Kunihiro Inai

Lipid rafts remodeling in estrogen receptor–negative breast cancer is reversed by histone deacetylase inhibitor

Recently, we have found dramatic overexpression of ecto-5′-nucleotidase (or CD73), a glycosylphosphatidylinositol-anchored component of lipid rafts, in estrogen receptor–negative [ER(−)] breast cancer cell lines and in clinical samples. To find out whether there is a more general shift in expression profile of membrane proteins, we undertook an investigation on the expression of selected membrane and cytoskeletal proteins in aggressive and metastatic breast cancer cells. Our analysis revealed a remarkably uniform shift in expression of a broad range of membrane, cytoskeletal, and signaling proteins in ER(−) cells. A similar change was found in two in vitro models of transition to ER(−) breast cancer: drug-resistant Adr2 and c-Jun-transformed clones of MCF-7 cells. Interestingly, similar expression pattern was observed in normal fibroblasts, suggesting the commonality of membrane determinants of invasive cancer cells with normal mesenchymal phenotype. Because a number of investigated proteins are components of lipid rafts, our results suggest that there is a major remodeling of lipid rafts and underlying cytoskeleton in ER(−) breast cancer. To test whether this broadly defined ER(−) phenotype could be reversed by treatment with differentiating agent, we treated ER(−) cells with trichostatin A, an inhibitor of histone deacetylase, and observed reversal of mesenchymal and reappearance of epithelial markers. Changes in gene and protein expression also included increased capacity to generate adenosine and altered expression profile of adenosine receptors. Thus, our results suggest that during transition to invasive breast cancer there is a significant structural reorganization of lipid rafts and underlying cytoskeleton that is reversed upon histone deacetylase inhibition. [Mol Cancer Ther 2006;5(2):238–45]

Differentiation and Reduction of Intracellular GTP Levels in Hl-60 and U937 Cells Upon Treatment with IMP Dehydrogenase Inhibitors

Inosine-5′-monophosphate dehydrogenase (IMP dehydrogenase, EC 1.1.1.205) is a rate-limiting enzyme of de novo guanylate biosynthesis and catalyzes the formation of XMP from IMP (1). IMP dehydrogenase have two isoforms. Type I transcript is the major form found in normal leukocytes (2), whereas type II is predominant in malignant cells (3), thus type II IMP dehydrogenase is a target for antineoplastic agents. In addition, a variety of human leukemic cells, including K562 erythroid leukemia cells (4), and HL-60 myeloid leukemia cells (5) as well as human breast cancer cells (6) which are treated with IMP dehydrogenase inhibitors, decreases in type II IMP dehydrogenase and the level of guanine nucleotide, especially GTP, are shown to be associated with cell maturation (7,8).

New radiosynthesis of 2-deoxy-2-[18F]fluoroacetamido-d-glucopyranose and its evaluation as a bacterial infections imaging agent

INTRODUCTION The diagnosis of infection and the ability to distinguish bacterial infection from nonbacterial inflammation by positron emission tomography (PET) have gained interest in recent years, but still few specific radiopharmaceuticals are available for use. In this study, we developed a new radiosynthesis method of 2-deoxy-2-[(18)F]fluoroacetamido-d-glucopyranose ([(18)F]FAG) by applying microwave irradiation and demonstrated that [(18)F]FAG could be a potential radiopharmaceutical to distinguish bacterial infection from nonbacterial inflammation. METHODS 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-bromoacetamido-d-glucopyranose was used as precursor, and labeling was performed under microwave irradiation conditions followed by alkaline hydrolysis and high-performance liquid chromatography (HPLC) purification. In vitro uptake of [(18)F]FAG by Escherichia coli was performed. Tissue biodistribution of [(18)F]FAG was performed in mice. Moreover, PET imaging acquisition of E. coli infection and nonbacterial inflammation models was performed in rats. Tissue radiotracer-accumulated sites were analyzed by hematoxylin and eosin staining and anti-E.coli immunostaining. RESULTS The radiosynthesis of [(18)F]FAG was achieved with microwave irradiation, and the radiochemical yield was 9.7%±2.8% end of bombardment (EOB); the radiochemical purity was more than 98%, and the total synthesis time was 62 min. Compared with control group, in vitro uptake of [(18)F]FAG by E. coli was significantly decrease in inhibition group (P<.05). Biodistribution studies in mice showed rapid clearance of [(18)F]FAG from the animal body. [(18)F]FAG clearly visualized the infection areas but not nonbacterial inflammation areas in PET studies. Quantitative analysis revealed that the uptake of [(18)F]FAG into infection areas was significantly higher than that of [(18)F]FAG into inflammation areas (P<.05). Histological analysis demonstrated the presence of bacterial cells at the sites of accumulation of [(18)F]FAG. CONCLUSIONS Using 1,3,4,6-tetra-O-acetyl-2-deoxy-2-bromoacetamido-d-glucopyranose as a precursor, the new radiosynthesis method of [(18)F]FAG was achieved in fewer steps and with a shorter synthesis time than previously reported. Furthermore, [(18)F]FAG was able to distinguish bacterial infection from nonbacterial inflammation.

Idarubicin and idarubicinol are less affected by topoisomerase II-related multidrug resistance than is daunorubicin

We investigated the cytotoxicity and cellular pharmacology of idarubicin (IDA), idarubicinol (IDAol) and daunorubicin (DNR) in K562/VP-H2 cells, which show topoisomerase II-related multidrug resistance but do not overexpress P-glycoprotein. K562/VP-H2 cells were less resistant to IDA and IDAol than to DNR. There was no significant difference in the accumulation of each drug between K562 and K562/VP-H2 cells. The cleavage of DNA induced by each drug was decreased in K562/VP-H2 cells, however, the decrease in cleavage in K562/VP-H2 cells was less with IDA and IDAol than with DNR. These results suggest that IDA and IDAol have more cytotoxic potency than DNR in topoisomerase II-related multidrug-resistant leukemia cells.

Induction of Cell Differentiation by IMPDH Antisense Oligomer in HL-60 and K562 Human Leukemia Cell Lines

Inosine-5’-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the enzyme that catalyzes the formation of xanthosine-5’-monophosphate from inosine-5’-monophosphate (IMP). In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is thus the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. Alterations in the activity of IMPDH and the levels of guanine nucleotides have been implicated in the regulation of cell growth and differentiation[1]. The addition of IMPDH inhibitors, such as mycophenolic acid and tiazofurin, to cultures induces terminal differentiation in a variety of human leukemia cells, including K562 erythroid leukemia cells, and HL-60 myeloid leukemia cells as well as human breast cancer cells and melanoma cells[2–4]. To verify the role of this enzyme in controlling differentiation, we determined the ability of specific IMPDH antisense oligomers to induce maturation in the K562 and HL-60 human leukemia cell lines. From these experiments, we concluded that a specific reduction in IMPDH results in inhibition of cell replication and induction of terminal differentiation in the two human leukemia cell lines.

Multiple inflammatory cytokine-productive ThyL-6 cell line established from a patient with thymic carcinoma

Thymic epithelial cells can produce many kinds of cytokines, and interleukin (IL)‐6‐producing thymic carcinoma cases have been reported. However, a cytokine‐producing human thymic tumor cell line has not previously been established. In this paper, we report a novel, multiple inflammatory cytokine‐productive cell line that was established from a patient with thymic carcinoma. This cell line, designated ThyL‐6, positively expressed epithelial membrane antigen, cytokeratins, vimentin intermediate filament and CD5, although hematological markers were not present in the cells. Cytokine antibody array analysis showed that the cells secreted several cytokines including IL‐1α, IL‐6, IL‐8, RANTES, soluble TNFα‐receptor 1, VEGF and CTLA into the culture medium. The addition of ThyL‐6‐cultured supernatant supported the growth of human myeloma ILKM‐3 cells, which require the presence of IL‐6 in the culture medium for the maintenance of cell growth, suggesting that the secreted IL‐6 from ThyL‐6 cells was biologically active. Chromosome analysis demonstrated that ThyL‐6 cells had complex karyotype anomalies, including der(16)t(1;16); the latter has been recognized in thymic squamous cell carcinoma and thymic sarcomatoid carcinoma cases, as well as in several other kinds of malignancies. Heterotransplantation of the cells into nude mice showed tumorigenesis with neutrophil infiltration and liquefactive necrosis. These findings suggest that ThyL‐6 cells will provide us with a new experimental tool for investigating not only the pathogenesis, biological behavior, chromo‐somal analysis and therapeutic reagents of human thymic carcinoma, but also for studying cytokine–chemokine network systems. (Cancer Sci 2008; 99: 1778–1784)

Frequent detection of multidrug-resistant pneumonia-causing bacteria in the pneumonia lung tissues of patients with hematological malignancies.

Pneumonia is a critical issue during the agonal phase, and often becomes lethal in the absence of pathogen detection. Autopsy is a powerful tool for analyzing the cause of a patient’s death, progression of the disease, and the therapeutic response. However, it is frequently limited to the identification of bacterial strains. To elucidate the pathogenesis during the agonal phase of pneumonia, intrapulmonary sputum was harvested by directly inserting a swab into a resected lung, and the bacterial composition was analyzed using both pathological and microbiological techniques from 15 patients with hematological malignancies, and the results were compared with those from 25 patients with other medical and surgical diseases. Among the 54 bacteria strains isolated from the 40 patients, multidrug-resistant strains were significantly more prevalent in hematological group than in other diseases (16/21 versus 11/33, P = .002). Enterococcus faecium was preferentially isolated from the hematological patients, whereas the methicillin-resistant Staphylococcus aureus was predominantly found in the nonhematological group. Two coagulase-negative Staphylococcus epidermidis strains in hematological diseases may be diagnosed as causative bacteria of pneumonia by both bacterial and pathological techniques. Although the results of this study may not be directly applicable for clinical diagnosis, this approach has a potential to become not only a diagnostic method for bacterial pneumonia, but may be also useful for the analysis of multidrug-resistant pathogens.

Feasibility of liver weight estimation by postmortem computed tomography images: An autopsy study

Although organ weight gives pathologists information about the pathogenesis of diseases at autopsy, the knowledge is rarely helpful in postmortem virtual autopsy by computed tomography (CT). To investigate the feasibility of liver weight estimation based on liver volume estimated from three‐dimensional CT images and the specific gravity of liver, thirty cadavers who died in the University of Fukui Hospital and whose family members agreed to postmortem CT and autopsy were prospectively enrolled. Mean specific gravity of liver was 1.054 ± 0.009 g/mL (95% confidence interval: 1.0507–1.0573 g/mL). The specific gravity was positively correlated to Hounsfield unit (HU) values of less than 40 (cases with moderate to severe fatty deposition) and remained stable between 1.05 to 1.065 g/mL for HU values greater than 40 (cases with mild or no fatty change). The liver weight estimated by our formula corresponded well to the actual liver weight, and the correlation coefficient was 0.96 (P < 1 × 10−13). The estimated liver weight calculated from estimated liver volume and the specific gravity of 1.055 g/mL was highly accurate, whereas the specific gravity should be reduced by 2%–3% in patients with an HU value less than 40 due to fatty deposition.

Binding of monosodium urate crystals with idiotype protein efficiently promote dendritic cells to induce cytotoxic T cells

Monosodium urate (MSU) crystals have been reported to evoke specific cell immunity and to work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DC). We focused on the application of MSU crystals as not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTL) efficiently in vitro. We selected human leukocyte antigen (HLA)‐A2 expressing the multiple myeloma IM‐9 cell line and its product idiotype (Id) protein as one of the best pairs of target cells and positively charged tumor‐specific antigen, respectively. Following the sensitization of DC derived from HLA‐A2‐positive volunteers pulsed with tumor‐specific monoclonal immunoglobulin G‐Fab fragments (IM‐9 Fab) attached to MSU crystals, the DC‐stimulated CD8+ T cells killed significantly more target cells (40.1 ± 1.7%) than those stimulated by DC pulsed with MSU crystals alone (6.2 ± 8.6%, P < 0.01) or IM‐9 Fab alone (4.7 ± 8.1%, P < 0.01). These cytotoxic effects of the DC‐stimulated CD8+ cells were reduced when MSU crystals were precoated with fetal bovine serum. In addition, we confirmed that MSU crystals facilitated human DC to express the maturation marker, CD83 and deliver (Fab′)2, attaching to the crystals by flow cytometer analysis. MSU crystals have distinct advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DC, as well as an adjuvant promoting DC maturation and inducing CTL. (Cancer Sci 2008; 99: 2268–2273)

Should prophylactic thrombolysis be routine in clinical practice? Evidence from an autopsy case of septicemia

BackgroundCentral venous catheters provide easy access for intravenous infusion and nutrition, but they can bring about complications such as catheter-related infections. Infected central venous catheters often cause nosocomial bloodstream infections with high morbidity and mortality. However, most of the morphological data that have been published are derived from in vitro and in vivo studies and few reports of direct evidence obtained from patient-derived samples have been described. Here we present visual evidence of catheter-related candidemia. To our knowledge, this is the first reported conventional histopathological evidence of a Candida-infected intraluminal thrombus in a patient’s central venous catheter.Case presentationA 62-year-old Japanese female with obstructive jaundice, gastrointestinal bleeding, and liver metastasis from pancreatic head cancer was given an implantable subcutaneous central venous port for nutrition and chemotherapy administration. High fever ensued on day 16 after the central venous port insertion and blood cultures revealed Candida albicans. Although the patient was given 300 mg/day of fosfluconazole according to the suggestion of the infection control team, she died from respiratory failure. Postmortem computed tomography revealed findings consistent with acute respiratory distress syndrome, suggesting that the patient’s course was complicated by catheter-related sepsis. Autopsy revealed a subcutaneous abscess around the port, from which C. albicans was cultured. However, no catheter-adherent thrombus, thrombosis of the great central veins, or endocardial vegetations were detected in the patient. Histological analysis revealed scattered abscesses in several organs including lungs and kidneys. Hyaline membrane formation and Candida colonies were found in the lungs. The central venous port tube, together with the part of the subclavian vein into which it had been inserted, was involved in an intraluminal fibrin thrombus containing neutrophils and macrophages, indicating that the thrombus existed while the patient was alive. Histopathological examination following use of the periodic acid-Schiff reagent and the Grocott stain revealed scattered Candida in the thrombus.ConclusionsProphylactic thrombolysis should be encouraged to prevent central venous catheter-related candidiasis in clinical practice.