Miyuki Yanaka

Continuous intake of a high-fat diet beyond one generation promotes lipid accumulation in liver and white adipose tissue of female mice

Lipid metabolism in a child may be altered when the mother has a high-fat diet (HFD), but it is unclear whether the lipid metabolism of future offspring (grandchildren) is also changed under these circumstances. In this study, we examined the influence of intake of an HFD beyond one generation on offspring in normal mice. Parent mice fed an HFD were bred and the resultant second and third generations were also fed an HFD. The diets used in the study had approximately 20% more energy than a standard chow diet. Changes in lipid metabolism were examined in each generation. Intake of an HFD from generation to generation promoted lipid accumulation in the white adipose tissue of female mice, increased lipid, glucose and insulin levels in the serum, increased the activities of enzymes associated with fatty acid metabolism in the liver, promoted lipid accumulation in hepatocytes and adipocytes and increased the mRNA levels of Cdkn1a in the liver and white adipose tissue. These results suggest that activation of Cdkn1a promoted lipid accumulation in the liver and white adipose tissue of third-generation female mice that were offspring from earlier generations fed HFDs. Moreover, intake of a high-energy diet beyond one generation led to offspring with obesity, fatty liver and hyperinsulinemia.

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.

Atopic dermatitis causes lipid accumulation in the liver of NC/Nga mouse

Various factors have been reported to influence lipid metabolism and cause metabolic syndrome. However, the influence of allergy on the liver that plays important role of lipid metabolism has not been clarified. The aim of this study was to examine the influence of allergy on lipid metabolism of liver. A model of atopic dermatitis was developed in the NC/Nga mouse using picryl chloride to induce allergy. Lipid metabolism parameters were measured and the mechanism of changes in these parameters was examined using DNA microarray analysis and quantitative reverse transcriptase PCR. Triacylglycerol accumulation was promoted in the liver in the mouse atopic dermatitis model despite reductions in food intake, body weight gain, and serum glucose. As this mechanism, it was thought that atopic dermatitis caused the suppression of fatty acid β-oxidation. These results suggest that atopic dermatitis causes lipid accumulation in the liver.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375–394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP−386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Establishment of P38Bf, a Core-Fucose-Deficient Mouse-Canine Chimeric Antibody Against Dog Podoplanin

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10−7, 5.2 × 10−9, and 6.5 × 10−9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.

Establishment of Monoclonal Antibody PMab-202 Against Horse Podoplanin

Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs, PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN (CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses. PMab-202 is expected to prove useful in investigating the function of horse PDPN.

Detection of Tiger Podoplanin Using the Anti-Cat Podoplanin Monoclonal Antibody PMab-52

Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies (mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, and cat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab-52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These results indicate that PMab-52 may be useful for the detection of tiger PDPN.

Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs.

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG2a-type, 47-mG2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.