Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region.

A novel GII.P17-GII.17 variant norovirus emerged as a major cause of norovirus outbreaks from December 2014 to March 2015 in Japan. Named Hu/GII/JP/2014/GII.P17-GII.17, this variant has a newly identified GII.P17 type RNA-dependent RNA polymerase, while the capsid sequence displays amino acid substitutions around histo-blood group antigen (HBGA) binding sites. Several variants caused by mutations in the capsid region have previously been observed in the GII.4 genotype. Monitoring the GII.17 variant's geographical spread and evolution is important.

Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors.

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Elevated PIN1 expression by C/EBPalpha-p30 blocks C/EBPalpha-induced granulocytic differentiation through c-Jun in AML.

The transcription factor CCAAT enhancer-binding protein alpha (C/EBPalpha) has an important role in granulopoiesis. The tumor suppressor function of C/EBPalpha is shown by the findings that loss of expression or function of C/EBPalpha in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPalpha mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPalpha (C/EBPalpha-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPalpha-p30 in AML. In the present study, we show that C/EBPalpha-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPalpha-p30 results in recruitment of E2F1 in the PIN1 promoter. We show that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPalpha mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also show that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination, and c-Jun blocks granulocyte differentiation mediated by C/EBPalpha. Our data suggest that the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPalpha mutation.

Lifestyle correlates of health perception and treatment satisfaction in a clinical cohort of men with prostate cancer.

There is a growing need to understand how nutritional and lifestyle practices may optimize quality of life (QOL) and health after diagnosis for the 1.5 million men living with prostate cancer in the United States. We are establishing a clinical cohort of men with prostate cancer at the University of California San Francisco. Men completed detailed dietary and lifestyle questionnaires annually and provided consent for blood and tissue specimens to be stored for research if they underwent radical prostatectomy. We examined the feasibility of establishing this cohort and analyzepreliminary baseline data on participant demographics, lifestyle habits, and QOL using c2 and t-tests and logistic regression models. Between February 2002 and July 2004, we enrolled 343 men with prostate cancer into the survey portion of this cohort. The response rate was approximately 85% via in-clinic enrollment and 30% via mail enrollment. Based on analysis of the first 193 men enrolled, there was a high level of treatment satisfaction in this population (88% of men were satisfied or extremely satisfied with treatment) and positive reports of general health perception (73% of men perceived themselves to be in excellent [34%] or very good [39%] health). Whether treatment interfered with diet was an independent predictor of health perception and treatment satisfaction. Use of dietary supplements was high (90%) in this well-educated population. In conclusion, we demonstrated good feasibility for conducting this longitudinal study and observed initial indications that diet and other lifestyle practices were important predictors of patient QOL.

Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its interaction with APC.

Phosphorylation on a serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism, and the conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Whereas the inhibition of Pin1 induces apoptosis, Pin1 is strikingly overexpressed in a subset of human tumours. Here we show that Pin1 regulates beta-catenin turnover and subcellular localization by interfering with its interaction with adenomatous polyposis coli protein (APC). A differential-display screen reveals that Pin1 increases the transcription of several beta-catenin target genes, including those encoding cyclin D1 and c-Myc. Manipulation of Pin1 levels affects the stability of beta-catenin in vitro. Furthermore, beta-catenin levels are decreased in Pin1-deficient mice but are increased and correlated with Pin1 overexpression in human breast cancer. Pin1 directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in beta-catenin, inhibits its interaction with APC and increases its translocation into the nucleus. Thus, Pin1 is a novel regulator of beta-catenin signalling and its overexpression might contribute to the upregulation of beta-catenin in tumours such as breast cancer, in which APC or beta-catenin mutations are not common.

Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1.

Phosphorylation on serines or threonines preceding proline (Ser/Thr-Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr-Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimer's disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c-Jun that is phosphorylated on Ser63/73-Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c-Jun towards the cyclin D1 promoter. Thus, Pin1 is up-regulated in human tumors and cooperates with Ras signaling in increasing c-Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas.

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.

Use of serial analysis of gene expression (SAGE) technology.

Serial analysis of gene expression, or SAGE, is an experimental technique designed to gain a direct and quantitative measure of gene expression. The SAGE method is based on the isolation of unique sequence tags (9-10 bp in length) from individual mRNAs and concatenation of tags serially into long DNA molecules for a lump-sum sequencing. The SAGE method can be applied to the studies exploring virtually any kinds of biological phenomena in which the changes in cellular transcription are responsible. SAGE is a highly competent technology that can not only give a global gene expression profile of a particular type of cell or tissue, but also help us identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions. In this review, we present an outline of the original method, several studies achieved by using the method as a major strategic tool, technological difficulties and intrinsic problems that emerged, and improvements and modifications of the method to cope with these drawbacks. We then present our modified SAGE procedure that generates longer sequence tags (14 bp) rather in detail, and the profile (80K profile) derived from HeLa cells that is composed of 80000 tags obtained from a single library. In addition, a series of smaller profiles (2, 4, 10, 20 and 40K) was made by dividing the 80K profile. When we compared these smaller profiles with respect to tag counts for a number of genes, it became apparent that counts of most gene tags increase stably and constantly as the size of profiles increase, while several genes do not. This may be another problem we have to keep in mind, when the profiles are compared for the identification of 'specific genes'.

Enhanced expression of translation factor mRNAs in hepatocellular carcinoma.

Several studies have demonstrated elevated expression of translation factor mRNAs in malignant tissues. In this study, using primary human hepatocellular carcinoma (HCC) tissues, we examined gene expression of translation factors, including 2 eukaryotic initiation factors (eIFs-4A1, -4E), 4 elongation factors (eEFs-1 alpha, -1 gamma, -1 delta, and -2) and 10 ribosomal proteins (Rps P1, P2, S10, L35, L5, L39, L9, L6, S3a and S17), whose mRNA expression has never been examined in HCC. Our results demonstrated that all the mRNAs examined were up-regulated in HCC tissues. Among 7 HCC tissues of different histological grades, the expression of these mRNAs remained at basal levels in a well to moderately differentiated (W/M-) HCC, was coordinately up-regulated in moderately differentiated (M-) HCCs. In moderately to poorly differentiated (M/P-) HCCs, the expression of eEFs-1 gamma, -1 delta, -2, Rps P0 and L9 mRNAs was further up-regulated along with the histological grading. These results therefore suggest that coordination and specific activation of translation factor genes might be involved in the process of liver carcinogenesis.

Identification and characterization of differentially expressed mRNAs in HIV type 1-infected human T cells.

We used a novel differential display (DD) technique to identify host factors involved in virus replication, pathogenesis, and host response in HIV-1-infected T cells. Thirteen cDNA fragments differentially expressed in HIV-1NL4-3-infected MT-4 cells prior to the occurrence of specific apoptotic cell death were sequenced and identified. Two of seven elevated genes were identical to HIV-1 sequences and the other five were MIP-1alpha, ACTE-III, CD11c, arginase I, and CCR5. The six downregulated genes included prothymosin-a, Jaw-1, proteasome subunit XAPC7, splicing factor 9G8, GA17 protein, and an unknown mRNA. Northern blot and RT-PCR analyses confirmed the altered gene expressions in MT-4 cells as well as in another T cell line, MOLT-4. We also revealed that the amount of MIP-1alpha in culture supernatant of HIV-1-infected cells was increased by more than 15-fold relative to control cells, and the expression of its receptor CCR5 was cooperatively upregulated on the surface of these cells. Furthermore, the upregulation of CD11c after HIV-1 infection was slightly inhibited by blocking the MIP-1alpha-mediated signal transduction. These results indicate that genes altered on HIV-1 infection may be mutually organized and play an important role in HIV-1-induced pathogenesis.

Serial analysis of gene expression in HIV-1-infected T cell lines.

The gene expression profile of the HIV-1 infection state was analyzed in the human T cell line MOLT-4. Using the serial analysis of gene expression (SAGE) method, a total of 142¿ omitted¿603 SAGE tags were sequenced and identified, representing 43¿ omitted¿581 unique mRNA species. Comparison of expression patterns revealed that 53 cellular genes were differentially expressed upon HIV-1 infection. Northern blot and RT-PCR analyses confirmed the altered expression of the genes in both MOLT-4 and MT-4 cells. Up-regulated genes were mainly composed of transcription factors and genes related to T cell activation, whereas down-regulated genes were comprised of mitochondrial proteins, actin-related factors and translational factors. These findings indicate that persistent T cell activation, which may accelerate HIV-1 replication, and the disruption of cellular housekeeping genes including those involved in anti-apoptotic systems, may play an important role in HIV-1-induced pathogenesis.

Serial analysis of gene expression in a microglial cell line.

We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology.

Identification and characterization of genes associated with human hepatocellular carcinogenesis.

Eight cDNAs encoding galectin 4 (Gal-4), UGT2B4 (UDP-glucuronosyltransferase), ribosomal phosphoprotein P0 (rpP0), dek, insulin-like growth factor binding protein (IGFBP) 1, vitronectin, retinoic acid-induced gene E (RIG-E), and CYP3A4 (cytochrome P450 nifedipine oxidase) were identified as differentially expressed genes between human hepatocellular carcinoma (HCC) and matched nontumorous liver tissues. Higher levels of UGT2B4, rpP0, dek, vitronectin, Gal-4, and IGFBP-1 mRNAs combined with a lower level of RIG-E mRNA were observed in at least four of five primary HCCs compared to matched nontumorous liver tissues. Furthermore, a pathological study suggested that the levels of UGT2B4, rpP0, dek, and vitronectin increased and the level of RIG-E decreased with the histological grading. On the other hand, the expression of CYP3A4 mRNA and CYP3A7 (P-450 Fla) mRNA, a transcript found in the fetus and highly homologous to CYP3A4, was higher in all nontumorous liver and some of the carcinoma tissues from five HCC patients, whereas it was significantly lower in normal liver tissues from two non-HCC patients. The examination using HCC cell lines HuH-7 and HepG2 under different growth conditions suggested that the expression of dek mRNA was growth-associated. In contrast, the expression of Gal-4, UGT2B4, IGFBP-1, and RIG-E mRNAs was regulated in a cell density-dependent manner: the levels of Gal-4, UGT2B4, and IGFBP-1 were undetectably low, whereas the level of RIG-E was high in rapidly proliferating, subconfluent HCC cells in 10% serum; however, the expression levels were reversed in dense, overcrowded cultures. In addition, IGFBP-1 and Gal-4 mRNAs were also induced by reducing the serum concentration to 0.1%. We also demonstrated that sodium butyrate, an inducer of differentiation, up-regulated and down-regulated RIG-E and dek mRNAs, respectively, in a dose-dependent manner in HuH-7 cells, supporting, in part, our pathological observation. In summary, therefore, high expression of Gal-4, UGT2B4, rpP0, dek, IGFBP-1, and vitronectin, together with low expression of RIG-E, was correlated with the malignant potential of HCC. CYP3A4 and CYP3A7 could be induced in HCC-bearing livers. These transcripts are differentially regulated depending on cell-cell contact, serum growth factors, growth and differentiation status, and/or other mechanisms in premalignant and malignant liver cells.

A method for analyzing the qualitative and quantitative aspects of gene expression: a transcriptional profile revealed for HeLa cells.

A number of strategies have been devised by which differentially expressed genes in different cell types or tissues can be identified. We here report an efficient method to analyze the qualitative and quantitative aspects of transcripts and to construct an extensive gene expression profile in any kind of cell or tissue of interest. This method enables us to analyze the composition of mRNA species, reflecting gene activities, by measuring the frequency of appearance of concatamerized 17mer cDNA mini-fragments, which are proportional to the abundance of mRNA. As compared with a related method previously described by others, we can analyze approximately 3-4 bp longer cDNA fragments derived from amounts of total RNA as small as 1 microg. Using this technique we examined 10 100 cDNA mini-fragments from HeLa cells and constructed a gene expression profile consisting of 3665 genes. This method should thus provide an overall indication of gene activities and a rational means for monitoring gene fluctuation in different cells or tissues at different stages of development, in normal and disease states.