Masahiko Shigemura

TRIM68 regulates ligand-dependent transcription of androgen receptor in prostate cancer cells.

The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is significantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer.

Effects of molecular structural variants on serum Krebs von den Lungen-6 levels in sarcoidosis

BackgroundSerum Krebs von den Lungen-6 (KL-6), which is classified as human mucin-1 (MUC1), is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis.MethodsWestern blot analysis using anti-KL-6 antibody was performed simultaneously on the bronchoalveolar lavage fluid (BALF) and serum obtained from 128 subjects with sarcoidosis.ResultsKL-6/MUC1 in BALF showed three bands and five band patterns. These band patterns were associated with the MUC1 genotype and the KL-6 levels. KL-6/MUC1 band patterns in serum were dependent on molecular size class in BALF. Significantly increased levels of serum KL-6, serum/BALF KL-6 ratio and serum soluble interleukin 2 receptor were observed in the subjects with influx of high molecular size KL-6/MUC1 from the alveoli to blood circulation. The multivariate linear regression analysis involving potentially relevant variables such as age, gender, smoking status, lung parenchymal involvement based on radiographical stage and molecular size of KL-6/MUC1 in serum showed that the molecular size of KL-6/MUC1 in serum was significant independent determinant of serum KL-6 levels.ConclusionsThe molecular structural variants of KL-6/MUC1 and its leakage behavior affect serum levels of KL-6 in sarcoidosis. This information may assist in the interpretation of serum KL-6 levels in sarcoidosis.

Myeloma cells produce sialyl salivary-type amylase

Figure 1 Immunoelectrophoresis of the patient’s serum. 1, normal serum; 2, patient’s serum; A-WS, antiserum against whole human serum; A-IgG, A, M, anti-immunoglobulin G, A, M serum; A-LØK, anti-light chain k serum; A-LØL, anti-light chain l serum. Monoclonal protein is indicated by the arrow. Masahiko Shigemura*, Takanori Moriyama, Tomoyuki Endo, Hitoshi Shibuya, Haruki Suzuki, Masaharu Nishimura, Hitoshi Chiba and Kazuhiko Matsuno 1 Clinical Laboratory, Hokkaido University Hospital, Sapporo, Japan 2 Division of Laboratory Technology, Department of Health Sciences, School of Medicine, Hokkaido University, Sapporo, Japan 3 The Second Department of Internal Medicine, Hokkaido University Hospital, Sapporo, Japan

Laboratory and clinical features of abnormal macroenzymes found in human sera

We report the analysis of unusual macroenzymes, performed in our laboratory, and review the relevant literature. In particular, we focused on macro AST, macroamylase, macro LD and macro CK. Macroenzymes are seen in healthy subjects, but can also be related to disease; thus, accurate detection is useful in day-to-day clinical practice. The macroenzyme is thought to be a specific antigen-antibody complex from the following findings: (1) the complex could be dissociated under acidic pH levels; (2) binding specificity of immunoglobulin in the complex was observed; (3) the binding site of immunoglobulin in the complex was Fab portion; and (4) the maternal IgG involved with macroenzyme was transferred to her children. This article is part of a Special Issue entitled: Medical Proteomics.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.

Total serum IgE levels and atopic status in patients with sarcoidosis.

To date, two studies have reported lower total serum immunoglobulin E (IgE) levels and lower prevalence of atopy in patients with sarcoidosis compared with healthy subjects. However, those reports did not consider age or gender differences between cases and controls. In addition, the association between total serum IgE levels and clinical manifestations of sarcoidosis has not been clarified. This study assessed total serum IgE levels and prevalence of atopy in patients with sarcoidosis after taking age and sex differences into account and evaluated associations between total serum IgE levels and clinical manifestations of sarcoidosis. Total serum IgE levels and prevalence of atopy on initial visits were compared between 189 patients with sarcoidosis and 378 age- and sex-matched controls. Associations between total serum IgE levels and involvement of each affected organ were evaluated. Changes in total serum IgE levels during the clinical course of sarcoidosis were also evaluated. Total serum IgE levels were significantly lower in patients with sarcoidosis than in controls, independent of atopic status (atopic subjects, p = 0.025; nonatopic subjects, p < 0.001). Total serum IgE levels did not differ according to the involvement of different organs. Total serum IgE levels decreased further, albeit only slightly, after disease remission (p < 0.001). Increased susceptibility to sarcoidosis may be attributable to several underlying genetic or environmental factors that result in lower total serum IgE levels.

The role of atopy in the clinical course of pulmonary sarcoidosis in the Japanese population

Sarcoidosis is a multisystem disorder characterized by a T-helper 1 (Th1)-mediated immune response. Conversely, atopy is characterized by the presence of a specific immunoglobulin E (IgE) E response in association with a Th2-type immune response. Several epidemiological studies have shown that atopic status influences disease activity and clinical course for several Th1-mediated diseases. The aim of this study was to evaluate associations between atopic status and clinical findings of sarcoidosis. We further evaluated the impact of atopic status on the clinical course of pulmonary sarcoidosis. We defined atopy as a positive specific IgE response to at least one common inhaled allergen (multiple antigen simultaneous test scores, lumicount of >1.01). Subjects comprised 134 patients given a diagnosis of sarcoidosis between 2000 and 2006, divided into atopic and nonatopic groups. Several clinical findings were compared between the two groups. Furthermore, 100 subjects observed 2 years after diagnosis were divided into resolving and persistent clinical course groups according to chest radiography and associations with atopic status were evaluated. Atopy was more prevalent among men than women (p = 0.009) and subjects with atopy were younger (p = 0.002) and showed less frequent lung parenchymal lesions (stages II and III; p = 0.018) compared with subjects without atopy. The prevalence of atopy was higher in the resolving clinical course group than in the persistent clinical course group (p = 0.002) and this association was independent of sex, age, presence of lung parenchymal lesions, and presence of extrapulmonary lesions (p = 0.037). Classification of sarcoidosis based on atopic status might be useful for predicting the clinical course of pulmonary sarcoidosis.

Serum KL-6 concentrations are associated with molecular sizes and efflux behavior of KL-6/MUC1 in healthy subjects

BACKGROUND Serum KL-6, a sialylated sugar chain on human MUC1, is used as a marker of interstitial lung diseases. We recently reported that efflux behavior of KL-6/MUC1 from the alveoli into the bloodstream assessed by molecular analysis differed according to genetically determined molecular sizes and influenced serum KL-6 concentrations in sarcoidosis. This study was designed to investigate associations between molecular size and efflux behavior of KL-6/MUC1, and factors contributing to serum KL-6 concentrations in healthy subjects. METHODS Western blot analysis using anti-KL-6 antibody was performed on serum obtained from 250 healthy subjects. RESULTS The efflux behavior of KL-6/MUC1 differed according to the genetically determined molecular sizes in healthy subjects. In subjects having low molecular size, there were significant associations between smoking status, aging, renal function and serum KL-6 concentrations. However, these associations were not significant in the subjects having higher molecular size and the efflux behavior of high molecular size was the only significant determinant of serum KL-6 concentrations. CONCLUSIONS This study showed an association between KL-6/MUC1 efflux based on molecular size and serum KL-6 concentrations in healthy subjects. We propose that the molecular size and efflux behavior of KL-6/MUC1 should be considered when interpreting serum KL-6 concentrations.

Clinical Course of Histologically Proven Multifocal Micronodular Pneumocyte Hyperplasia in Tuberous Sclerosis Complex: A Case Series and Comparison with Lymphangiomyomatosis

Background: Multifocal micronodular pneumocyte hyperplasia (MMPH) is a rare pulmonary manifestation of tuberous sclerosis complex (TSC). Because of its rarity, no previous study has described the detailed clinical course of this disease. Objectives: This study aimed to clarify the longitudinal clinical characteristics of subjects with MMPH. Methods: Nine patients with MMPH diagnosed at Hokkaido University Hospital were retrospectively analyzed. Changes in computed tomography findings and pulmonary function were compared during the follow-up period. Serum levels of KL-6, surfactant protein (SP)-A, and SP-D were measured to clarify their potentials as blood biomarkers of the disease. Fourteen cases of lymphangiomyomatosis (LAM) were also included to compare their clinical characteristics with those of subjects with MMPH. Results: Of the 9 patients, 7 were female and 2 were male. The median age at diagnosis was 43 years (range, 19–56), and all cases were diagnosed following incidental abnormal radiographic findings. During the follow-up, 1 patient died of lung cancer, but others were radiographically stable and had stable pulmonary function. Serum levels of SP-A in 5 patients (mean, 146.4 ng/mL) and SP-D in 6 patients (mean, 337.3 ng/mL) were elevated in subjects with MMPH, whereas KL-6 levels were within the reference range (mean, 230 U/mL) in all patients. Levels of SP-A and SP-D were significantly higher in subjects with MMPH than those with LAM (p < 0.05). Conclusions: Radiographic findings and pulmonary function were stable in all cases of MMPH. Serum SP-A and SP-D, but not KL-6, may be useful markers for suspicion of the presence of MMPH in patients with TSC.

P-136 Establishment and characterization of amylase-producing lung adenocarcinoma cell line, IMEC-2

BACKGROUND Few studies have successfully established an amylase-producing lung cancer cell line or have examined its cytological, biochemical and biological features. PATIENTS AND METHODS Cancer cells, isolated from pleural effusion using a gradient method, were cultivated. RESULTS Amylase production from the newly established cell line was confirmed by positive staining for alpha-amylase and increased amylase levels in the culture supernatant. Electron microscopy revealed zymogen granule-like structures. Sialylation of salivary-type amylase was confirmed directly from the cell line by examining the neuraminidase sensitivity and amylase elution profile under high-performance liquid chromatography. Neither EGFR or KRAS mutation were found. CONCLUSION This cell line offers a useful tool for analyzing the pathogenesis and pathophysiology of amylase-producing lung cancers. Moreover, it might be useful for probing the metastasis and invasiveness of lung cancer cells and for developing an early diagnostic method based on sialylated salivary-amylase production.