Hair Follicle Damage after 100 mGy Low-Dose Fractionated X-Ray Irradiation and the Protective Effects of TEMPOL, a Stable Nitroxide Radical, against Radiation.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a non-irradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

Neutrophil-Derived MicroRNA-1290 Promotes Keratinocyte Proliferation in Psoriasis.

The pathological hallmark of psoriasis is the infiltration of neutrophils into the skin. Some neutrophil-derived microRNAs (miRNAs) serve as biomarkers for various diseases, but none have been reported for psoriasis. In this study, we investigated the involvement of miRNAs released from neutrophils in psoriasis pathogenesis. We compared the expression of miRNAs in the sera of patients with psoriasis with that in healthy individuals and found that the expression of 2 miRNAs-miR-223 and miR-1290-was significantly upregulated in the sera of patients with psoriasis. The serum levels of these miRNAs positively correlated with the PASI and CRP levels. We used all-trans retinoic acid to induce the differentiation of human promyelocytic leukemia HL-60 cells into neutrophil-like cells and found that the release of both miRNAs increased during differentiation. Furthermore, the release of miR-1290 was increased by TNF-α in neutrophil-like cells and human neutrophils. Treatment with the miR-1290 precursor promoted the proliferation of human keratinocytes, increased the proportion of S-phase cells, and upregulated the phosphorylation of extracellular signal-regulated kinase 1/2. These results suggest that miR-1290 plays a vital role in regulating neutrophil differentiation and keratinocyte proliferation and could be a serum marker of psoriasis severity.

Establishment and Characterization of a TFE3-rearranged Renal Cell Carcinoma Cell Line (FU-UR-2) With the PRCC-TFE3 Fusion Transcript.

BACKGROUND/AIM: Xp11.2-RCC was classified into molecularly defined renal carcinomas and named TFE3-rearranged renal cell carcinoma (TFE3-rRCC) in the 2022 World Health Organization classification of renal tumors. MATERIALS AND METHODS: In this study, we established and characterized a TFE3-rRCC cell line from a right-sided renal tumor of a 35-year-old female patient and named it FU-UR-2. FU-UR-2 had been initially diagnosed as a papillary RCC because the patient was 35 years old, a routine immunohistochemical staining for TFE3 was negative, and its morphology was papillary. The G-band analysis revealed an X-chromosome aberration, thus we performed immunohistochemical re-staining for TFE3 and examined the aberration in the TFE3 gene by reverse-transcriptase polymerase chain reaction and fluorescence in situ hybridization. RESULTS: FU-UR-2 was confirmed as a TFE3-rRCC with a PRCC-TFE3 fusion transcript. CONCLUSION: Cultured FU-UR-2 cells continuously propagated over 90 passages and may provide a new permanent culture model to study pathogenetic mechanisms, investigate biological behavior, and develop new treatments such as molecular-targeting antitumor agents or immunological drugs for TFE3-rRCCs.

Establishment and Characterisation of Human Clear Cell Sarcoma and Malignant Melanoma Cell Lines.

BACKGROUND/AIM: Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. MATERIALS AND METHODS: We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. RESULTS: FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). CONCLUSION: We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non-small-cell lung cancer cells.

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6ß4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.

Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles.

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.

Application of high-mannose-type glycan-specific lectin from Oscillatoria Agardhii for affinity isolation of tumor-derived extracellular vesicles.

Tumor cells secrete membrane vesicles of various sizes, termed extracellular vesicles (EVs), which have gained increasing attention as potential tumor diagnostic markers. Tumor-derived EVs are enriched with high-mannose-type glycans. Here, we report the affinity isolation of EVs from human melanoma A375 cells by using high-mannose-type glycan-specific agglutinin from Oscillatoria Agardhii (OAA). Glycan analysis of melanoma EVs revealed the presence of high-mannose-type glycans with structural units preferred by OAA. We showed that in solution, OAA binds to melanoma EVs in a high-mannose-type glycan-dependent manner. Furthermore, OAA-immobilized beads were found to capture 60% of the particles and most proteinous components from melanoma EVs. Major EV glycoproteins that potentially interact with OAA were identified to be cluster of differentiation 109 (CD109), integrin α6 and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). In addition to melanoma EVs, OAA captured EVs from human lung cancer, glioblastoma and colon cancer cells, but not those from endothelial cells and fibroblasts. These results indicate that OAA-immobilized beads may serve as a novel platform for affinity-capture of tumor-derived EVs.

E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases.

E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial-mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell-extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased ß-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for ß-catenin expression and ß-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration.

Generation of the heterogeneity of extracellular vesicles by membrane organization and sorting machineries.

BACKGROUND: Cells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains. METHODS: Membrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments. RESULTS: We identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met. CONCLUSION: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. GENERAL SIGNIFICANCE: This study provides novel insights into important aspects of EV biogenesis.

The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma.

AIMS: Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC. RESULTS: In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4-/y), and human PRDX4 transgenic (hPRDX4+/+) mice were given a weekly intraperitoneal injection of diethylnitrosamine for 25 weeks. The HCC incidence was higher in PRDX4-/y mice than in WT or hPRDX4+/+ mice. Intrahepatic and circulating oxidative stress and inflammatory cell infiltration in the liver were obviously decreased in hPRDX4+/+ mice, compared with WT mice. Furthermore, in our cohort study, human HCC specimens with low expression of PRDX4 had higher ROS levels and a highly malignant phenotype, which was associated with a reduced overall survival, compared with those with high PRDX4 expression. However, in human HCC cell lines, PRDX4 knockdown led to a rapidly increased intracellular ROS level and suppressed cell proliferation, inducing cell death. Innovation and Conclusion: Our results clearly indicate that PRDX4 has an inhibitory effect in the initiation of HCC, but a dual (inhibitory or promoting) role in the progression of HCC, suggesting the potential utility of PRDX4 activators or inhibitors as therapy for different stages and phenotypes of HCC.

High-fat diet exacerbates imiquimod-induced psoriasis-like dermatitis in mice.

Psoriasis, a chronic inflammatory skin disease, is closely related to systemic metabolism. An elevated body mass index (BMI) is a risk factor for psoriasis; inflammasomes are activated by adipose tissue macrophages in obese subjects. We hypothesized that hyperlipidaemia is involved in the pathogenesis of psoriasis and examined the role of a high-fat diet (HFD) in the development of psoriasis in imiquimod (IMQ)-treated mice. The body weight and serum level of cholesterol were significantly higher in mice fed an HFD than in a regular diet (RD). HFD mice had higher psoriasis skin scores, and the number of neutrophils infiltrating into the lesional skin was elevated. IL-17A mRNA expression was significantly increased in the skin of IMQ-treated HFD mice; the expression of IL-22, IL-23 and TNF-α mRNA was not enhanced. Caspase-1 and IL-1ß were activated in the skin of IMQ-treated HFD mice, and their serum level of IL-17A, TNF-α and IL-1ß was significantly upregulated. Our findings strongly suggest that hyperlipidaemia is involved in the development and progression of psoriasis via systemic inflammation and inflammasome activation.

Epstein-Barr virus infection and gene promoter hypermethylation in rheumatoid arthritis patients with methotrexate-associated B cell lymphoproliferative disorders.

We analyzed CpG-island hypermethylation status in 12 genes of paraffin-embedded tissues from 38 rheumatoid arthritis (RA) patients with methotrexate (MTX)-associated large B cell lymphoproliferative disorder (BLPD), 11 RA patients with non-MTX-associated BLPD (non-MTX-BLPD), 22 controls with diffuse large B cell lymphoma (DLBCL), and 10 controls with Epstein-Barr virus (EBV)+ DLBCL. Among them, tumor cells from EBV+ MTX-BLPD patients and control EBV+ DLBCL patients had significantly lower median incidence of CpG island methylator phenotype (CIMP) than those from non-MTX-BLPD and control DLBCL groups (2.3 and 1.7 vs. 4.3 and 4.4; P < 0.01 for each). In the MTX-BLPD group, EBV+ patients showed lower median CIMP than EBV- patients (2.3 vs. 3.2); they also had significantly lower hypermethylation incidence in four apoptosis-related genes, especially death-associated protein kinase (14 vs. 55 %), higher incidence of massive tumor necrosis (86 vs. 27 %), and lower BCL2 protein expression (19 vs. 86 %) than did the control DLBCL group (P < 0.01 for all). In all clinical stages, EBV+ MTX-BLPD patients had better prognoses than the EBV- MTX-BLPD (P = 0.011), non-MTX-BLPD (P = 0.002), and control DLBCL groups (P = 0.015). MTX-BLPD patients without hypermethylated RAS-associated domain family-1A (RASSF1A) or O 6 -methyl guanine-DNA methyltransferase (MGMT) had significantly better prognosis than those with hypermethylation of those genes (P = 0.033). We conclude that in RA patients with MTX-BLPD, EBV infection is associated with a lower incidence of CIMP, apoptosis-related gene hypermethylation, and BCL2 expression, which can induce tumor regression by MTX withdrawal and lead to better prognoses.

Molecular cytogenetic characterization of two established ESFT cell lines.

Ewing's sarcoma/primitive neuroectodermal tumor/Askin's tumor (Ewing`s sarcoma family of tumors: ESFT) is the most common type of malignant tumor of bone and soft tissue in children and young adults, and morphologically is a member of a group of small round cell tumors. We report, here, on the establishment of two human ESFT cell lines, FU-PNET-3 and FU-PNET-4, from the iliac and the chest wall, respectively, the cells of both cell lines were tumorigenic in immunodeficient mice. Histologically, both original and xenograft tumors and cultured cells were composed of small round cells with positive immunoreactivity for CD99 and Nkx2.2. Molecular biological examination demonstrated chimeric transcripts of EWSR1 exon 7 to FLI1 exon 6 in FU-PNET-3 cells, and EWSR1 exon 10 to FLI1 exon 6 in FU-PNET-4 cells. Cytogenetic analysis revealed chromosome translocation t(11;22)(q24;q12) and some secondary changes in both cultured cells. These histological, molecular biological, and cytogenetical findings indicate ESFT in both cell lines. ESFT is well studied, but its recurrent fusion genes are heterogeneous and its biological behaviors are unclear. The FU-PNET-3 and FU-PNET-4 cell lines have been well examined and may become useful tools for studying the genetic and biological behavioral properties of ESFT.

Cleaved CD147 shed from the surface of malignant melanoma cells activates MMP2 produced by fibroblasts.

BACKGROUND: Cluster of differentiation 147 (CD147)/basigin on the malignant tumor cell surface is critical for tumor proliferation, invasiveness, metastasis, and angiogenesis. CD147 expressed on malignant melanoma cells can induce tumor cell invasion by stimulating the production of matrix metalloproteinases (MMPs) by surrounding fibroblasts. Membrane vesicles, microvesicles and exosomes have attracted attention, as vehicles of functional molecules and their association with CD147 has been reported. Cleaved CD147 fragments released from tumor cells were reported to interact with fibroblasts. We investigated the intercellular mechanisms by which CD147 stimulates fibroblasts to induce MMP2 activity. MATERIALS AND METHODS: CD147 was knocked-down using short hairpin RNA (shRNA). The stimulatory effect of CD147 in cell culture supernatants, microvesicles, and exosomes on the enzymatic activity of MMP2 was examined by gelatin zymography. RESULTS: Supernatants from A375 control cells induced increased enzymatic activity of fibroblasts; such activity was significantly lower in CD147 knock-down cells. CONCLUSION: Cleaved CD147 plays a pivotal role in stimulating fibroblasts to induce MMP2 activity.

Abcb10 role in heme biosynthesis in vivo: Abcb10 knockout in mice causes anemia with protoporphyrin IX and iron accumulation.

Abcb10, member 10 of the ABC transporter family, is reportedly a part of a complex in the mitochondrial inner membrane with mitoferrin-1 (Slc25a37) and ferrochelatase (Fech) and is responsible for heme biosynthesis in utero. However, it is unclear whether loss of Abcb10 causes pathological changes in adult mice. Here, we show that Abcb10(-/-) mice lack heme biosynthesis and erythropoiesis abilities and die in midgestation. Moreover, we generated Abcb10(F/-); Mx1-Cre mice, with Abcb10 in hematopoietic cells deleted, which showed accumulation of protoporphyrin IX and maturation arrest in reticulocytes. Electron microscopy images of Abcb10(-/-) hematopoietic cells showed a marked increase of iron deposits at the mitochondria. These results suggest a critical role for Abcb10 in heme biosynthesis and provide new insights into the pathogenesis of erythropoietic protoporphyria and sideroblastic anemia.

Snail modulates cell metabolism in MDCK cells.

Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of key enzymes. This results in enhanced glucose dependency and leads to cell death under low-glucose conditions. On the other hand, the reduced requirements for oxygen and nutrients from the surrounding environment, might confer the resistance to cell death induced by hypoxia and malnutrition.

Impaired neural differentiation of induced pluripotent stem cells generated from a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in ß-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.

p53R2 is a prognostic factor of melanoma and regulates proliferation and chemosensitivity of melanoma cells.

BACKGROUND: The treatment of melanoma, an aggressive, chemo-resistant skin cancer characterized by rapid metastasis and a poor prognosis, requires the development of innovative therapies with improved efficacy. The p53R2 gene that encodes the ribonucleotide reductase small subunit 2 homologue is induced by several stress signals including DNA-damaging agents that activate p53. The p53R2 gene product increases the deoxynucleotide triphosphate pool in the nucleus; this facilitates DNA repair and synthesis. OBJECTIVE: We examined the expression of p53R2 in melanoma and evaluated whether p53R2 is involved in the growth and proliferation of melanoma cells. Methods We examined the clinicopathological significance of p53R2 in melanoma. To investigate the role of p53R2 in melanoma we used KHm5 and KHm6 melanoma cells that express p53R2, and p53R2-targeting small interfering (si) RNA. RESULTS: p53R2 expression was detected immunohistochemically in 56 of 78 patients (71.8%). The expression of p53R2 was significantly correlated with the depth of invasion and the tumor stage. p53R2-targeting siRNA successfully knocked down p53R2 and significantly inhibited the growth of KHm5 and 6 cells. Moreover, The degree of KHm5 and 6 cell growth inhibition was greater in the presence of both p53R2-targeting siRNA and nimustine (ACNU) than with ACNU alone, suggesting that p53R2 silencing enhanced the chemosensitivity of KHm5 and 6 cells to ACNU. CONCLUSIONS: We propose p53R2 as a therapeutic target to enhance the effectiveness of chemotherapy in patients with p53R2-positive melanoma.

Fabry disease: biochemical, pathological and structural studies of the α-galactosidase A with E66Q amino acid substitution.

Recently, male subjects harboring the c.196G>C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G>C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G>C is not a pathogenic mutation but is a functional polymorphism.