. Furthermore, Western immunoblot analysis of HIT cell extract against GG R G R G R GG antibodies strongly immunoreacted with the 20-kDa protein. These results suggested that methylation of the endogenous 20-kDa protein might play some role in insulin secretion.

Protein-arginine methylation is one of the modifications that yields mono and dimethyl (asymmetric or symmetric) arginine residues in proteins. Previously, we found that asymmetric arginine methylation is decreased proportionately with a decrease of cell proliferation potential of cells, and such arginine methylation is greatest in immortalized cells, followed by normal young cells, and lowest in replicatively senescent cells. Using an asymmetric dimethyl-arginine-specific antibody, we identified arginine-methylated proteins in these cell types by immunoprecipitation and 2-D immunoblotting followed by MS. As a result, arginine methylation of chaperone molecules and RNA-binding proteins was differentially regulated between immortalized or young cells and senescent cells. Immortalized cells had significantly higher levels of methyl-accepting proteins, such as cleavage stimulation factor 2 (CstF2) and heterogeneous nuclear ribonucleoprotein (hnRNP) R, than young cells. However, senescent cells contained hypomethylated CstF2, hnRNP K, and chaperone containing TCP1 subunit 7, as well as decreased hnRNP R level. Further, significant reduction of arginine modification in CstF2 and chaperone containing TCP1 subunit 7 was observed in prematurely senescent fibroblasts, induced by treatment with adenosine dialdehyde, a transmethylation inhibitor, or subcytotoxic concentration of H(2)O(2). These results suggest that asymmetric modification of RNA-binding proteins and molecular chaperones plays an essential role in maintaining cell proliferation capability.

Protein arginine methylation is one of the post-translational modifications which yield monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. In the present study, we investigated the status of protein arginine methylation during human diploid fibroblast senescence. When the expression of protein arginine methyltransferases (PRMTs), namely PRMT1, PRMT4, PRMT5 and PRMT6 was examined, a significant reduction was found in replicatively senescent cells as well as their catalytic activities against histone mixtures compared with the young cells. Furthermore, when the endogenous level of arginine-dimethylated proteins was determined, asymmetric modification (the product of type I PRMTs including PRMT1, PRMT4 and PRMT6) was markedly down-regulated. In contrast, both up- and down-regulations of symmetrically arginine-methylated proteins (the product of type II PRMTs including PRMT5) during replicative senescence were found. Furthermore, when young fibroblasts were induced to premature senescence by sub-cytotoxic H 2 O 2 treatment, results similar to replicative senescence were obtained. Finally, we found that SV40-mediated immortalized WI-38 and HeLa cell lines maintained a higher level of asymmetrically modified proteins as well as type I PRMTs than young fibroblasts. These results suggest that the maintenance of asymmetric modification in the expressed target proteins of type I PRMTs might be critical for cellular proliferation.

The pressure during hyperbaric oxygen treatment may increase oxygen toxicity via an augmented oxygen pressure in the gas. Nevertheless, only a few reports have been published on the effect of cells grown under 2 atmospheric absolute (ATA) pressure. To evaluate the effect of pressure on oxygen toxicity and to study effects in addition to oxygen toxicity, we designed an experiment to compare the effects of normobaric mild hyperoxia (NMH, 40% oxygen) and hyperbaric air condition (HA, air with 2 ATA) on human diploid fibroblasts (HDF) in a hyperbaric incubator. HDFs in both the NMH and the HA condition had a similar oxidative stress response and exhibited premature senescence. To investigate differences in gene profiling in cells grown in the NMH and HA conditions, samples from cells exposed to each condition were applied to microarrays. We found no expression difference in genes related to aging and deoxyribonucleic acid damage, but the expression of genes including cell adhesion, stress response, and transcription were significantly increased in fibroblasts that were responsive to pressure. Among 26 statistically reliable genes, the expression of apoptosis related genes such as ADAM22, Bax, BCL2L14, and UBD, as well as tumor suppressor-related genes like Axin2 and ATF, and also mitogen-activated protein kinase-related genes like mitogen-activated protein kinase kinase kinase 1, histamine receptor, and RAB24, were significantly changed in cells responsive to pressure-induced oxidative stress.

Protein-arginine methylation is a posttranslational modification which yields monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. We investigated the expressions of PRMT1 and PRMT5 in relation to their catalytic activities in rat liver during growth and differentiation as well as in the pancreas. Western immunoblot analysis revealed that both PRMT1 and PRMT5 proteins were expressed in the cytosol of liver and pancreas with molecular mass of about 42 kDa and 72 kDa, respectively. However, on molecular sieve chromatography, the enzyme activities were eluted at about 500 kDa for PRMT5 and 440 kDa for PRMT1, indicating that the multimer complex of these expressed monomers were catalytically active. While the 500 kDa complex methylated predominantly myelin basic protein (MBP), the 440 kDa complex methylated hnRNP A1 protein. In fetal rat liver, the amount of expressed 42 kDa PRMT1 protein and the enzyme activity to methylate hnRNPA1 protein were 2- to 3-fold and 4- to 5-fold higher, respectively, than those of post-natal livers. While the 72 kDa PRMT5 protein was consistently expressed, its activity varied only about 2-fold. However, PRMT5 to methylate MBP showed one distinct peak at around the 20th day post-natal. Furthermore, while the PRMT1 enzyme activity increased more than 10-fold after 3 days of 70% partial hepatectomy, the amount of expressed PRMT1 protein was only about 3.2-fold higher than the control livers. In summary, we observed that PRMTs are catalytically active only in the form of multimers, but not as a dimer or tetramer of the expressed subunit. Furthermore, the amount of expressed PRMT protein, determined by Western immunoblot, did not correlate with the amount of their catalytic activity, and thus, some uncharacterized additional factor(s) may multimerize PRMTs to express catalytic activities in vivo.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters. Graphical Abstract Keywords: TCDD, ABCG2, AhR, Acquired Cisplatin Resistance, Drug Transporters INTRODUCTION The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins. TCDD is persistence in the environment as a poisonous chemical and generates as a byproduct of many industrial processes such as metal production and fuel combustion (1). Exposure to TCDD induces a wide range of adverse health effects in the reproductive, immune, and endocrine systems, and the liver (2, 3). The mechanism of TCDD toxicity involves activation of the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor and a member of the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) superfamily (3). The activated receptor heterodimerizes with the AhR nuclear translocator (ARNT) in the nucleus and binds xenobiotic response elements (XREs) (4), inducing expression of many target genes, including Phase III drug transporters (5). Phase III drug transporters are divided into the following subgroups: ATP-binding cassette (ABC) -transporters that utilize energy generated by ATP hydrolysis, and organic cation transporters (OCT) and organic anion-transporting polypeptides (OATP) that utilize the energy in the proton gradient (6). Accumulating evidence suggests that TCDD induces expression of the ABC-transporter genes and proteins, such as ABCC4 in HepG2 cells (7), P-gP, MRP2, and ABCG2 in the blood-brain barrier (8), and ABCC/MRP 2, -3, -5, and -6 mRNA expressions in the liver (9). In our previous study, expression of the ABCG2 gene was significantly induced by TCDD in HepG2 cells (10), suggesting that the ABCG2 gene is high sensitive to TCDD exposure. The ABC subfamily G 2 (ABCG2) transporter is critically involved in multidrug resistance of human cancer (11). These transporters mediate ATP-dependent drug efflux, and are thereby associated with reduction of intracellular drug accumulation. Overexpression of ABCG2 was shown to underlie cancer cell resistance to mitoxantrone, doxorubicin, paclitaxel, and etoposide (12). However, there is lack of knowledge about the acquired anti-cancer drug resistance conferred by TCDD through induction of the ABCG2. In this study, cisplatin (cis-diamminedichloroplatinum, CDDP), one of the most effective anticancer agents to treat solid tumors, was used as a prototype anticancer drug because of its ability to induce acquired resistance (13). A number of drug transporters, including copper uptake transporter (CTR1), copper efflux transporting P-type ATPases (ATP7A, ATP7B) (14, 15), and multidrug-resistance-related protein (MRP2) (16) contributes to cisplatin resistance. Other solute carrier (SLC) transporter subfamilies, such as organic cation transporter and multidrug and toxin extrusion types of transporters (17), are also involved in cisplatin resistance. Until present, the mechanism of acquired cisplatin resistance in cancer cells through induction of the ABCG2 gene in the presence of cisplatin has not been described. Therefore, in this study, we investigated whether induction of ABCG2 gene expression by TCDD treatment caused human cancer cells to acquire resistance to cisplatin. Previous studies have reported that inducing transcription of the ABCG2 gene requires the AhR-signaling pathway (18, 19). It has been reported that constitutive activation of AhR leads to ABCG2 up-regulation in cisplatin-resistant esophageal carcinoma cells, which cisplatin resistance originated from parental cells (20). However, it is still unknown whether activation of the AhR-signaling pathway may be implicated in cisplatin resistance acquired in cancer cells after exposure to TCDD. The aim of this study was to investigate the effect of TCDD pretreatment on the cisplatin responsiveness of human cancer cells by assessing expression of the ABC-drug transporter genes in TCDD-treated cancer cells with acquired cisplatin resistance. In particular, we examined whether the AhR-signaling pathway was the principal pathway involved in cisplatin resistance acquired after TCDD pretreatment. Our results demonstrate that pretreatment with TCDD confers cisplatin resistance to cancer cells, especially colon cancer LS180 cells through AhR-dependent induction of the ABCG2 gene. However, the TCDD-induced acquired cisplatin resistance was shown to be cancer cell-type-specific and additional experiments are required to further elucidate the molecular mechanisms of acquired resistance to cisplatin in each cell types.