Distinct expression of interferon-induced protein with tetratricopeptide repeats (IFIT) 1/2/3 and other antiviral genes between subsets of dendritic cells induced by dengue virus 2 infection.

Dengue virus (DENV) infection is an emerging public health hazard threatening inhabitants of the tropics and sub-tropics. Dendritic cells (DCs) are one of the major targets of DENV and the initiators of the innate immune response against the virus. However, current in vitro research on the DENV-DC interaction is hampered by the low availability of ex vivo DCs and donor variation. In the current study, we attempted to develop a novel in vitro DC model using immature DCs derived from the myeloid leukaemia cell line MUTZ-3 (IMDCs) to investigate the DENV-DC interaction. The IMDCs morphologically and phenotypically resembled human immature monocyte-derived dendritic cells (IMMoDCs). However, the permissiveness of IMDCs to DENV2 was lower than that of IMMoDCs. RT-PCR arrays showed that a group of type I interferon (IFN) -inducible genes, especially IFIT1, IFITM1, and IFI27, were significantly up-regulated in IMMoDCs but not in IMDCs after DENV2 infection. Further investigation revealed that IFIT genes were spontaneously expressed at both transcriptional and protein levels in the naive IMDCs but not in the naive IMMoDCs. It is possible that the poor permissiveness of IMDCs to DENV2 was a result of the high basal levels of IFIT proteins. We conclude that the IMDC model, although less permissive to DENV2, is a useful platform for studying the suppression mechanism of DENV2 and we expand the knowledge of cellular factors that modulate DENV2 infection in the human body.

Current and future molecular diagnostics in non-small-cell lung cancer.

The molecular investigation of lung cancer has opened up an advanced area for the diagnosis and therapeutic management of lung cancer patients. Gene alterations in cancer initiation and progression provide not only information on molecular changes in lung cancer but also opportunities in advanced therapeutic regime by personalized targeted therapy. EGFR mutations and ALK rearrangement are important predictive biomarkers for the efficiency of tyrosine kinase inhibitor treatment in lung cancer patients. Moreover, epigenetic aberration and microRNA dysregulation are recent advances in the early detection and monitoring of lung cancer. Although a wide range of molecular tests are available, standardization and validation of assay protocols are essential for the quality of the test outcome. In this review, current and new advancements of molecular biomarkers for non-small-cell lung cancer will be discussed. Recommendations on future development of molecular diagnostic services will also be explored.

Trehalose, an mTOR independent autophagy inducer, alleviates human podocyte injury after puromycin aminonucleoside treatment.

Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

Disease-specific target gene expression profiling of molecular imaging probes: database development and clinical validation.

Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2-deoxy-2-d-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/.

Distinct effects of Disabled-2 on transferrin uptake in different cell types and culture conditions.

Iron uptake by the transferrin (Tf)-transferrin receptor (TfR) complex is critical for erythroid differentiation. The mechanisms of TfR trafficking have been examined, but the adaptor proteins involved in this process are not fully elucidated. We have investigated the role of the adaptor protein, Disabled-2 (Dab2), in erythroid differentiation and Tf uptake in the cells of hematopoietic lineage. Dab2 was upregulated in a time-dependent manner during erythroid differentiation of mouse embryonic stem cells and human K562 erythroleukemic cells. Attenuating Dab2 expression in K562 cells diminished TfR internalization and increased surface levels of TfR concomitantly with a decrease in Tf uptake and erythroid differentiation. Dab2 regulated Tf uptake of the suspended, but not adherent, cultures of K562 cells. In contrast, Dab2 is not involved in TfR trafficking in the HeLa cells with epithelial origin. These differential effects are Dab2-specific because attenuating the expression of adaptor protein 2 µ subunit inhibited the uptake of Tf regardless of culture condition. We offer novel insight of Dab2 function in iron uptake and TfR internalization for the suspended culture of hematopoietic lineage cells.

Current and future molecular diagnostics in colorectal cancer and colorectal adenoma.

Colorectal cancer (CRC) is one of the most prevalent cancers in developed countries. On the other hand, CRC is also one of the most curable cancers if it is detected in early stages through regular colonoscopy or sigmoidoscopy. Since CRC develops slowly from precancerous lesions, early detection can reduce both the incidence and mortality of the disease. Fecal occult blood test is a widely used non-invasive screening tool for CRC. Although fecal occult blood test is simple and cost-effective in screening CRC, there is room for improvement in terms of the accuracy of the test. Genetic dysregulations have been found to play an important role in CRC development. With better understanding of the molecular basis of CRC, there is a growing expectation on the development of diagnostic tests based on more sensitive and specific molecular markers and those tests may provide a breakthrough to the limitations of current screening tests for CRC. In this review, the molecular basis of CRC development, the characteristics and applications of different non-invasive molecular biomarkers, as well as the technologies available for the detection were discussed. This review intended to provide a summary on the current and future molecular diagnostics in CRC and its pre-malignant state, colorectal adenoma.

Advanced technologies for studying circulating tumor cells at the protein level.

Metastasis is the main cause of cancer death. As the tumor progresses, cells from the primary tumor site are shed into the bloodstream as circulating tumor cells (CTCs). Eventually, these cells colonize other organs and form distant metastases. It is therefore imperative that we gain a better understanding of the biological characteristics of CTCs for development of novel treatment modalities to minimize metastasis-associated cancer deaths. In recent years, rapid developments in technologies for the study of CTCs have taken place. We now have a variety of tools for the isolation and examination of CTCs which were not available before. This review introduces some commonly used protein markers in CTC investigations and summarizes a few advanced technologies which have been successfully applied for studying CTC biology at the protein level.

A novel interaction of nucleophosmin with BCL2-associated X protein regulating death evasion and drug sensitivity in human hepatoma cells.

UNLABELLED: Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently, we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells-particularly those bearing inactivated p53 gene (Huh7, Hep3B, and Mahlavu)-to ultraviolet irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not changed further, as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of the nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation revealed a direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, up-regulation of NPM was significantly associated with advanced tumor stage and poor prognosis. CONCLUSION: By directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential cotarget in combination with other therapies for HCC, particularly those that harbor inactivated p53 gene. Our findings are of clinical significance because NPM up-regulation and p53 mutations are usually found in advanced human cancers, including HCC.

Involvement of nucleophosmin/B23 in the cellular response to curcumin.

Nucleophosmin (NPM/B23) is a nucleolar phosphoprotein involved in cellular response to many different stimuli. Herein, we studied the molecular mechanism of NPM/B23 induction by curcumin, a natural AP-1 inhibitor with antitumor properties. Exposure to 5-30 µM curcumin significantly and dose-dependently increased the level of NPM/B23 in non-transformed NIH 3T3 cells but not HeLa cells and F9 cells. Besides, the transformed F9 and HeLa cells are more sensitive to curcumin-induced cell death and growth inhibition than NIH 3T3 cells. Overexpression of c-Jun, but not c-Fos, decreased ∼40% of NPM/B23 and enhanced the sensitivity of NIH 3T3 cells to 30 µM curcumin. Furthermore, down-regulation of NPM/B23 by transfection with NPM/B23 antisense plasmid enhanced the sensitivity to curcumin-induced cell death and growth inhibition. These results indicated that NPM/B23 expression regulates cellular sensitivity to curcumin. Besides, NPM/B23 knockdown may facilitate as a novel strategy to promote the sensitivity of cancer cells to curcumin.

Dephosphorylation of nucleophosmin by PP1ß facilitates pRB binding and consequent E2F1-dependent DNA repair.

Nucleophosmin (NPM) is an important phosphoprotein with pleiotropic functions in various cellular processes. Although phosphorylation has been postulated as an important functional determinant, possible regulatory roles of this modification on NPM are not fully characterized. Here, we find that NPM is dephosphorylated on various threonine residues (Thr199 and Thr234/237) in response to UV-induced DNA damage. Further experiments indicate that the serine/threonine protein phosphatase PP1ß is a physiological NPM phosphatase under both the genotoxic stress and growth conditions. As a consequence, NPM in its hypophosphorylated state facilitates DNA repair. Finally, our results suggest that one possible mechanism of this protective response lies in enhanced NPM-retinoblastoma tumor suppressor protein (pRB) interaction, leading to the relief of the repressive pRB-E2F1 circuitry and the consequent transcriptional activation of E2F1 and several downstream DNA repair genes. Thus, this study unveils a key phosphatase of NPM and highlights a novel mechanism by which the PP1ß-NPM pathway contributes to cellular DNA damage response.

Bikunin loss in urine as useful marker for bladder carcinoma.

PURPOSE: We searched for bladder tumor markers by analyzing urine samples from patients with bladder cancer and normal individuals. MATERIALS AND METHODS: Proteins in urine samples of patients with cancer and normal subjects were systematically examined by 2-dimensional electrophoresis combined with matrix assisted laser desorption ionization time-of-flight mass spectrometry. Of the proteins bikunin expression was confirmed by Western blot analysis and further evaluated. To correlate urinary bikunin levels with clinical significance we examined urine samples from patients with bladder cancer and normal controls for bikunin expression in parallel with pro-urolinase-plasminogen activator, which was previously shown to be associated with advanced bladder carcinoma. RESULTS: A significant relationship was established between the low level and absence of bikunin, and pro-urolinase-plasminogen activator in urine samples from patients with bladder tumors. CONCLUSIONS: Analysis of urinary proteomes may be a feasible, noninvasive and efficient strategy for searching for potential bladder tumor biomarkers. We identified bikunin loss in urine as a potential bladder carcinoma marker.

Association of nucleophosmin/B23 with bladder cancer recurrence based on immunohistochemical assessment in clinical samples.

AIM: To investigate the possible correlation of nucleophosmin/B23 expression with bladder carcinoma recurrence. METHODS: Surgically-resected bladder tumors staged pTa to pT4 were examined for nucleophosmin/B23 expression by immuno-histochemistry. The study group consisted of 132 consecutive patients surgically treated at Chang Gung Memorial Hospital between December 1998 and November 1999. The mean follow up was 72 months (range: 48-84 months). RESULTS: Nuclear nucleophosmin/B23 staining was detected in 96% of advanced stage and poorly-differentiated tumors. Higher nucleophosmin/B23 levels were linked to more advanced tumor stages, grades, poor prognosis, and likelihood of recurrence (P<0.05). The Cox multivariate analysis indicated the nucleophosmin/B23 expression as an independent indicator for tumor recurrence (P=0.009). CONCLUSION: The results suggest that nucleophosmin/B23 is a favorable prognostic indicator for bladder cancer. Nucleophosmin/B23 could be a useful molecular tumor marker for predicting bladder cancer recurrence.

Oncogenic role of nucleophosmin/B23.

Nucleophosmin/B23 was first identified as a nucleolar protein expressed at higher levels in cancer cells compared to normal cells. Nucleophosmin/B23 has long been thus thought to have a role in tumor formation. With our efforts and others in the last 15 years, nucleophosmin/B23 has proven to have an oncogenic role. In this review, we provide evidence suggesting that nucleophosmin/B23 may be a crucial gene in regulation of cancer growth and discuss how nucleophosmin/B23 can contribute to tumorigenesis.

Can probability of genetic mutation be an indicator of clinical relevance?

NPM1 gene mutation evaluated on a population basis is a valuable and realistic tool to reflect the pathophysiological relevance of cancer. In a comparison of the NPM1 cDNA of human bladder cancer with its consensus sequence, we have found that a higher NPM1 sequence identity in a population is consistent with poor tumor differentiation, advanced tumor stage, and likelihood of recurrence. These data imply that "probability" of NPM1 mutation is an indicator of status of malignancy.

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells.

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.

Searching cell-secreted proteomes for potential urinary bladder tumor markers.

To search for biomarkers critical for bladder carcinoma diagnosis and prognosis, secreted proteomes of highly malignant U1 and pre-malignant U4 cell lines were initially analyzed. Proteins in the culture media of the U1 and U4 cell lines were systematically examined by SDS-PAGE combined with MALDI-TOF MS. Among them, expression of pro-u-plasminogen activator (pro-u-PA) was confirmed by Western blot analysis and further evaluated. In analyzing urine samples from bladder cancer patients and normal subjects, we established a statistically significant relationship between the low level and absence of pro-u-PA in urine with high stages and grades of the tumor samples. Constitutive expression of Ras dominant negative protein led to increased expression of pro-u-PA in culture media, indicating that the loss of pro-u-PA is associated with oncogenic transformation. Analysis of cancer-secreted proteomes can be a feasible, non-invasive and efficient strategy for searching potential bladder tumor biomarkers. Our work also has identified the loss of pro-u-PA in urine as potential marker of more advanced bladder carcinoma.

Ras-dependent recruitment of c-Myc for transcriptional activation of nucleophosmin/B23 in highly malignant U1 bladder cancer cells.

U1 bladder cancer cells of high malignancy exhibited higher proliferation capacity than U4 premalignant cells. Higher expression of Ras, c-Myc, and nucleophosmin/B23 and greater c-Myc transactivation and nucleophosmin/B23 promoter activities were detected in U1 cells compared with U4 cells. Moreover, c-Myc and nucleophosmin/B23 were increased in U1 but not in U4 cells upon serum stimulation from quiescence. Likewise, only in U1 cells could serum stimulate transcriptional activity of nucleophosmin/B23 promoter and c-Myc response element. The increase of nucleophosmin/B23 promoter activity could be abrogated by mitogen-activated protein kinase/extracellular signal-regulated kinase activating kinase inhibitor and was associated with recruitment of c-Myc to the promoter. U1 cells constitutively expressing dominant-negative Ras reduced the levels of Ras, nucleophosmin/B23, and p-ERK, and consequently abolished the serum-induced up-regulation of nucleophosmin/B23 promoter activity and c-Myc promoter recruitment. Our results indicate that Ras and c-Myc play important roles in the up-regulation of nucleophosmin/B23 during proliferation of cells associated with a high degree of malignancy, thus outlining a signaling cascade involving these factors in the cancer cells.

Nucleophosmin/B23 regulates transcriptional activation of E2F1 via modulating the promoter binding of NF-kappaB, E2F1 and pRB.

Expression of nucleophosmin/B23 and E2F1 and E2F1-dependent transcription increased in U1 bladder cancer cells upon serum stimulation from quiescence. Nucleophosmin/B23-siRNA treatment abrogated such increase of E2F1-dependent transcriptional activity. In identifying physiologically important factors that may occupy E2F1 promoter and regulate its activity in vivo, we found that the pattern of NF-kappaB, E2F1 and pRB recruitment to E2F1 promoter changed in a strikingly dynamic fashion as cells progressed from quiescence into serum-stimulated growth. E2F1 promoter activity in quiescent cells was associated with recruitment of NF-kappaB. NF-kappaB was replaced largely by E2F1 in concert with gene activation during the early stage (12 h) of serum stimulation. At late stage (24 h) of serum stimulation, pRB was then recruited to the E2F1-promoter complex to counterbalance its activity. Upon siRNA-mediated reduction of intracellular nucleophosmin/B23, E2F1 and pRB were recruited to the promoter with the dissociation of NF-kappaB concomitant with gene inactivation. Based on immunoprecipitation experiments, nucleophosmin/B23 was found to be associated with NF-kappaB in cells grown in serum-supplemented but not in serum-deprived medium. Furthermore, nucleophosmin/B23 could also be co-immunoprecipitated with ppRB at the early stage (12 h) but not at the late stage (24 h) of serum stimulation. The results demonstrate a novel mechanism for transcriptional regulation of E2F1 and identify the functional role of nucleophosmin/B23 in modulating the binding of NF-kappaB, E2F1 and pRB to activate E2F1 promoter.

Nucleophosmin/B23-binding peptide inhibits tumor growth and up-regulates transcriptional activity of p53.

The Rev peptide that binds to nucleophosmin/B23 with the highest affinity exhibited the greatest cytotoxicity on Ras-3T3 cells and inhibited tumor growth most effectively in nude mice. The efficiency of colony formation in soft agar of Ras-3T3 cells was significantly inhibited by treatment with Rev peptide. In addition, Rev peptide could potentiate the doxorubicin-induced decrease of cellular viability in U1 bladder cancer cells and inhibition of tumor growth in nude mice. Treatment of Rev peptide increased protein expression and transcriptional activity of p53 and inhibited the nucleophosmin/B23-mediated PCNA promoter activation. Peptides having high affinity of binding to molecular targets such as nucleophosmin/B23 represent a potentially useful approach to anti-cancer biotherapeutics.

Identification of differentially expressed genes in oral squamous cell carcinoma (OSCC): overexpression of NPM, CDK1 and NDRG1 and underexpression of CHES1.

To identify cellular genes that could potentially serve as predictive molecular markers for human oral cancer, we employed differential display analysis to compare the gene expression profiles between oral squamous cell carcinoma (OSCC) and histopathologically normal epithelium tissues. Comparative real-time RT-PCR was used to confirm the gene expression in 52 OSCC patients, and a 2-fold difference was defined as over- or underexpression. A total of 7 genes were identified: NPM, CDK1, NDRG1, HMGCR, EF1A, NAC and CHES1. In the cancer tissues, NPM, CDK1 and NDRG1 were significantly overexpressed (an average of 7.6-, 17.2- and 12.9-fold, respectively), and CHES1 was underexpressed (15-fold). The frequencies of the differential expression were 40, 56, 67 and 46%, respectively in NPM, CDK1, NDRG1 and CHES1. In Western blot analysis, the protein expressions of NPM, CDK1 and NDRG1 were also increased in the cancer tissues, consistent with the mRNA expression results. To further evaluate clinicopathological associations in these genes, Pearson chi-square analysis was employed. Levels of CDK1 and NDRG1 were associated with poorly differentiated tumors (p = 0.043 and 0.023), suggesting that these genes participate in the mechanism of tumor transformation. Expressions of CDK1 and NDRG1, and CDK1 and CHES1 were mutually statistically correlated (p = 0.001 and 0.014), indicating that these genes share a very close regulatory relationship or interact synergistically in oncogenesis. In conclusion, we identified 7 genes that are differentially expressed in OSCC, and we provide the first evidence that NPM, CDK1 and NDRG1 are overexpressed and CHES1 is underexpressed in oral cancer. These results serve as a fundamental base for employing these genes in future clinical applications.