Stress-activated protein kinases (SAPKs), members of a mitogen-activated protein kinase (MAPK) subfamily, are highly conserved among eukaryotes. Studies of yeasts demonstrated that SAPKs play pivotal roles in survival responses to high osmolarity, oxidative stress, and heat shock. Here we report a novel physiological role of the fission yeast Spc1 SAPK in cellular resistance to certain cations, such as Na(+), Li(+), and Ca(2+). Strains lacking Spc1 or its activator, Wis1 MAPK kinase, are hypersensitive to these cations. Spc1 positively regulates expression of sod2(+) encoding a Na(+)/H(+) antiporter through Atf1 and other transcription factors. In addition, we have identified a novel Spc1-interacting protein, Hal4, which is highly homologous to the budding yeast Sat4/Hal4 protein kinase. Like its budding yeast counterpart, the fission yeast Hal4 kinase is essential for cellular resistance to Na(+), Li(+), and Ca(2+). The hal4-null phenotype is complemented by overexpression of the Trk1 potassium transporter or increased K(+) in the growth medium, suggesting that Hal4 promotes K(+) uptake, which consequently increases cellular resistance to other cations. Interestingly, the Spc1-Hal4 interaction appears to be required for cellular resistance to Ca(2+) but not Na(+) and Li(+). We propose that Spc1 SAPK and Hal4 kinase cooperatively function to protect cells from the toxic cations.

BACKGROUND

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

RESULTS

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

CONCLUSIONS

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.

Angiomatoid fibrous histiocytoma is a neoplasm of uncertain histogenesis, which most commonly arises in the subcutaneous tissue of the extremities of children and young adults. We report the first case of a calcifying sclerosing variant of this entity. This case arose in bone-a site where there has been just 1 previously published case of typical (nonsclerosing/mineralizing) angiomatoid fibrous histiocytoma. The patient presented with the classical paraneoplastic syndrome that can occur with this tumor type and, apart from the described extracellular matrical features, displayed typical histologic features. Due to the rarity of angiomatoid fibrous histiocytoma at this site and the presence of matrical sclerosis/mineralization, this case raised the important differential diagnosis of osteosarcoma, both histologically and radiologically. The diagnosis was confirmed by molecular analysis; fluorescence in situ hybridization for EWSR1 gene disruption, and reverse transcription-polymerase chain reaction using newly designed primers on both frozen and decalcified, paraffin-embedded tissue samples showing an EWSR1-ATF1 translocation. It is important to recognize that this relatively indolent tumor can arise in bone and that it can contain focally mineralized hyalinized sclerotic matrix to avoid making the serious misdiagnosis of high-grade osteosarcoma and administering aggressive systemic chemotherapeutic treatment.

Flaxseed (FS), a dietary oilseed, contains a variety of anti-inflammatory bioactives, including fermentable fiber, phenolic compounds (lignans), and the n-3 polyunsaturated fatty acid (PUFA) α-linolenic acid. The objective of this study was to determine the effects of FS and its n-3 PUFA-rich kernel or lignan- and soluble fiber-rich hull on colitis severity in a mouse model of acute colonic inflammation. C57BL/6 male mice were fed a basal diet (negative control) or a basal diet supplemented with 10% FS, 6% kernel, or 4% hull for 3 wk prior to and during colitis induction via 5 days of 2% (wt/vol) dextran sodium sulfate (DSS) in their drinking water (n = 12/group). An increase in anti-inflammatory metabolites (hepatic n-3 PUFAs, serum mammalian lignans, and cecal short-chain fatty acids) was associated with consumption of all FS-based diets, but not with anti-inflammatory effects in DSS-exposed mice. Dietary FS exacerbated DSS-induced acute colitis, as indicated by a heightened disease activity index and an increase in colonic injury and inflammatory biomarkers [histological damage, apoptosis, myeloperoxidase, inflammatory cytokines (IL-6 and IL-1β), and NF-κB signaling-related genes (Nfkb1, Ccl5, Bcl2a1a, Egfr, Relb, Birc3, and Atf1)]. Additionally, the adverse effect of the FS diet was extended systemically, as serum cytokines (IL-6, IFNγ, and IL-1β) and hepatic cholesterol levels were increased. The adverse effects of FS were not associated with alterations in fecal microbial load or systemic bacterial translocation (endotoxemia). Collectively, this study demonstrates that although consumption of a 10% FS diet enhanced the levels of n-3 PUFAs, short-chain polyunsaturated fatty acids, and lignans in mice, it exacerbated DSS-induced colonic injury and inflammation.

Geraniol produced by grape is the main precursor of terpenols which play a key role in the floral aroma of white wines. We investigated the fate of geraniol during wine fermentation by Saccharomyces cerevisiae. The volatile compounds produced during fermentation of a medium enriched with geraniol were extracted by Stir-bar sorptive extraction and analysed by GC-MS. We were able to detect and quantify geranyl acetate but also citronellyl- and neryl-acetate. The presence of these compounds partly explains the disparition of geraniol. The amounts of terpenyl esters are strain dependant. We demonstrated both by gene overexpression and gene-deletion the involvement of ATF1 enzyme but not ATF2 in the acetylation of terpenols. The affinity of ATF1 enzyme for several terpenols and for isoamyl alcohol was compared. We also demonstrated that OYE2 is the enzyme involved in geraniol to citronellol reduction. Fermenting strain deleted from OYE2 gene produces far less citronellol than wild type strain. Moreover lab strain over-expressing OYE2 allows 87% geraniol to citronellol reduction in bioconversion experiment compared to about 50% conversion with control strain.

The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.

The Tup family corepressors contribute to critical cellular responses, such as the stress response and differentiation, presumably by inducing repressive chromatin, though the precise repression mechanism remains to be elucidated. The Schizosaccharomyces pombe fission yeast Tup family corepressors Tup11 and Tup12 (Tup11/12), which are orthologs of Tup1 in Saccharomyces cerevisiae budding yeast and Groucho in Drosophila, negatively control chromatin and the transcriptional activity of some stress-responsive genes. Here, we demonstrate that Tup11/12 repress transcription of a gluconeogenesis gene, fbp1⁺, by three distinct mechanisms. First, Tup11/12 inhibit chromatin remodeling in the fbp1⁺ promoter region where the Atf1 and Rst2 transcriptional activators bind. Second, they repress the formation of an open chromatin configuration at the fbp1⁺ TATA box. Third, they repress mRNA transcription per se by regulating basic transcription factors. These inhibitory actions of Tup11/12 are antagonized by three different types of transcriptional activators: CREB/ATF-type Atf1, C₂H₂zinc finger-type Rst2, and CBF/NF-Y-type Php5 proteins. We also found that impaired chromatin remodeling and fbp1⁺ mRNA transcription in php5Δ strains are rescued by the double deletions of tup11⁺ and tup12⁺, although the distribution of the transcription start sites becomes broader than that in wild-type cells. These data reveal a new mechanism of precise determination of the mRNA start site by Tup family corepressors and CBF/NF-Y proteins.

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biological potential, predominantly occurring in the extremities of children and young adults. It has only recently been reported as a primary lung tumor. We describe 2 cases arising endobronchially harboring EWSR1 gene rearrangements by fluorescence in situ hybridization and, respectively, EWSR1-CREB1 and EWSR1-ATF1 gene fusions by reverse transcription polymerase chain reaction. Histologically, both tumors showed classical features of AFH, comprising multiple nodules of bland spindle to epithelioid cells surrounded by lymphoplasmacytic inflammation and at least a partial fibrous capsule. Both tumors showed focal but strong desmin immunoreactivity, with focal pancytokeratin and epithelial membrane antigen in 1 case. The lung is now a recognized site of AFH occurrence, but tumors arising here can be associated with different gene fusions. It is important to recognize AFH in the pulmonary region, as its behavior at other sites is generally relatively indolent; however, it may be mistaken for metastatic or more aggressive primary lung tumors. It is likely that cases of AFH in the lung may have been previously missed because of their morphologic and genetic overlap with other pulmonary lesions.

Little is known about how liver fibrosis influences lobular zonation. To address this question, we used three mouse models of liver fibrosis, repeated CCl₄ administration for 2, 6 and 12 months to induce pericentral damage, as well as bile duct ligation (21 days) and mdr2^-/- mice to study periportal fibrosis. Analyses were performed by RNA-sequencing, immunostaining of zonated proteins and image analysis. RNA-sequencing demonstrated a significant enrichment of pericentral genes among genes downregulated by CCl₄; vice versa, periportal genes were enriched among the upregulated genes. Immunostaining showed an almost complete loss of pericentral proteins, such as cytochrome P450 enzymes and glutamine synthetase, while periportal proteins, such as arginase 1 and CPS1 became expressed also in pericentral hepatocytes. This pattern of fibrosis-associated 'periportalization' was consistently observed in all three mouse models and led to complete resistance to hepatotoxic doses of acetaminophen (200 mg/kg). Characterization of the expression response identified the inflammatory pathways TGFβ, NFκB, TNFα, and transcription factors NFKb1, Stat1, Hif1a, Trp53, and Atf1 among those activated, while estrogen-associated pathways, Hnf4a and Hnf1a, were decreased. In conclusion, liver fibrosis leads to strong alterations of lobular zonation, where the pericentral region adopts periportal features. Beside adverse consequences, periportalization supports adaptation to repeated doses of hepatotoxic compounds.

Malignant gastrointestinal neuroectodermal tumor (GNET) is an aggressive rare tumor, primarily occurring in young adults with frequent local-regional metastases and recurrence after local control. The tumor is characterized by the presence of EWSR1-ATF1 or EWSR1-CREB1 and immunohistochemical positivity for S-100 protein without melanocytic marker positivity. Due to poor responses to standard sarcoma regimens, GNET has a poor prognosis, and development of effective systemic therapy is desperately needed to treat these patients. Herein, we present a patient with a small bowel GNET who experienced recurrent hepatic and skeletal metastases after a primary resection. Comprehensive genomic profiling (CGP) in the course of clinical care with DNA and RNA sequencing demonstrated the presence of an exon 7 to exon 6 EWSR1-CREB1 fusion in the context of a diploid genome with no other genomic alterations. In a clinical trial, the patient received a combination of 250 mg crizotinib with 600 mg pazopanib quaque die and achieved partial response and durable clinical benefit for over 2.8 years, and with minimal toxicity from therapy. Using a CGP database of over 50,000 samples, we identified 11 additional cases that harbor EWSR1-CREB1 and report clinicopathologic characteristics, as these patients may also benefit from such a regimen.

In eukaryotic organisms, stress-activated mitogen-activated protein kinases (MAPK) play crucial roles in transmitting environmental signals to regulate gene expression for cellular stress adaptation. Here we report that, in the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 MAPK and the Atf1 transcription factor regulate the stress-induced expression of Pmp3, a ubiquitous small membrane protein implicated in the modulation of the plasma membrane potential. The pmp3 null mutant, as well as the spc1 and atf1 mutants, is hypersensitive to the cationic antibiotic hygromycin B. Transcriptional regulation of the Pmp3-like genes by the stress-activated MAPK may also be conserved in other eukaryotes, including plants.

Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3×10(7) HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes by proteomic methods.

IL-2 stimulates the proliferative response of various lymphoid cells. Previous studies showed an increase in intracellular levels of cAMP concomitant with an increase in phosphorylation of discrete proteins by protein kinase A at late G1 phase in mitogen-stimulated lymphocytes. Thus, experiments were undertaken to study nuclear proteins that bind to the cAMP-responsive enhancer (CRE) in cloned T lymphocytes stimulated with IL-2. With the use of a 32P-labeled CRE consensus sequence in a DNA binding gel mobility shift assay, we showed that IL-2 stimulation resulted in the induction of two major DNA-protein complexes at late G1/S during the cell cycle. This binding was competed in a dose-dependent manner by a nonlabeled CRE oligonucleotide but was not competed by a nonlabeled AP-1 oligonucleotide. Rapamycin, a potent immunosuppressant, which arrests IL-2-stimulated T lymphocytes at G1/S, inhibited the IL-2-induced CRE binding activities concomitantly with inhibition of DNA synthesis. By using specific Abs in a gel mobility shift assay, we identified two known CREB/ATF transcription factors in the IL-2-induced CRE complexes: the CRE binding factor (CREB), and ATF1. The induction of CREB binding by IL-2 was not associated with an increase in its abundance but was associated with a major increase in CREB phosphorylation that was particularly prominent at late G1/S. However, we found that G1/S progression induced by IL-2 was not associated with an increase in the intracellular levels of cAMP. These results suggest that 1) the transcription factors CREB and ATF1 and possibly other CRE binding proteins may have an important role in the modulation of specific gene expression at G1/S during cell cycle progression induced by IL-2. 2) The involvement of these CRE binding transcription factors in IL-2-stimulated cells is regulated via a mechanism that is not cAMP dependent.

Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1 RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.

In this study, we mainly focused on how aldosterone regulates Nox1, a catalytic subunit of NADPH oxidase (NOX) in vascular smooth muscle cells (VSMC). We found that aldosterone can induce the expression of Nox1, which is upregulated by the activation of the Src and activating transcription factor 1 (ATF1), but can not be suppressed by the inhibitors of the epidermal growth factor receptor (EGFR) or Matrix Metalloproteinase (MMP). Aldosterone triggers ATF1 phosphorylation in dose dependent fashion, but this effect is not blocked by actinomycin D, suggesting a non-genomic effect of aldosterone. On the other hand, aldosterone induced Nox1 expression can be suppressed by the gene silencing of the ATF1 using RNA interference. Furthermore, silencing ATF1 can also attenuate aldosterone-induced O(2)(-) production and protein synthesis, and inhibit hypertrophy in this vascular cell lineage. In short, our results primarily unveiled the relationship between aldosterone and Nox1 expression and the regulation mechanism of their signal pathways in the hypertrophy of vascular smooth muscle cell. Src, ATF1, Nox1 and MR are likely efficient targets in the treating of vascular diseases but need more study.

Protein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position -45 to -38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.

Clear cell sarcoma (CCS), malignant melanoma of soft parts, is a rare malignant tumor with a poor prognosis. In this study, a CCS cell line, designated MP-CCS-SY, was established from a metastatic tumor of a 17-year-old Japanese girl that originated in the left Achilles tendon. A small number of melanosomes were detected in the cytoplasm by electron microscopy. The melanosomes immunoreacted with two melanoma-associated antibodies, HMB45 and Melan-A. A Western blot demonstrated the existence of a Melan-A antigen in this cell line. Although a t(12;22)(q13;q12), which is characteristic of CCS, was not identified by a chromosomal analysis with conventional banding techniques, fluorescence in situ hybridization analysis with painting probes of chromosomes 12 and 22 revealed the insertion of a chromosome 12 fragment into one of the long arms of chromosome 22. The chimeric EWS/ATF1 transcript was detected by the reverse transcriptase polymerase chain reaction. Extra copies and structural abnormalities of chromosome 8 were observed. Overexpression of c-myc mRNA was detected by Northern blot analysis and may have a role in malignant progression of CCS. The availability of this MP-CCS-SY cell line will help to understand the molecular biology of this malignancy and should be useful as a tool for developing an immunotherapy.

The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.

BACKGROUND

Clear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines.

METHODS

Hewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo.

RESULTS

Hewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling.

CONCLUSIONS

CCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism.

Angiomatoid fibrous histiocytoma is an uncommon soft tissue tumor most frequently affecting the deep dermis and subcutis of the extremities in children and young adults. We report the first case presenting as a primary pulmonary tumor in a 46-year-old man. Histologically, the tumor was composed of multiple cellular nodules surrounded by a fibrous pseudocapsule and peritumoral lymphoplasmacytic infiltrates. The nodules were composed of histiocytoid cells with a diffuse, whorled, or vague storiform pattern, with the intervening areas densely packed with plasma cells and lymphocytes. The tumor cells were immunoreactive for epithelial membrane antigen, and focally desmin, CD68, and CD163. Fluorescence in-situ hybridization revealed EWS gene translocation, which was further confirmed on polymerase chain reaction to result from EWS/ATF1 gene fusion. It is important to recognize that angiomatoid fibrous histiocytoma can occur in the lung because its histologic features are rather nondescript and thus can be mistaken for other tumors such as meningioma, inflammatory myofibroblastic tumor, and follicular dendritic cell sarcoma.