The CUG-translated WT1, not AUG-WT1, is an oncogene.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

p38α MAPK is required for arsenic-induced cell transformation.

Arsenic exposure has been reported to cause neoplastic transformation through the activation of PcG proteins. In the present study, we show that activation of p38α mitogen-activated protein kinase (MAPK) is required for arsenic-induced neoplastic transformation. Exposure of cells to 0.5 µM arsenic increased CRE and c-Fos promoter activities that were accompanied by increases in p38α MAPK and CREB phosphorylation and expression levels concurrently with AP-1 activation. Introduction of short hairpin (sh) RNA-p38α into BALB/c 3T3 cells markedly suppressed arsenic-induced colony formation compared with wildtype cells. CREB phosphorylation and AP-1 activation were decreased in p38α knockdown cells after arsenic treatment. Arsenic-induced AP-1 activation, measured as c-Fos and CRE promoter activities, and CREB phosphorylation were attenuated by p38 inhibition in BALB/c 3T3 cells. Thus, p38α MAPK activation is required for arsenic-induced neoplastic transformation mediated through CREB phosphorylation and AP-1 activation.

Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K.

Recombinant Human IL-32θ Induces Polarization Into M1-like Macrophage in Human Monocytic Cells.

The tumor microenvironment (TME) is formed by several immune cells. Notably, tumor-associated macrophages (TAMs) are existed in the TME that induce angiogenesis, metastasis, and proliferation of cancer cells. Recently, a point-mutated variant of IL-32θ was discovered in breast cancer tissues, which suppressed migration and proliferation through intracellular pathways. Although the relationship between cancer and IL-32 has been previously studied, the effects of IL-32θ on TAMs remain elusive. Recombinant human IL-32θ (rhIL-32θ) was generated using an Escherichia coli expression system. To induce M0 macrophage polarization, THP-1 cells were stimulated with PMA. After PMA treatment, the cells were cultured with IL-4 and IL-13, or rhIL-32θ. The mRNA level of M1 macrophage markers (IL-1ß, TNFα, inducible nitric oxide synthase) were increased by rhIL-32θ in M0 macrophages. On the other hand, the M2 macrophage markers (CCL17, CCL22, TGFß, CD206) were decreased by rhIL-32θ in M2 macrophages. rhIL-32θ induced nuclear translocation of the NF-κB via regulation of the MAPK (p38) pathway. In conclusion, point-mutated rhIL-32θ induced the polarization to M1-like macrophages through the MAPK (p38) and NF-κB (p65/p50) pathways.

Raepenol™ Cream, a Complex of Natural Compounds, Promotes Wound Healing and Relieves Pruritus In Vivo.

BACKGROUND/AIM: Skin wound healing is a physiological process restoring the structural and functional integrity of injured skin. During this process, wound management preventing bacterial infection and complications is important for the regeneration of skin layers and adnexa, as well as the protective function of the skin. Therefore, the development of an effective ointment to promote wound healing without complications is beneficial. MATERIALS AND METHODS: This study developed Raepenol™ cream, comprising a base cream and natural compounds including paeonol, D-panthenol and extract of Centella asiatica, and assessed its therapeutic effect in wound healing. A rat model of skin wound healing and a mouse model of imiquimod-induced pruritus were employed. The effect of Raepenol™ cream was evaluated by wound size and histological analysis, including the integrity of skin structures and inflammatory response. RESULTS: Raepenol™ cream treatment effectively restored the structural integrity of the skin in rats, including wound closure, regeneration of skin adnexa, and reconstitution of collagen, comparable to commercial ointment. Additionally, Raepenol™ cream significantly suppressed pruritus by inhibiting mast cell infiltration or retention in the inflammatory site of mouse ears. CONCLUSION: Raepenol™ cream effectively promoted wound healing and relieved pruritus in animal models. These results suggest that it could be a promising option for wound care and pruritus relief, offering potential advantages over current ointments.

Polycomb (PcG) proteins, BMI1 and SUZ12, regulate arsenic-induced cell transformation.

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 µm) of arsenic trioxide (As(2)O(3)) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16(INK4a) and p19(ARF) mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16(INK4a) and p19(ARF) was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.

Phosphorylation of caspase-7 by p21-activated protein kinase (PAK) 2 inhibits chemotherapeutic drug-induced apoptosis of breast cancer cell lines.

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

Phosphorylation of histone H2B serine 32 is linked to cell transformation.

Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together, our results demonstrate a novel role for H2B phosphorylation in cell transformation and show that H2BS32 phosphorylation is critical for controlling activator protein-1 activity, which is a major driver in cell transformation.

Role of NEK6 in tumor promoter-induced transformation in JB6 C141 mouse skin epidermal cells.

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Histone XH2AX is required for Xenopus anterior neural development: critical role of threonine 16 phosphorylation.

A role for histone H2AX, one of the variants of the nucleosome core histone H2A, has been demonstrated in DNA repair, tumor suppression, apoptosis, and cell cycle checkpoint function. However, the physiological function and post-translational modification of histone H2AX during vertebrate development have not been elucidated. Here, we provide evidence showing that Xenopus histone H2AX (XH2AX) has a role in the anterior neural plate for eye field formation during Xenopus embryogenesis. A loss-of-function study clearly demonstrated a critical role of XH2AX in anterior neural specification. Through a differentiation assay with Xenopus animal cap embryonic stem cells, we confirmed that XH2AX is required for the activin-induced anterior neural specification of the ectoderm. Furthermore, we found that Chk1 is an essential kinase to phosphorylate histone XH2AX at Thr(16), which is involved in the biological function of this histone. Taken together, our findings reveal that XH2AX has a specific role in anterior neural formation of Xenopus, which is mediated through phosphorylation of XH2AX at Thr(16) by Chk1.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Honokiol Inhibits Melanoma Growth by Targeting Keratin 18 in vitro and in vivo.

Honokiol, a natural compound, derived from Magnolia officinalis, has been shown to have anti-cancer effect in several cancer types. However, the underlying molecular mechanism associated with its anti-cancer properties has not been fully elucidated. In the current study, we showed that honokiol inhibited the growth of melanoma cells in a dose and time-dependent manner. Mechanistically, it directly interacts with keratin 18 (KRT18) protein and induces its degradation through ubiquitination. Furthermore, the expression of KRT18 was found to be higher in melanoma tissues compared to the normal skin tissues. In addition, KRT18 overexpression significantly promoted melanoma cell proliferation and growth. Our results showed that honokiol treatment significantly decreased KRT18 protein level and suppressed the tumor growth in melanoma cell-derived xenograft mice models. Hence, KRT18 plays an oncogenic role in melanoma and honokiol can be an inhibitor for KRT18.

Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation.

The ribosomal S6 kinase 2 (RSK2), a member of the p90(RSK) (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2(-/-) mouse embryonic fibroblasts (MEFs) compared with RSK2(+/+) MEFs. Moreover, RSK2(-/-) MEFs accumulated at the G(1) phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (Ras(G12V))-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser(10)). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA.

T-lymphokine-activated killer cell-originated protein kinase functions as a positive regulator of c-Jun-NH2-kinase 1 signaling and H-Ras-induced cell transformation.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is overexpressed in highly proliferating tumors such as leukemias and myelomas, and seems to play a key role in tumorigenesis or metastasis. However, the precise role and regulatory mechanism explaining the effects of TOPK on tumor cells still remain elusive. Here, we reported that TOPK regulates UVB-induced c-Jun-NH2-kinase 1 (JNK1) activity, and is essential for H-Ras-induced activator protein-1 activity and cell transformation. We showed that TOPK associated with and phosphorylated JNK1 following UVB irradiation in vitro or in vivo. Moreover, UVB-induced JNK1 activity was greatly augmented in mouse epidermal JB6 Cl41 cells that stably expressed TOPK cDNA. On the other hand, JNK1 activity was markedly attenuated by stable expression of small interfering RNA against TOPK in malignant melanoma RPMI 7951 cells. Interestingly, TOPK interacted with JNK-interacting protein 1 and caused an elevation of JNK-interacting protein 1 scaffolding activity, thereby enhancing JNK1 activity. Furthermore, JNK1 was required for TOPK-mediated activator protein-1 transcriptional activity and transformed foci induced by UVB or H-Ras. Taken together, these findings showed that TOPK positively modulated UVB-induced JNK1 activity and played a pivotal role in JNK1-mediated cell transformation induced by H-Ras. These studies might also provide a novel molecular mechanism for the role of TOPK in UVB-mediated skin carcinogenesis.

Carnosol suppresses patient-derived gastric tumor growth by targeting RSK2.

Carnosol is a phenolic diterpene that is isolated from rosemary, sage, and oregano. It has been reported to possess anti-oxidant, anti-inflammatory, and anti-cancer properties. However, the molecular mechanism of carnosol's activity against gastric cancer has not been investigated. Herein, we report that carnosol is an RSK2 inhibitor that attenuates gastric cancer growth. Carnosol reduced anchorage-dependent and -independent gastric cancer growth by inhibiting the RSKs-CREB signaling pathway. The results of in vitro screening and cell-based assays indicated that carnosol represses RSK2 activity and its downstream signaling. Carnosol increased the G2/M phase and decreased S phase cell cycle and also induced apoptosis through the activation of caspases 9 and 7 and inhibition of Bcl-xL expression. Notably, oral administration of carnosol suppressed patient-derived gastric tumor growth in an in vivo mouse model. Our findings suggest that carnosol is an RSK2 inhibitor that could be useful for treating gastric cancer.

Targeting PRPK and TOPK for skin cancer prevention and therapy.

Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar-simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization, and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1, and MMP-9. This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.

Targeting PRPK Function Blocks Colon Cancer Metastasis.

The biological functions of the p53-related protein kinase (PRPK) remain unclear. We have previously demonstrated that PRPK is phosphorylated by the T-LAK cell-originated protein kinase (TOPK) and that phosphorylated PRPK (p-PRPK) promotes colon cancer metastasis. Here, we analyzed colon adenocarcinomas from 87 patients and found that higher expression levels of p-PRPK were associated with later stages of metastatic dissemination (stage III and IV) as compared with earlier stages (stages I and II). Indeed, levels of p-PRPK were higher in metastatic versus malignant human colon adenocarcinomas. Knocking down PRPK expression attenuated colorectal liver and lung metastasis of colon cancer cells in vivo An in vitro kinase assay indicated that active PRPK does not phosphorylate p53 directly. We found that PRPK phosphorylates survivin, a regulator of colon cancer metastasis. PRPK phosphorylates survivin at Thr34, which is important for survivin stability. Taken together, our data strongly suggest that the PRPK signaling pathway promotes colon cancer metastasis by modulating survivin stability, and that PRPK could be a new prognostic marker for the survival of colon cancer patients. In addition, we identified an FDA-approved bacteriostatic antibiotic, fusidic acid sodium salt (fusidic acid or FA) as an inhibitor of PRPK, and show that FA combined with 5-fluorouracil (5-FU) inhibited PRPK activity and colon cancer metastasis to the lung in mice. We contend that the combination of FA with 5-FU could be an alternative therapeutic strategy to traditional chemotherapy for colon cancer patients with poor prognosis. Mol Cancer Ther; 17(5); 1101-13. ©2018 AACR.

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway.

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.

The fission yeast gene encoding monothiol glutaredoxin 5 is regulated by nitrosative and osmotic stresses.

Glutaredoxin (Grx) is a small, heat-stable redox protein acting as a multi-functional glutathione (GSH)-dependent disulfide oxidoreductase. We have cloned the monothiol Grx5 gene from the genomic DNA of the fission yeast Schizosaccharomyces pombe. It has 1,904 bp, with one intron, and encodes a putative protein of 146 amino acids with a molecular mass of 16.5 kDa. Recombinant Grx5 produced functional Grx in S. pombe cells. NO-generating sodium nitroprusside (SNP, 1.0 and 2.0 mM) and potassium chloride (KCl, 0.2 and 0.5 M) increased the synthesis of beta-galactosidase from a Grx5-lacZ fusion gene, and transcription of Grx5 was also enhanced by SNP and KCl. Synthesis of beta-galactosidase from the Grx5-lacZ fusion was lower in Pap1-negative TP108-3C cells than in wild type KP1 cells, and when Pap1 was overproduced in KP1 cells, the level of beta-galactosidase increased. We also found that Pap1 is involved in the induction of Grx5 by SNP and KCl. S. pombe Grx5 may play a crucial role in responses to nitrosative and osmotic stresses.

Up-regulation of glutathione biosynthesis in NIH3T3 cells transformed with the ETV6-NTRK3 gene fusion.

The ETV6-NTRK3 gene fusion, first identified in the chromosomal translocation in congenital fibrosarcoma, encodes a chimeric protein tyrosine kinase with potent transforming activity. ETV6-NTRK3-dependent transformation involves the joint action of NTRK3 signaling pathways, and aberrant cell cycle progression resulting from activation of Mek1 and Akt. The level of glutathione (GSH) was found to be markedly increased in ETV6-NTRK3-transformed NIH3T3 cells. The activities of the two GSH biosynthetic enzymes as well as of glutathione peroxidase, together with their mRNAs, were also higher in the transformed cells. The transformed cells were able to grow in the presence of GSH-depleting agents, whereas the control cells were not. L-Buthionine-(S,R)-sulfoximine (BSO) inhibited activation of Mek1 and Akt in the transformed NIH3T3 cells. These observations imply that up-regulation of GSH biosynthesis plays a central role in ETV6-NTRK3-induced transformation.