DNA methylation of GJA1, BMP2 and BMP4 in a human cementoblast cell line induced by lipopolysaccharide.

AIM: To examine DNA methylation of GJA1, BMP2 and BMP4 in human cementoblasts (HCEM) induced by lipopolysaccharide (LPS). METHODOLOGY: HCEM were cultured in osteoinduction medium. After 24 h, Escherichia coli LPS (1 µg/mL) was added to the medium, which was changed every 2-3 days. Untreated samples were used as controls. Messenger RNA was extracted after 4 weeks, and quantitative real-time polymerase chain reaction (qRT-PCR) for GJA1, BMP2, BMP4 and DNMT1 was performed. Genomic DNA was extracted after 4 weeks, and quantitative methylation-specific polymerase chain reaction was carried out for GJA1, BMP2 and BMP4. To detect mineralization, alizarin red and alkaline phosphatase staining were performed. The cells were also treated with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza) and examined. The significance of differences amongst groups was assessed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with P < 0.05 being significant. RESULTS: Decreased expression of mRNA was seen in GJA1, BMP2 and BMP4 after 4 weeks (P < 0.05). DNA hypermethylation was detected in GJA1, BMP2 and BMP4 (P < 0.05). Alizarin red staining and alkaline phosphatase staining revealed decreased mineralization levels in HCEM stimulated with LPS. 5Aza abolished the effects of DNA methylation in HCEM stimulated with LPS. CONCLUSIONS: These results suggest that long-term LPS stimulation induces DNA methylation of GJA1, BMP2 and BMP4 in HCEM.

Alkaline extracellular conditions promote the proliferation and mineralization of a human cementoblast cell line.

AIM: To investigate the proliferation and mineralization of a human cementoblast cell line under alkaline conditions. METHODOLOGY: A human cementoblast cell line was cultured in alkaline media with several pHs (pH 7.6, 8.0 and 8.4) without CO2 . Cell numbers, phospho-p44/42 expression, alkaline phosphatase (ALP) activity and mineralization were evaluated. The significance of differences between groups was assessed using two-way analysis of variance 15 (ANOVA) followed by Bonferroni's multiple comparison test (α = 0.01). RESULTS: Cell numbers increased in a time-dependent manner in the high pH medium groups. Western blot analysis revealed the upregulated expression of phospho-p44/42 under alkaline conditions. ALP activity was also increased at pH 8.0 and 8.4. Alizarin red staining revealed increased mineralization in the high pH medium groups. The incorporation of the transient receptor potential ankyrin subfamily member 1 (TRPA1) antagonist HC030031 markedly negated the effect on proliferation and mineralization. CONCLUSIONS: Extracellular alkaline conditions promoted the proliferation and mineralization of human cementoblasts in vitro via TRPA1.

The Great East Japan Earthquake (2011): using the One Health approach to minimise the impact on the livestock industry and human health.

Supplying safe livestock products made from healthy animals is the primary purpose of the agriculture industry, making it essential to include agriculture in the One Health approach to disaster preparedness and response. After the Great East Japan Earthquake of 11 March 2011, and the following crisis at the Fukushima Nuclear Complex, producing and supplying safe livestock products became a challenging issue, because the area was highly polluted with radiation leaks from the nuclear plants. To produce livestock products that satisfied the safety standards for radioactive materials in food for humans, it was necessary to create feeding management guidelines and set standard limits for radioactive materials in animal feeds. The Ministry of Agriculture, Forestry and Fisheries (MAFF) established provisional maximum limits on radioactive caesium in feeds in order to secure safe food for the nation. Furthermore, there were other issues that Japan's livestock industry had to tackle. The authors outline key measures taken by the Livestock Industry Department of the MAFF to reconstruct the livestock industry, which was a small but important part of the whole reconstruction plan. They also discuss the measures implemented to protect companion animals.

La vocation première du secteur de l'élevage est de fournir au consommateur des produits d'origine animale sans danger et issus d'animaux en bonne santé, ce qui suppose d'intégrer le secteur agricole dans les activités de préparation et de réponse aux catastrophes naturelles sur la base d'une approche Une seule santé. Après le grand séisme survenu le 11 mars 2011 sur la côte Pacifique du Tohoku au Japon et la catastrophe du complexe nucléaire de Fukushima qui a suivi, la question de l'innocuité de la production et distribution d'animaux et de produits d'origine animale est devenue un enjeu majeur en raison du taux élevé de contamination de la zone par les émissions radioactives qui s'étaient échappées des centrales nucléaires. Afin de garantir la conformité des produits de l'élevage aux normes de sécurité au regard du risque de contamination radioactive, il a fallu élaborer des lignes directrices en matière de gestion de l'alimentation animale et fixer une valeur limite à la contamination radioactive des aliments destinés aux animaux. Le ministère japonais en charge de l'agriculture, des forêts et des pêches (MAFF) a fixé à titre provisoire une limite de concentration maximale admissible pour le césium radioactif dans les aliments destinés aux animaux afin de garantir l'innocuité des denrées alimentaires distribuées à la nation. Ce problème n'est pas le seul auquel a dû faire face le secteur japonais de l'élevage suite au séisme de 2011. Les auteurs font le point sur les principales mesures adoptées par le département de l'élevage du MAFF pour restaurer le secteur, volet mineur mais néanmoins essentiel du plan de reconstruction nationale. Les mesures adoptées pour protéger les animaux de compagnie sont également présentées.

El objetivo primordial de la industria agropecuaria estriba en abastecer a la población de productos inocuos, obtenidos a partir de animales sanos. Ello hace indispensable incluir la agricultura en toda labor de preparación y respuesta para casos de catástrofe que se efectúe desde los planteamientos de Una sola salud. Después del llamado Gran terremoto del Japón Oriental que tuvo lugar el 11 de marzo de 2011, y tras la crisis del complejo nuclear de Fukushima, la producción y el suministro de productos ganaderos inocuos pasaron a ser una cuestión muy espinosa, pues la zona estaba sumamente contaminada por fugas radiactivas de las centrales nucleares. Para obtener productos ganaderos que cumplieran las normas de inocuidad relativas a la presencia de material radiactivo en los alimentos destinados al consumo humano fue necesario establecer directrices de gestión de alimentos y fijar límites normalizados de material radiactivo en los piensos animales. A fin de garantizar que el país gozara de un suministro de alimentos inocuos, el Ministerio de Agricultura, Bosques y Pesca estableció límites máximos provisionales de cesio radiactivo en los piensos. Pero este no era el único problema con que debía lidiar la industria ganadera del Japón. Los autores exponen a grandes líneas las principales medidas adoptadas por el Departamento de Industria Ganadera del Ministerio para reconstruir la industria pecuaria, en lo que representaba una parte pequeña, pero importante, del plan de reconstrucción global. Los autores examinan asimismo las medidas instituidas para proteger a los animales de compañía.

Value of 100 kVp scan with sinogram-affirmed iterative reconstruction algorithm on a single-source CT system during whole-body CT for radiation and contrast medium dose reduction: an intra-individual feasibility study.

AIM: To perform an intra-individual investigation of the usefulness of a contrast medium (CM) and radiation dose-reduction protocol using single-source computed tomography (CT) combined with 100 kVp and sinogram-affirmed iterative reconstruction (SAFIRE) for whole-body CT (WBCT; chest-abdomen-pelvis CT) in oncology patients. MATERIALS AND METHODS: Forty-three oncology patients who had undergone WBCT under both 120 and 100 kVp protocols at different time points (mean interscan intervals: 98 days) were included retrospectively. The CM doses for the 120 and 100 kVp protocols were 600 and 480 mg iodine/kg, respectively; 120 kVp images were reconstructed with filtered back-projection (FBP), whereas 100 kVp images were reconstructed with FBP (100 kVp-F) and the SAFIRE (100 kVp-S). The size-specific dose estimate (SSDE), iodine load and image quality of each protocol were compared. RESULTS: The SSDE and iodine load of 100 kVp protocol were 34% and 21%, respectively, lower than of 120 kVp protocol (SSDE: 10.6±1.1 versus 16.1±1.8 mGy; iodine load: 24.8±4versus 31.5±5.5 g iodine, p<0.01). Contrast enhancement, objective image noise, contrast-to-noise-ratio, and visual score of 100 kVp-S were similar to or better than of 120 kVp protocol. CONCLUSION: Compared with the 120 kVp protocol, the combined use of 100 kVp and SAFIRE in WBCT for oncology assessment with an SSCT facilitated substantial reduction in the CM and radiation dose while maintaining image quality.

Response of porcine epithelial rests of Malassez to stimulation by interleukin-6.

AIM: To investigate the proliferation and migration of epithelial cell rests of Malassez (ERM) after stimulation with IL-6. METHODOLOGY: Porcine-derived ERM were seeded on Dulbecco's modified Eagle's Medium, and IL-6 (100 pg mL-1 ) was incorporated into the culture medium. The WST-1 assay was performed to evaluate cell proliferation, and absorption was measured at 450 nm. A wound-healing assay and immunofluorescence assay for integrin α3 were conducted to investigate migration. The Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni correction were used to analyse data of WST-1 and wound-healing assays. RESULTS: Cell proliferation following the stimulation by IL-6 increased over time, with a significant increase being observed at 6 h (P < 0.05), but not in a concentration-dependent manner. Cell proliferation was significantly greater in IL-6-treated ERM than in nontreated ERM (P < 0.05). The results of the wound-healing assay revealed earlier closure in IL-6-treated ERM (P < 0.05). In the immunofluorescence assay, integrin α3 was detected at the edge of cell processes adjacent to the wound area. A neutralized antibody abrogated the effects of the IL-6 stimulation in cell proliferation and migration. CONCLUSION: IL-6 promoted the proliferation and migration of porcine ERM in vitro.

Actin stabilization induces apoptosis in cultured porcine epithelial cell rests of Malassez.

AIM: To test whether actin stabilization by jasplakinolide induces inhibition of cell viability and apoptosis in epithelial cell rests of Malassez (ERM). METHODOLOGY: ERM derived from porcine were spread in a 96-well dish (5 × 10(4) /well) using Dulbecco's modified Eagle's medium. The actin-specific stabilization reagent, jasplakinolide, was incorporated into the culture medium and incubated for 24 h. To evaluate cell viability, the WST-1 assay was carried out and absorption (450 nm) was measured. To detect apoptotic cells, monoclonal antibody to single-strand DNA (ssDNA) was used and absorption (405 nm) was measured. Actin stabilization and apoptosis induced by jasplakinolide were morphologically investigated by staining with Alexa Fluor 568 phalloidin and observed under a fluorescent microscope. As a negative control, DMSO was used instead of jasplakinolide. Differences between the jasplakinolide-treated group and the control group were analysed statistically using the Student's t-test. RESULTS: Cell viability decreased in a concentration-dependent manner, and cell viability in the jasplakinolide-treated ERM was lower than that in nontreated ERM (n = 16, P < 0.01). Apoptotic cells in the jasplakinolide-treated ERM were more frequently detected compared to that in nontreated ERM (n = 16, P < 0.01). Morphologically, shrinkage, irregular forms and fragmentation of nuclei suggesting apoptotic bodies were observed in jasplakinolide-treated ERM, whilst actin filaments were extended in non-treated ERM. CONCLUSION: Actin stabilization by jasplakinolide inhibited cell viability and induced apoptosis in epithelial cell rests of Malassez.

Analysis of T cell hybridomas. II. Comparisons among three distinct types of monoclonal suppressor factors.

Five hybridoma T cell lines were prepared by fusion of Ts3 cells with the BW 5147 thymoma. The culture supernatants from these T cell hybrids contained a factor, TsF3, which specifically suppressed 4-hydroxy-3-nitrophenyl acetyl hapten (NP(-hapten cutaneous sensitivity responses. The properties of this new series of hybridoma factors was compared with those of two previously characterized types of NP-specific suppressor factors (TsF1 and TsF2). TsF3 activity was only observed if the factor was administered during the effector phases of the immune response. TsF3 bears I-J and C57BL anti-NP antibody idiotypic determinants and has binding specificity for the NP hapten. Furthermore, TsF3 does not suppress H-2 (I-J)-incompatible mice. In addition to this H-2 restriction, the monoclonal TsF3 factors also demonstrated an Igh genetic restriction. Finally, the TsF3 factors could be distinguished by their ability to suppress cyclophosphamide-treated recipients.

Acute antibody-mediated rejection in living ABO-incompatible kidney transplantation: long-term impact and risk factors.

The impact of acute antibody-mediated rejection (AAMR) on the long-term outcome on ABO-incompatible (ABOI) kidney transplantation is not well understood. We retrospectively analyzed the long-term impact of AAMR and risk factors for AAMR in 57 consecutive recipients performed between 1999 and 2004. Nineteen patients (33%) who developed AAMR within 3 months posttransplantation constituted of the AMR group. The graft survival rate was significantly lower in the AMR group (AMR vs. non-AMR, respectively; 5 years: 84% vs. 95%; 8 years: 45% vs. 95%; p = 0.009). The prevalence of transplant glomerulopathy at 1 year posttransplantation was significantly higher in the AMR group (AMR 64% vs. non-AMR 3%, p < 0.001). Multivariate analysis demonstrated that anti-blood group IgG antibody titers of 1:32 at the time of transplantation (OR, 9.52; p = 0.041) and donor-specific anti-HLA antibodies (DSHA) detected by Luminex single bead method (OR, 5.68; p = 0.015) were independent risk factors for AAMR regardless of baseline anti-blood group IgG antibody titers. Our results indicate that AAMR has a heavy impact on the long-term outcome and preoperative DSHA appears to have a more significant association with poor graft outcomes than anti-blood group antibodies, even in ABOI kidney transplantation.

hst-1 transforming protein: expression in silkworm cells and characterization as a novel heparin-binding growth factor.

A protein encoded by an hst-1 transforming gene was expressed in silkworm-derived BmN cells and secreted into the culture medium using a recombinant baculovirus vector. The strong affinity for heparin of the secreted protein made it possible to purify the hst-1 protein to homogeneity in a two-step procedure. The purified hst-1 protein has a molecular weight of 18,000 and stimulates both DNA synthesis in NIH3T3 cells and human umbilical vein endothelial cell proliferation. In addition, morphological changes and anchorage-independent growth of NIH3T3 cells are induced by this product. These results show that the hst-1 transforming protein is a novel heparin-binding growth factor as predicted by nucleotide sequence analysis.

Allograft glomerulitis: histologic characteristics to detect chronic humoral rejection.

The aim of this study was to clarify the histopathologic significance of allograft glomerulitis in chronic allograft nephropathy (CAN). Review of our renal allograft biopsy files revealed 140 specimens with CAN among 115 selected patients. They were classified into two groups: one had CAN with glomerulitis (group G), and the other was free of this finding (group NG). We evaluated the clinicopathologic parameters as follows: levels of serum creatinine and proteinuria in the biopsy; presence of circulating anti-donor antibodies; allograft failure rate; history of biopsy-proven acute cellular rejection (ACR) and acute humoral rejection (AHR); complications of ACR and chronic rejection (CR); and results of immunofluorescence studies for C4d and HLA-DR. The glomerulitis group showed a significantly greater incidence of CR complications, the presence of circulating anti-donor antibodies, and C4d deposition in peritubular and glomerular capillaries. This group also showed higher levels of serum creatinine and proteinuria, higher graft loss rate, and increased AHR incidence, although the differences were not significant. There was also no statistical significance in the HLA-DR expression on tubular epithelial cells. The present results strongly suggest that humoral factors may play an important role in the progression of glomerulitis in CAN. Therefore, we suspect that glomerulitis in CAN is one of the main histologic markers for CR. The presence of glomerulitis may represent humoral factor-dependent inflammation. It should be considered an important diagnostic criterion for CR in addition to double-contour formation and elastica disruptions with or without subendothelial inflammation (Banff '97).

A fluoroquinolone (DR-3355) protects human lymphocyte cell lines from HIV-1-induced cytotoxicity.

HIV-1 infection of human CD4+ lymphocyte cell lines results in cell death. Treatment, but not pretreatment, of infected cells, with a fluoroquinolone antibiotic, DR-3355, protects a significant subfraction of cells from HIV-1-mediated cytolysis. All surviving cells have lost expression of the CD4 antigen, but do (MT-4) or do not (CEM) express viral antigens and produce infective virus. The rescued CEM and MT-4 cells are phenotypically stable and do not require continuous exposure to the drug for survival.

Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method.

Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes.

An immunization method using antigen entrapped in erythrocyte ghosts.

Intraperitoneal injection into mice of dinitrophenol-conjugated ovalbumin entrapped in autologous erythrocyte ghosts gave rise to an increase of anti-dinitrophenol antibody production without use of artificial adjuvants. This technique could be useful for vaccination or other immuno-therapeutic purposes.

MR evaluation of radiation otomastoiditis.

PURPOSE: The purpose of this study was to evaluate the incidence of radiation otomastoiditis, on using T2-weighted magnetic resonance (MR) imaging, in relation to radiation fields, doses, intervals, and clinical symptoms after radiotherapy that included the temporal bone in the fields. METHODS AND MATERIALS: We performed follow-up MR examinations at various intervals after radiotherapy including the temporal bones for 270 ears of 114 patients with various diseases of the head and neck and intracranial regions. The middle ear and mastoid air cells on T2-weighted images were scored as follows; showing no high signal intensity, a local high signal intensity area, or a high signal intensity area occupying the entire middle ear and all mastoid air cells. The radiation fields as depicted on the lateral simulator films classed as anterior to the clival line, posterior to the clival line, or both. RESULTS: The incidence of radiation otomastoiditis depicted on T2-weighted MR images increased in the patients who had received irradiation doses of 50 Gy or more. In the patients with doses of less than 50 Gy, the incidence was 18% within 6 months following radiotherapy, 13% at between 6 and 12 months, and 8% after 12 months, whereas it was more than 50% at any period after radiotherapy in the patients with 50 Gy or more. The incidence of radiation otomastoiditis was quite high in the patients whose radiation fields included region both anterior and posterior to the clival line. CONCLUSION: The incidence of radiation otomastoiditis as demonstrated on T2-weighted MR images is increased at irradiation doses of 50 Gy or more. To reduce the incidence of severe radiation otomastoiditis, the irradiation fields of the temporal bone when the dose is 50 Gy or more should be limited to as small as possible. The clival line is considered to be a good landmark in reducing the irradiation field when doses of 60-70 Gy are delivered in curative radiotherapy.

Optimal image resolution for digital storage of radiotherapy-planning images.

PURPOSE: To evaluate the quality of digitized radiation-planning images at different resolution and to determine the optimal resolution for digital storage. METHODS AND MATERIALS: Twenty-five planning films were scanned and digitized using a film scanner at a resolution of 72 dots per inch (dpi) with 8-bit depth. The resolution of scanned images was reduced to 48, 36, 24, and 18 dpi using computer software. Image qualities of these five images (72, 48, 36, 24, and 18 dpi) were evaluated and given scores (4 = excellent; 3 = good; 2 = fair; and 1 = poor) by three radiation oncologists. An image data compression algorithm by the Joint Photographic Experts Group (JPEG) (not reversible and some information will be lost) was also evaluated. RESULTS: The scores of digitized images with 72, 48, 36, 24, and 17 dpi resolution were 3.8 +/- 0.3, 3.5 +/- 0.3, 3.3 +/- 0.5, 2.7 +/- 0.5, and 1.6 +/- 0.3, respectively. The quality of 36-dpi images were definitely worse compared to 72-dpi images, but were good enough as planning films. Digitized planning images with 72- and 36-dpi resolution requires about 800 and 200 KBytes, respectively. The JPEG compression algorithm produces little degradation in 36-dpi images at compression ratios of 5:1. CONCLUSION: The quality of digitized images with 36-dpi resolution was good enough as radiation-planning images and required 200 KBytes/image.

Role of dynamic MRI in the evaluation of head and neck cancers treated with radiation therapy.

PURPOSE: To study the usefulness of dynamic magnetic resonance imaging in the evaluation of head and neck cancers treated with radiation therapy. METHODS AND MATERIALS: Seventy-six patients (58 males and 18 females; ages 20-82) with head and neck cancers (10 nasopharyngeal carcinomas, 22 mesopharyngeal carcinomas, 10 hypopharyngeal carcinomas, 16 oral cavity carcinomas, 11 lingual carcinomas, and 7 laryngeal carcinomas) were treated by radiation therapy combined with concomitant low-dose cisplatinum. Magnetic resonance imaging (MRI) was performed before and 2 weeks after the irradiation in all cases. After bolus administration of gadopentetate dimeglumine (Gd-DTPA) (0.1 mmol kg), images were obtained every 30 s (repetition time 200 ms, echo time 16 ms) using a 1.5 or 0.5-T superconductive unit. Biopsy or surgery was performed after radiation therapy and the histologic findings were correlated with the MRI findings (T1, T2, dynamic, and enhanced T1). RESULTS: Complete remission, partial response, and no response were obtained in 18, 36, and 7 cases, respectively. Dynamic MRI correctly diagnosed 17 of the 18 complete remission cases, 33 of the 36 partial response cases, and all of the 7 no-response cases. The accuracy of dynamic MRI, T1-weighted image, T2-weighted image, and Gd-enhanced T1-weighted image was 94.4%, 68%, 82%, and 86%, respectively. CONCLUSION: Dynamic MRI proved to be useful in the evaluation of the radiation therapy of head and neck cancers.

The effect of azelastine on acute radiation dermatitis in mice models.

PURPOSE: In our previous report we described the clinical value of azelastine, an oral antiallergic agent, as an inhibitor of radiation dermatitis. Here we studied the effect of azelastine on normal skin and tumor size after irradiation in a mouse model. METHODS AND MATERIALS: The modifying effects of azelastine on both the degree of radiation dermatitis and antitumoral effect of radiation therapy were investigated in the normal skin as well as in SCC VII tumors of C3H/He mice. The right hind legs, with or without tumors, were irradiated with 20-60 Gy at 0.62 Gy/min. Azelastine was administered via the mouse chow, and acute skin reactions and tumor growth curves were compared between the azelastine and control groups. RESULTS: The acute skin reactions of the azelastine group were significantly less prominent than those of the control group (p < 0.01). At a dose of 40 Gy the dose modification factors were 1.19-1.25. The tumor growth curves of the azelastine and control groups were almost identical, indicating that the treatment response of irradiation was not affected by administration of azelastine. CONCLUSIONS: Application of azelastine reduces the degree of acute radiation dermatitis without affecting the antitumoral effect of radiation therapy.

On the origin of alpha-glucosidase in human urine.

The recent findings that alpha-glucosidase from human kidney was identical with one component (F1) of the alpha-glucosidases found in human urine suggested the idea that this enzyme might originate in the kidneys. The present study was performed to test this idea by immunological methods. Urine alpha-glucosidase F1 was isolated in the electrophoretically homogeneous state, and the antibody prepared in rabbits was purified by affinity chromatography after the antisera were fractionally precipitated with ammonium sulfate and chromatographed on diethylamino ethyl (DEAE)-cellulose. The staining of human kidney tissue sections was performed by the indirect method, using alpha-glucosidase F1 antibody and fluorescein-conjugated anti-rabbit immunoglobulin goat sera. The proximal convoluted portion (proximal tubules) with brush border and Henle's loops (late proximal) were stained clearly. Preincubation of intact antibody with purified antigen prevented specific staining of the proximal convoluted portion and Henle's loops. In contrast, all other tissues of kidney were stained less positively or negatively. These results indicate that alpha-glucosidase F1 originates in the kidney, and that glucosidase is specifically localized in the proximal convoluted portion and Henle's loops.

Expression of xenoantigen transformed human cancer cells to be susceptible to antibody-mediated cell killing.

The carbohydrate epitope, alphaGal epitope, is known as a major xenoantigen. The epitope exists abundantly in non-primate mammals and has recently been found on C-type retroviruses. Humans and Old World monkeys have anti-alphaGal antibody, a natural antibody. The present study was performed to examine if the alphaGal epitope could be used as a new target of gene therapy against human cancer. Bovine alpha1-3 galactosyltransferase (alpha1-3 GT) cDNA which produces the alphaGal epitope was electrophoretically transfected into the human pancreatic cancer cell line, MIA PaCa-2 and the human hepatocellular carcinoma cell line, huH7. The expression of the alphaGal epitope was confirmed by flow cytometry, using specific binding with IB4 lectin conjugated with fluorescein isothiocyanate. Transfected MIA PaCa-2 cells and huH7 cells showed a positive log shift for the alphaGal epitope. Next, we examined whether human cancer cells expressing the alphaGal epitope could be lysed by natural antibodies using the complement-dependent cytotoxic cross-match test. The results showed that transfected MIA PaCa-2 cells and huH7 cells were effectively lysed by human natural antibodies, and the killing was observed with any serum irrelevant of blood type. The results indicate that transfection of the functional alpha1-3 GT gene into human cancer cells can lead to their transformation, making them susceptible to lysis by natural antibodies, and, thus, useful for gene therapy.

AMY-1 is a trigger for the erythrocyte differentiation of K562 cells.

We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from cytoplasm to nuclei in a c-Myc-dependent manner. Here, the role of AMY-1 in cell differentiation was examined. AMY-1 expression was up-regulated after differentiation induction of human K562 cells to erythrocyte cells by AraC, while c-Myc expression was rapidly down-regulated. K562 cell lines expressing exogenous AMY-1 were established, and these cells expressed a high level of epsilon-globin mRNA, a marker gene necessary for erythrocyte cell differentiation, without differentiation induction. The addition of AraC rapidly initiated differentiation in these cell lines, which continued to differentiate to erythrocyte cells possessing a high level of hemoglobin even after the decrease in AMY-1 expression. These results suggest that AMY-1 is a trigger for K562 cells to differentiate to erythrocyte cells and that AMY-1 may have a function independent of or different from c-Myc.