Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate.

The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.

Prophylactic and therapeutic effects of Acanthopanax senticosus Harms extract on murine collagen-induced arthritis.

Evidences are accumulating that extract of Acanthopanax senticosus Harms (ASH; syn Eleutherococcus senticosus [Rupr. & Maxim.] Maxim), a shrub native to Northeastern Asia, has antiinflammatory effects. In this study, we examined prophylactic and therapeutic effects of ASH extract (ASHE) on rheumatoid arthritis using collagen-induced arthritis (CIA) mouse model. Acanthopanax senticosus Harms extract was administered before the onset of arthritis in the prophylaxis model. In the therapeutic model, ASHE was administered after the onset of arthritis with or without anti-TNF-α antibody. The ASHE treatment showed efficacy before onset of CIA but there was no effect after CIA was established. The ASHE treatment delayed the onset and decreased severity of CIA. In vitro examinations showed that ASHE is an antioxidant and that ASHE suppresses TNF-α and interleukin-6 production in human peripheral blood mononuclear cells. The combination therapy with ASHE and anti-TNF-α antibody reduced the severity of arthritis compared with anti-TNF-α antibody alone. The present study shows that ASHE has prophylactic effect against CIA and support therapeutic effect of anti-TNF-α antibody.

Identification of autoantibodies expressed in acquired aplastic anaemia.

Acquired aplastic anaemia (aAA) is recognized as an autoimmune disorder; however, the autoantigens and target cells involved remain elusive. Expression of autoantibodies and their target cells were examined using the haematopoietic cell line K562 and bone marrow stromal cell line hTS-5; 43·5% and 21·7% of aAA expressed autoantibody against K562 and hTS-5 cells, respectively. The autoantigens were identified by serological identification of antigens through recombinant cDNA expression cloning. This study indicates that haematopoietic cells are the targets of immune abnormality in aAA. These autoantibodies may be utilized to distinguish patients associated with immune abnormality from bone marrow failure syndrome.

Essential role of protein kinase C zeta in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP.

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) zeta was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCzeta in SASH1 cells, myristoylated PKCzeta peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

Comparison of four direct homogeneous methods for the measurement of low-density lipoprotein cholesterol.

BACKGROUND: The primary lipoprotein risk factor is low-density lipoprotein cholesterol (LDL-C), and medication is targeted at lowering LDL-C values. To clarify the usefulness of direct homogeneous assays for LDL-C measurement, we compared the values obtained by various reagents to those obtained by the Friedewald equation and analyzed different reactivity to IDL/VLDL and LDL. METHODS: Serum samples were collected from 55 patients with hypercholesterolemia. The LDL-C concentrations were determined by four direct homogeneous assays using reagent A (Kyowa Medex), B (Sekisui Medical), C (Denka Seiken), and D (Sysmex), which are commercially available. RESULTS: Significant correlation was observed in LDL-C values obtained by the homogeneous assays and the Friedewald equation. However, there were two discrepancies in reagents B and C, respectively. These assays showed 40% and 55% lower LDL-C values than those calculated by the Friedewald equation, respectively. Reactivity to the IDL fraction in reagents B and C was lower than in reagents A and D. CONCLUSIONS: Direct homogeneous assays for LDL-C are suitable for routine laboratory examination. However, it was shown that attention should be given to the different reactivity to IDL and LDL among reagents in some clinical samples.

Down-regulation of hTERT expression plays an important role in 15-deoxy-Delta12,14-prostaglandin J2-induced apoptosis in cancer cells.

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) has been shown to possess antineoplastic activity in human cancers of various origins. However, the mechanism of the antineoplastic activity of 15d-PGJ2 remains to be completely elucidated. It has been reported that inhibiting the expression of human telomerase reverse transcriptase (hTERT), a major determinant of telomerase activity, induces rapid apoptosis in cancer cells. In this study, we investigated the effect of 15d-PGJ2 on hTERT expression. Treatment with 30 microM 15d-PGJ2 for 72 h induced apoptosis in the colon cancer cells LS180. 15d-PGJ2 treatment decreased hTERT protein expression in a dose-dependent manner. Down-regulation of hTERT expression by hTERT-specific small inhibitory RNA induced apoptosis. These results indicate that the down-regulation of hTERT expression by 15d-PGJ2 plays an important role in its proapoptotic properties. Since 15d-PGJ2 reduced hTERT mRNA expression, we examined the effect of 15d-PGJ2 on the DNA-binding activity of c-Myc, specificity protein 1 (Sp1) and estrogen receptor (ER) to the hTERT gene promoter using an electrophoretic mobility shift assay. 15d-PGJ2 attenuated the DNA-binding of all three transcriptional factors. Further, we observed that 15d-PGJ2 inhibited the DNA binding of these factors by different mechanisms; suppressed c-Myc mRNA expression, enhanced Sp1 protein degradation via the ubiquitin-proteasome pathway and inhibited ERbeta phosphorylation at serine residues. We conclude that hTERT down-regulation by 15d-PGJ2 plays an important role in its proapoptotic properties. Furthermore, 15d-PGJ2 inhibits the transcriptional activity of c-Myc, Sp1 and ER by three different mechanisms and results in the transcriptional repression of the hTERT gene.

Diagnostic importance of overexpression of Bmi-1 mRNA in early breast cancers.

Target molecules for a highly sensitive and specific diagnosis of breast cancer in its early clinical stages have not yet been identified. Here, we show the first evidence for diagnostic performance of the molecule B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) in breast cancer patients. Only 5 out of 46 non-cancerous samples were positive for Bmi-1 mRNA expression resulting in a sensitivity and specificity of 72.0 and 91.3%, respectively. The mRNA expression was estimated using the cut-off value obtained from the receiver operating characteristic curve analysis. Further, Bmi-1 mRNA expression was found to be elevated in 97.8% (45/46) of cancerous tissues in comparison to the expression in paired cancerous tissues and non-cancerous tissues obtained from identical patients. Bmi-1 mRNA was found to be highly expressed even in the early clinical stages of breast cancer. Our results suggest that Bmi-1 mRNA might be a new tool to support the diagnosis of breast cancers, irrespective of the clinical stage.

Diagnostic relevance of overexpressed Nanog gene in early lung cancers.

Currently, no target molecules have been identified that enable the diagnosis of lung cancer with high sensitivity and specificity, especially in the early clinical stages of cancer. Recently, Nanog has been reported to play an important role in the self-renewal and regeneration of ES cells by maintaining these cells in the undifferentiated state and by accelerating cell proliferation. Here, we compared the degree of Nanog mRNA expression in lung cancer tissues with that in non-cancerous tissues. Nanog mRNA was detected in 84.8% (39/46) of lung cancer tissues. The sensitivity and specificity of this diagnostic technique was 80.4 and 93.3%, respectively, as estimated using the cut-off obtained from the analysis of the receiver operating characteristic curve. Further, comparison of paired cancerous and non-cancerous tissues from the same patient revealed elevated Nanog mRNA levels in all patients. No obvious correlations were detected between the clinicopathological factors and Nanog mRNA expression; however, Nanog mRNA was expressed at high levels even in the early clinical stages of the cancer. In addition, the transduction of Nanog siRNA in lung carcinoma cells resulted in growth inhibition. These results suggest that Nanog mRNA might be a new tool to support the diagnosis of lung cancers, irrespective of the clinical stage.

Regulation of programmed cell death by the p53 pathway.

The p53 pathway is targeted for inactivation in most human cancers either directly or indirectly, highlighting its critical function as a tumor suppressor gene. p53 is normally activated by cellular stress and mediates a growth-suppressive response that involves cell cycle arrest and apoptosis. In the case of cell cycle arrest, p21 appears sufficient to block cell cycle progression out of G1 until repair has occurred or the cellular stress has been resolved. The p53-dependent apoptotic response is more complex and involves transcriptional activation of multiple proapoptotic target genes, tissue, and signal specificity, as well as additional events that are less well understood. In this chapter, we summarize the apoptosis pathway regulated by p53 and include some open questions in this field.

What are caspases 3 and 7 doing upstream of the mitochondria?

Apoptosis is a cell suicide program that is initiated after cells are exposed to cytotoxic stresses including UV, IR irradiation, chemotherapeutic drugs, hypoxia, serum deprivation and TRAIL. Caspases are the central components of this process. In mammals, caspases involved in apoptotic responses are classified into two groups according to their function and structure. The first group is termed initiator caspases (caspase-2, 8, 9, 10) that contain N-terminal adapter domains which allow for auto-cleavage and activation of downstream caspases. The second group is termed effector or executioner caspases (caspase-3, 6, 7) that lack N-terminal adapter domains and are cleaved and activated by initiator caspases. Lakhani et al. (Science 2006, 311:847-51) have reported that caspase-3 and -7 regulate mitochondrial events in the apoptotic pathway. In this journal club, we summarize the results of the article and include some open questions left in the study.

Amelioration of murine dextran sulfate sodium-induced colitis by ex vivo extracellular superoxide dismutase gene transfer.

BACKGROUND: Although the etiology of inflammatory bowel disease has not been fully clarified, reactive oxygen species is speculated to be involved. Extracellular superoxide dismutase (EC-SOD), an isozyme of SODs, is known to function mainly in body fluids. We investigated the efficacy of an ex vivo EC-SOD gene transfer into dextran sulfate sodium (DSS)-induced colitis mice. MATERIALS AND METHODS: Experimental colitis was induced by providing Balb/c mice with DSS in sterile distilled water provided as desired. The syngenic fibroblasts were obtained from Balb/c mice embryos and retrovirally transduced with the hEC-SOD gene. These engineered cells were confirmed to secrete EC-SOD in culture medium by enzyme-linked immunosorbent assay and were inoculated subcutaneously in the backs of DSS-treated mice. Mucosal injury of the colon was evaluated by the disease activity index (DAI: body weight, rectal bleeding, and stool consistency), grading of histologic disease severity, and levels of cytokine (tumor necrosis factor-alpha, interleukin-1beta) production. 8-Hydroxydeoxyguanosine (8-OHdG) levels in the mucosal tissue were assessed by immunohistochemical staining. Malondialdehyde (MDA) was measured using a colorimetric assay. RESULTS: A significant improvement was observed in DAI score and histologic severity as well as in mucosal tissue levels of inflammatory cytokines, 8-OHdG, and MDA of mice treated with the EC-SOD gene as compared with those without gene therapy, not only in a mild colitis model but also in a severe colitis model. Survival of treated mice in these models was significantly prolonged. CONCLUSIONS: Ex vivo transfer of the EC-SOD gene was feasible for treatment of DSS-induced colitis.

A patient with TP53 germline mutation developed Bowen's disease and myelodysplastic syndrome with myelofibrosis after chemotherapy against ovarian cancer.

Here we report a case of myelodysplastic syndrome (MDS) with myelofibrosis associated with Bowen's disease. A female patient had undergone an operation and chemotherapy for ovarian cancer when she was 65 years old, and she developed MDS at the age of 70 years old. PCR-single strand conformation polymorphism (SSCP) analysis of peripheral blood mononuclear cells, a Bowen's disease lesion, and normal skin showed an abnormal peak in TP53 exon5. Direct sequencing revealed that they all had missense mutation in codon 175 (G to A) of arginine switched to histidine, suggesting a germline mutation of TP53. It was speculated that p53 function was lost by TP53 germline mutation with the loss of a wild type allele induced by the chemotherapy against ovarian cancer, leading to the development of MDS. No therapeutic effects of low dose melphalan or cyclosporine A on MDS were observed, however one month of 30 mg/day prednisolone administration induced a hematological response.

[Essential thrombocythemia associated with incomplete type intestinal Behçet disease during hydroxyurea treatment].

A 77-year-old man was diagnosed as having essential thrombocythemia in 1992. Treatment with hydroxyurea was started in 1997, which stabilized the platelet count. The patient then suffered from pharyngalgia and rhinitis with a high fever, immediately after which he developed tarry stools and anemia and was admitted to our hospital. The physical examination revealed splenomegaly, oral aphthous ulcers, genital ulcers and skin lesions on the lower limbs. His hematological and biochemical tests revealed anemia and increased level of C-reactive protein. He also had an HLA-B51 phenotype. The findings of gastro-intestinal and colon fiberoscopy showed a duodenal ulcer and multiple ulcers on ascending colon. He was thus diagnosed as having intestinal tract-type Behçet disease. After withdrawal of the hydroxyurea administration, the intestinal ulcers, oral aphthous ulcers and genital ulcers improved.

[Two cases of primary skeletal muscle lymphoma, and a review of the literature].

Here we report two cases of primary skeletal muscle lymphoma. The first patient was an 82-year-old man. On April 2004, he was referred to our hospital because of swelling of the right upper arm. Magnetic resonance imaging (MRI) showed an area which was isointense on T1 and hyperintense on T2-weighted imaging compared with normal skeletal muscle. The size of the tumor was 5 x 7 x 13 cm. Following pathological, flow cytometric and genetic analyses of the specimen, we diagnosed the tumor as a non-Hodgkin lymphoma of the T-cell rich diffuse large B-cell type. The second patient was an 87-year-old man. He was admitted to our hospital on July 2004, under the chief complaint of swelling of the right thigh. MRI revealed a giant tumor mass of the right thigh which was isointense on T1 and hyperintense on T2 imaging. The patient was diagnosed by open biopsy as having diffuse large B-cell lymphoma. We could find only 62 cases of primary skeletal muscle lymphoma through a MEDLINE search. We report on our two cases with a review of the literature.

Significance of SALL4 as a drug­resistant factor in lung cancer.

We examined first evidence for the significance of SALL4, a transcription factor essential for embryonic development and the self-renewal of embryonic stem (ES) cells, as a natural resistance factor against anticancer drugs in lung cancer. To determine the significance of SALL4 expression in lung cancer cells, small inhibitory RNA (siRNA) against SALL4 was transduced into A549 and SBC-3 cells, resulting in increased sensitivity towards anticancer drugs [cisplatin (CDDP), carboplatin (CBDCA), and paclitaxel (PTX)]. SALL4 cancer tissues from 31 lung cancer patients were used to assess clinical significance. The analysis showed differences in SALL4 expression corresponding to the therapeutic outcomes. SALL4 expression measured before adjuvant chemotherapy was significantly higher in the patients showing recurrence of cancer than in the disease-free patients. In addition, the period until recurrence was shorter in the patients showing high SALL4 expression. These results indicate that SALL4 overexpression acts as a natural resistance factor and may be involved in the recurrence of lung cancer after adjuvant chemotherapy.

Diagnostic relevance of autoantibody detection against inhibitors of apoptosis proteins in colon cancer and colon adenoma.

Autoantibodies against cancer-related antigens may be detected in the sera of patients with various types of cancer, although their clinical utility has not yet been established. In this study, we aimed to demonstrate the diagnostic relevance of autoantibody detection against inhibitor of apoptosis protein (IAP) family members in colon cancer, as compared to anti-p53 antibody, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9. We established an ELISA system using original recombinant proteins of IAP family members (survivin, livin and X-linked IAP) and measured the expression levels in the sera of 62 healthy donors, 250 patients with colon polyps (adenoma) and 176 patients with colon cancer. When the cutoff value was set as the mean value + 2 standard deviations in healthy donors, anti-survivin exhibited the highest positivity rate (24.4%) among IAP autoantibodies in cancer patients. Furthermore, the anti-survivin antibody exhibited a high positivity rate in early-stage carcinoma and adenoma. In the combination assay, reflecting the significantly high positivity rate of CEA in stage IV tumors, the positivity rate was highest when combining the detection of anti-survivin antibody and CEA in cancer patients (50.0%), indicating that this combination may not be useful for the diagnosis of early-stage cancers. By contrast, reflecting the complete independencE of anti-survivin and anti-p53 antibodies, the combination of detecting these two antibodies resulted in the highest positivity rate (35.6%) in early-stage disease (stage 0-I). These results suggest that the combined measurement of anti-survivin and anti-p53 antibodies may be useful for the detection of early-stage colon cancer.

Two cases of inverted hyperplastic polyps of the colon and association with adenoma.

Here we report two cases of inverted hyperplastic polyps of the colon. The first patient showed three inverted hyperplastic polyps in the ascending colon, one of which was associated with adenoma. We immunostained this adenoma-associated polyp using anti-beta-catenin antibody and found accumulation of beta-catenin in the cytoplasm of the adenomatous lesion but not in the inverted hyperplastic polyp. This suggested an adenomatous polyposis coli (APC) mutation in the adenomatous region but not in the inverted hyperplastic polyp. The inverted hyperplastic polyp in the second patient was located at the caecum and was studied using magnifying colonoscopy. The polyp appeared to be flat and elevated with a depressed pit in the centre. After spraying with methylene blue dye, the pit pattern of the lesion was observed and small asteroid pits on the polyp were found, consistent with a hyperplastic gland pattern. From these results, we diagnosed inverted hyperplastic polyp of the colon by colonoscopy.

[T-cell chronic lymphocytic leukemia associated with adenocarcinoma of colon].

A 72-year-old woman had been diagnosed in 1998 as having colonic adenocarcinoma associated with lymphocytosis, and had undergone an operation. Three years later she was referred to our hospital with the chief complaint of blood stools. She again developed adenocarcinoma of the sigmoid colon. Blood chemistry showed a leukocyte count of 26,850/ml without anemia or thrombocytopenia. Bone marrow aspiration gave a nucleated cell count of 109,600/ml with 50.2% lymphocytes. Lymphocytes surface marker showed T-cell characteristics with CD4+/CD-. The serological test showed negative anti-HTLV-1 antibody and the TCRab chain was rearranged in her bone marrow aspirate. From these results she was diagnosed as having T-CLL.

[Successful induction and complete improvement of myelofibrosis and erythema nosodum with cladribine in a case of hairy cell leukemia].

The patient was a 45-year-old male who had been diagnosed with pancytopenia in 1998 at another hospital, where he continued treatment for idiopathic thrombocytopenic purpura (ITP) as an outpatient. After atypical lymphocytes were detected in his peripheral blood, he was admitted to our hospital for further examination in April 2002. Tests results revealed the additional existence of pancytopenia, splenomegaly, bone marrow fibrosis and erythema nodosum, in addition to the findings of surface marker. Tartrate resistant acid phosphatase (TRAP) staining and scanning electron microscopy of peripheral blood lymphocytes indicated hairy cell leukemia. After administration of cladribine (0.09 mg/kg) for 7 days, complete remission was obtained, with bone marrow fibrosis and erythema nodosum also being completely improved.

Stanniocalcin-1 promotes metastasis in a human breast cancer cell line through activation of PI3K.

Stanniocalcin-l (STC-1) is a secreted glycoprotein hormone that regulates calcium and phosphate homeostasis. STC-1 expression is upregulated in several cancers including breast cancer, and has been shown to be prognostic. Although these clinical observations implicate STC-1 as a potential tumor marker, it is still unclear whether STC-1 confers a malignant phenotype. In this study, this question was addressed by overexpressing STC-1 in the human breast cancer cell line MDA-MB-231 and examining the resultant phenotype in vitro and in vivo. Overexpression of STC-1 enhanced invasiveness of MDA-MB-231 cells in vitro and promoted their lung metastasis in vivo, while having no effect on proliferation, adhesion, or proteinase activity. The addition of soluble STC-1 to MDA-MB-231 cultures resulted in the activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, suggesting a mechanistic basis for the observed increases in cell motility and metastasis. Taken together, it was indicated that secreted STC-1 promotes metastatic potential of breast cancer cells via activation of PI3K/AKT.