Hiroko Natsume

High Incidence of Positive Autoantibodies against Thyroid Peroxidase and Thyroglobulin in Patients with Sarcoidosis

OBJECTIVE Although abnormalities of the humoral immune system, such as increased immunoglobulin production, are known in sarcoidosis, the relationship between sarcoidosis and autoimmune disorders is uncertain. We studied the incidence of thyroid autoantibodies and the prevalence of Hashimoto’s thyroiditis in patients with sarcoidosis.

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

BackgroundGenomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.ConclusionThese results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Urinary excretion of pyridinoline and deoxypyridinoline measured by immunoassay in hypothyroidism

OBJECTIVE We measured pyridinium cross‐links, markers of bone resorption, by an enzyme‐linked immunosorbent assay (ELISA) in hypothyroid patients to see whether bone resorption was reduced in hypothyroidism and whether it increased with T4 treatment.

Alterations in the enzyme activity and protein contents of protein disulfide isomerase in rat tissues during fasting and refeeding

Protein disulfide isomerase (PDI) is an enzyme that participates in the formation of disulfide bonds. It is also known to be the subunits of some enzymes and the membrane-associated thyroid hormone-binding protein. In this study, we measured the quantitative distribution of PDI protein in rat tissues and examined the relationship between protein level and enzyme activity in PDI during fasting and refeeding. Western blotting with specific anti-PDI antiserum detected the PDI protein band of 55 kd. Among several tissues, liver contained the largest amount of PDI protein, followed by kidney and fat, in which one-third to one-fourth of the hepatic PDI protein existed. The PDI protein band was also detected in heart and muscle. Fasting for 3 days decreased PDI protein levels in rat liver by 40%; control levels were recovered after 3 days of refeeding. The same change was observed in kidney. PDI activity, measured by the scrambled ribonuclease method, did not show the parallel alteration to PDI protein level in liver and kidney. Isomerase activity decreased to 50% of control values during fasting, but did not recover by refeeding. Thyroidal status did not affect either PDI protein level or isomerase activity. These findings show that fasting and refeeding affect PDI protein and enzyme activity, and that PDI protein level does not always reflect PDI activity.

Difference in dominant negative activities between mutant thyroid hormone receptors α1 and β1 with an identical truncation in the extreme carboxyl-terminal tau4 domain

Although different expression patterns of thyroid hormone receptor (TR) alpha1 and beta1 have been reported, no essential distinction has been established in their functions. Unlike the TR beta gene, a mutation in the TR alpha1 gene has never been found in patients with resistance to thyroid hormone (RTH). Previously we found a mutant TR beta with an 11-carboxyl (C)-terminal amino acid truncation (betaF451X) in a girl with severe RTH. BetaF451X is a natural mutant with disruption of the transactivation domain, tau4, and it had very strong dominant negative activities. Based on the fact that the 46 amino acid sequence in the extreme C-terminal region is identical in TR alpha1 and TR beta, except for a C-terminal three amino acid extension of TR alpha1, we constructed a mutant TR alpha1 (alphaF397X) with the identical C-terminal truncation to betaF451X, to study functional differences between TR alpha1 and beta1. Both betaF451X and alphaF397X had negligible T3 binding and transcriptional activities even with 1 microM T3. The dominant negative activities of the mutant TRs were remarkable and T3 response element (TRE)-dependent. Co-expression of betaF451X decreased the CAT activity of either wild-type TR alpha1 or beta1 at 100 nM T3 by approximately 90% on the TRE-pal2 and 70% on DR4. AlphaF397X inhibited the transcriptional activities of both wild-type TR alpha1 and beta1 by approximately 50% on TRE-pal2 and by 60% on DR4. The dominant negative potency of betaF451X was significantly stronger than that of alphaF397X on the TRE-pal2, -DR4 and chicken lysozyme silencer F2, but similar on TRE-myosin heavy chain alpha and malic enzyme. No partiality for the TR subtypes was found in the dominant negative effects of betaF451X and alphaF397X. Co-expression with RXR enhanced the dominant negative effects of alphaF397X, but not of betaF451X. The results indicate that there are different dominant negative properties between alphaF397X and betaF451X, which are TRE-dependent, despite their identical C-terminal truncation. Deletion in the tau4 domain might affect the receptor structures of TR alpha1 and beta1 differently.

EPH-EPHRIN in human gastrointestinal cancers

Ever since its discovery two decades ago, the erythropoietin-producing hepatoma (EPH)-EPHRIN system has been shown to play multifaceted roles in human gastroenterological cancer as well as neurodevelopment. Over-expression, amplification and point mutations have been found in many human cancers and many investigators have shown correlations between these up-regulations and tumor angiogenesis. Thus, the genes in this family are considered to be potential targets of cancer therapy. On the other hand, the down-regulation of some members as a result of epigenetic changes has also been reported in some cancers. Furthermore, the correlation between altered expressions and clinical prognosis seems to be inconclusive. A huge amount of protein-protein interaction studies on the EPH-EPHRIN system have provided a basic scheme for signal transductions, especially bi-directional signaling involving EPH-ERPHRIN molecules at the cell membrane. This information also provides a manipulative strategy for harnessing the actions of these molecules. In this review, we summarize the known alterations of EPH-EPHRIN genes in human tumors of the esophagus, stomach, colorectum, liver and pancreas and present the perspective that the EPH-EPHRIN system could be a potential target of cancer therapy.

Abstract 3682: Overexpression and cell proliferation-regulating function of CRKL protein in gastric cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. To identify previously undiscovered amplified genes in gastric cancer, a genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers. Twenty-two genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, which has not been previously characterized in gastric cancer and the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. A relatively high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization, and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. These results suggested that CRKL protein is overexpressed in a subset of gastric cancers, that it regulates gastric cell proliferation, and that it has the potential to be a molecular therapy target for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3682. doi:1538-7445.AM2012-3682