Regression of gastric de novo diffuse large B-cell lymphoma following Helicobacter pylori eradication: a case report.

We report a case of primary gastric diffuse large B-cell lymphoma (DLBCL), de novo DLBCL without the features of mucosa-associated lymphoid tissue (MALT) lymphoma, which regressed after Helicobacter pylori (HP) eradication. A 27-year-old Japanese female with epigastralgia was revealed to have ulcerated lesions in the angle and antral regions on gastroscopy. Biopsy specimen was consistent with a diagnosis of DLBCL without MALT lymphoma component, indicating de novo development. Her clinical staging on the Lugano system was Stage I. HP was positive on a rapid urease test, and she received HP eradication therapy twice, because the first therapy was not successful. On gastroscopy performed 1 month after the second HP eradication therapy, no ulcerated lesion was noted, and the lymphoma cells had regressed histopathologically. (Acta gastro-enterol. belg., 2016, 79, 367-369A).

Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells.

Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target.

Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells through increase of receptor tyrosine kinase MET.

Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3'-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, co-treatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemoresistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy.

Involvement of EphA2-mediated tyrosine phosphorylation of Shp2 in Shp2-regulated activation of extracellular signal-regulated kinase.

Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase-defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases.

Heterogeneity of colorectal cancers and extraction of discriminator gene signatures for personalized prediction of prognosis.

Dissected specimens of colorectal cancer (CRC) have been intensively studied using molecular sketches (gene signatures) to obtain a set of discriminator gene signatures for accurate prognosis prediction in individual patients. The discriminators obtained so far are not universally applicable, as the gene sets reflect the method and site of the study. In this study, we show that dissected stage II and III CRC samples are significantly heterogeneous in molecular sketches, and are not appropriate sources for discriminator extraction unless handled individually. To search for an accurate discriminator gene set for prediction of metastases, we need to start with less heterogeneous stage II CRC. We examined 198 (92 stage II and 106 stage III) CRC dissected samples for the predictability of discriminator gene signatures by analyzing stage II CRC alone, stage III alone, or in combination. The best predictive power of discriminator genes was obtained only when these genes were extracted and validated with stage II CRC samples. An accurate discriminator gene set for the prediction of CRC metastases can be obtained by focusing on stage II CRC samples.

Role of secretory leukocyte protease inhibitor in the development of subclinical emphysema.

Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. In some chronic airway diseases, the level of SLPI is decreased in sputum, leading to the continuation of neutrophil inflammation. In this study, the role of SLPI in subclinical pulmonary emphysema was examined. Sequential bronchoalveolar lavage was performed in an attempt to separately evaluate the levels of SLPI in the large airways and in the peripheral airways for two groups of smokers. One group had subclinical emphysema by computed-tomography scans and one group did not. SLPI localized in alveolar macrophages (AM) was also assessed. The level of SLPI was significantly elevated in the peripheral airways from the subjects with emphysema compared to those without emphysema (1589.2+/-353.9 versus 729.1+/-31.0 ng x mL(-1)), although it was similar in the large airways (3442.3+/-499.6 versus 2535.7+/-578.8 ng x mL(-1)). There was a trend for higher amount of SLPI to be released from AM in subjects with subclinical emphysema, although this did not reach statistical significance. In conclusion, there is compensatory upregulation of secretory leukocyte protease inhibitor in peripheral airways in subclinical pulmonary emphysema, which is in sharp contrast to the decreased level of secretory leukocyte protease inhibitor reported in some chronic airway diseases.

Increased levels of interleukin-8 in BAL fluid from smokers susceptible to pulmonary emphysema.

BACKGROUND: It has previously been shown that smokers with computed tomographic (CT) evidence of subclinical emphysema have signs of neutrophil activation, despite having no appreciable increase in the number of neutrophils in their bronchoalveolar lavage (BAL) fluid. METHODS: The levels of the following chemoattractants in BAL fluid from 61 community based older volunteers classified into four groups according to current smoking status and the presence or absence of emphysema were determined: interleukin 8 (IL-8), epithelial neutrophil activating protein 78 (ENA-78) and leukotriene B(4) (LTB(4)) which are primarily chemotactic for neutrophils; monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) which are predominantly chemotactic for mononuclear leucocytes. RESULTS: Of the five chemoattractants studied, only the level of IL-8 in BAL fluid clearly distinguished between subjects with and without emphysema among current smokers (median values 34.7 and 12.2 pg/ml, respectively, p<0.01). In addition, the levels of IL-8 and neutrophil elastase-alpha(1) protease inhibitor complex in BAL fluid were significantly correlated (r=0.65, p<0.01). There was no difference in either the release of IL-8 from cultured alveolar macrophages at 24 hours or the expression of IL-8 messenger RNA of alveolar macrophages in the two groups of current smokers with and without emphysema. CONCLUSION: An accelerated response of IL-8 to chronic smoking is a factor that characterises those smokers who are susceptible to pulmonary emphysema, although the cellular source of IL-8 remains to be determined.

Effect of naturally occurring E2F-4 alterations on transcriptional activation and proliferation in transfected cells.

E2F is a family of transcription factors implicated in the regulation of gene expression required for progression through the G(1)-S transition. We have previously detected tumor-specific mutations at a trinucleotide repeat coding sequence of E2F-4 gene in a subset of human sporadic colorectal cancers. The purpose of this study was to investigate the potential functional consequences of these E2F-4 mutations. We transfected NIH3T3 fibroblasts with expression constructs containing wild-type as well as mutant E2F-4 cDNA, and the effect of the E2F-4 mutations on proliferation was examined. Alteration in transactivation of the E2F consensus promoter sequence was also examined by transient cotransfection of a E2F-4 with a DP-2 construct into cultured human cells. Transfected cell clones overexpressing mutant E2F-4 grew more rapidly and showed higher proliferative activity by increased immunohistochemical staining for proliferating cell nuclear antigen (PCNA). All three mutant forms of E2F-4 showed elevated transactivation of the E2F consensus promoter sequence. Thus, expression of mutant E2F-4s confers a growth advantage in vivo, and this effect may be related to the acquisition of a neoplastic phenotype.

Functional suppression of integrin beta 4-mediated adhesion caused by in vivo sequential selection for cancer cell intravasation.

Intravasation is essential for hematogenous metastasis in cancer cells, but its cellular determinants have not been well elucidated because of a lack of suitable experimental cell systems. Int-3LL was originally developed by in vivo sequential selection for intravasation from Lewis lung carcinoma (3LL) cells. Here, we found that these variant cells showed a highly penetrating ability in vitro as well as an augmented intravasating potential in vivo. In three-dimensional collagen-gel, Int-3LL cells formed diffusive colonies with less plating efficiency than their parental cells. Despite these properties, Int-3LL cells showed an ability of invasive migration in vitro similar to parental cells. On the other hand, a reduced adhesiveness and less spreading on extracellular matrices were revealed in Int-3LL cells. Analyses using anti-integrin antibodies indicated that the dysadhesion phenotype in Int-3LL cells was associated with integrin beta 4 dysfunction, which is known to produce epithelial detachment. Also, the types and the levels of integrins were not indistinguishable between Int-3LL and parental 3LL cells. Thus, the impaired function of integrin beta 4-mediated adhesion is considered to be an important factor in intravasation during metastasis.

A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma.

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.

[A comparative study of computed tomographic techniques for the detection of emphysema in middle-aged and older patient populations].

Helical-scan computed tomography (CT) is now widely utilized as a mass screening procedure for lung cancer. By adding 3 slices of high-resolution CT (HRCT) to the standard screening procedure, we were able to compare the efficacy of helical-scan CT and HRCT in detecting pulmonary emphysema. Additionally, the prevalence of emphysema detected by HRCT was examined as a function of patient age and smoking history. The subjects (106 men and 28 women) were all community-based middle-aged and older volunteers who participated in a mass lung cancer screening program. Based on visual assessments of the CT films, emphysema was detected in 29 subjects (22%) by HRCT, but in only 4 (3%) by helical-scan CT. Although the prevalence of emphysema was higher among subjects with a higher smoking index, no correlations with age were observed. We concluded that the efficacy of helical scan CT in detecting pulmonary emphysema can be significantly improved with the inclusion of 3 slices of HRCT, and confirmed that cigarette smoking is linked to the development of pulmonary emphysema.

Decline in FEV(1) in community-based older volunteers with higher levels of neutrophil elastase in bronchoalveolar lavage fluid.

BACKGROUND: Neutrophil elastase (NE) is thought to be one of the key proteinases in the development of chronic obstructive pulmonary disease (COPD). Previously, we have shown that the NE-alpha1-proteinase inhibitor (NE-alpha1PI) complex in bronchoalveolar lavage (BAL) fluid was markedly elevated in asymptomatic smokers who had subclinical emphysema on CT scans. We proposed that excessive NE-alpha1PI complex in BAL fluid was a factor which might differentiate smokers who were developing emphysema from others. OBJECTIVE: In this study, we addressed the question of whether elevated levels of the NE-alpha1PI complex in BAL fluid are linked to the accelerated decline in pulmonary functions in those subjects. METHODS: We conducted a follow-up study to analyze the decline in FEV(1) for 4.3 years on average for 26 community-based volunteers who had received pulmonary function tests, CT scans and BAL. The levels of the NE-alpha1PI complex in BAL fluid and in plasma was measured. RESULTS: Neither pulmonary function measurements nor the presence of emphysema on CT scans could predict the decline in FEV(1). The number of inflammatory cells in BAL fluid was also not an indicator of progression. By contrast, subjects with higher levels of the NE-alpha1PI complex in BAL fluid had a significantly accelerated decline in FEV(1) compared to those with lower levels. CONCLUSION: These data seem to support the hypothesis that NE in the lung is related to the onset and/or progression of COPD.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Citrus limonoids obacunone and limonin inhibit azoxymethane-induced colon carcinogenesis in rats.

Obacunone and limonin are bitter limonoids in citrus. Their modifying effects on the development of aberrant crypt foci (ACF), the activity of detoxification enzymes, glutathione S-transferase (GST) and quinone reductase (QR), and cell proliferation activity were investigated in male F344 rats treated with azoxymethane (AOM). Obacunone and limonin were administered in the diet, during the initiation (for 4 weeks) or postinitiation phase (for 4 weeks) of AOM-induced tumorigenesis. Feeding of obacunone and limonin (0.02% or 0.05%) caused significant reduction (55-65% by "initiation" feeding and 28-42% by "postinitiation" feeding) in the yield of ACF. The ability to reduce the proliferating cell nuclear antigen-labeling index in crypts and correlated well with the prevention of ACF. In a subsequent long-term experiment (38 weeks), in which rats were initiated with AOM and fed 0.05% obacunone or 0.05% limonin during the initiation or post-initiation phase, both compounds in diet caused significant reduction (65%-92% inhibition) in the incidence of colonic adenocarcinoma. Thus, citrus bitter limonoids obacunone and limonin possess chemopreventive effects on chemically induced rat colon carcinogenesis.

Transforming activity of the RL-akt gene, a c-akt gene activated by long terminal repeat insertion in murine leukemia RL(male symbol)1 cells.

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL(male symbol)1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL(male symbol)1 cells.

[Chronic pulmonary thromboembolism diagnosed on the basis of characteristic mosaic patterns on lung computed tomograms].

We report on a 59-year-old woman who presented with characteristic findings on lung computed tomographic (CT) scans and was therefore suspected to have chronic pulmonary thromboembolism. She visited our hospital because of worsening exertional dyspnea over the preceding year, and because she was dissatisfied with an earlier diagnosis made by another institution. Chest roentgenograms disclosed nonhomogeneous hyperlucency in both lungs associated with linear and bundle shadows, dullness of the right costophrenic angle, and dilatation of the descending branch of the right pulmonary artery. The patient experienced moderate hypoxemia even at rest. Pulmonary function tests demonstrated a restrictive ventilatory pattern associated with reduced diffusion capacity. The lung CT scans disclosed a mosaic pattern of attenuation in the lung parenchyma, which probably reflected scattered areas of low perfusion. The unique characteristics of such CT findings drew our attention to chronic pulmonary thromboembolism as a possible diagnosis. We eventually confirmed the diagnosis on the basis of enhanced CT scans, pulmonary perfusion and ventilation scintigrams, and digital subtraction angiography. In our view, chronic pulmonary thromboembolism should be kept in mind as a possible differential diagnosis of the mosaic patterns of attenuation on lung CT scans.