Identification of compounds that preferentially suppress the growth of T-cell acute lymphoblastic leukemia-derived cells.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.

DEAD box protein DDX1 promotes colorectal tumorigenesis through transcriptional activation of the LGR5 gene.

DDX1, a member of the DEAD box RNA helicase family, plays a critical role in testicular tumors. However, it remains to be clarified whether DDX1 is involved in other types of malignant tumors such as colorectal cancer. We disrupted the DDX1 gene in a human colorectal cancer cell line LoVo using the CRISPR/Cas9-mediated gene-targeting system. DDX1-KO LoVo cells exhibited a much slower growth rate, produced fewer colonies in soft agar medium, and generated smaller solid tumors in nude mice than parental LoVo cells. Such phenotypes of the DDX1-KO cells were mostly reversed by exogenous expression of DDX1. These results indicate that DDX1 is required for tumorigenicity of colorectal cancer cells. In the DDX1-KO cells, the cancer stem cell marker genes LGR5, CD133, ALDH1 and SOX2 were markedly suppressed. Among them, expression of LGR5, which is essential for tumorigenicity of colorectal cancer cells, was restored in the DDX1-transfected DDX1-KO cells. Consistently, the DDX1-KO cells lost sphere-forming capacity in a DDX1-dependent fashion. Reporter and chromatin immunoprecipitation assays revealed that DDX1 directly bound to the -1837 to -1662 region of the enhancer/promoter region of the human LGR5 gene and enhanced its transcription in LoVo cells. Repression of LGR5 by DDX1 knockdown was observed in 2 other human colorectal cancer cell lines, Colo320 and SW837. These results suggest that LGR5 is a critical effector of DDX1 in colorectal cancer cells. The DDX1-LGR5 axis could be a new drug target for this type of malignant cancer.

CXCL14 Acts as a Specific Carrier of CpG DNA into Dendritic Cells and Activates Toll-like Receptor 9-mediated Adaptive Immunity.

CXCL14 is a primordial chemokine that plays multiple roles in tumor suppression, autoimmune arthritis, and obesity-associated insulin resistance. However, the underlying molecular mechanisms are unclear. Here, we show that CXCL14 transports various types of CpG oligodeoxynucleotide (ODN) into the endosomes and lysosomes of bone marrow-derived dendritic cells (DCs), thereby activating Toll-like receptor 9 (TLR9). A combination of CpG ODN (ODN2395) plus CXCL14 induced robust production of IL-12 p40 by wild-type, but not Tlr9-knockout, DCs. Consistent with this, ODN2395-mediated activation of DCs was significantly attenuated in Cxcl14-knockout mice. CXCL14 bound CpG ODN with high affinity at pH7.5, but not at pH6.0, thereby enabling efficient delivery of CpG ODN to TLR9 in the endosome/lysosome. Furthermore, the CXCL14-CpG ODN complex specifically bound to high affinity CXCL14 receptors on DCs. Thus, CXCL14 serves as a specific carrier of CpG DNA to sensitize TLR9-mediated immunosurveillance.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells.

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit(-)Tie2(-)CD41(+) Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41(+)CD42b(+)CD61(+) platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future.

Detection of hepatitis B and C viruses in almost all hepatocytes by modified PCR-based in situ hybridization.

Although PCR-based in situ hybridization (PCR-ISH) can be used to determine the distribution and localization of pathogens in tissues, this approach is hampered by its low specificity. Therefore, we used a highly specific and sensitive PCR-ISH method to reveal the lobular distribution and intracellular localization of hepatitis B virus (HBV) and HCV in chronic liver disease and to clarify the state of persistent HBV and HCV infection in the liver. HBV genomic DNA was detected in almost all hepatocytes, whereas HBV RNA or protein was differentially distributed only in a subset of the HBV DNA-positive region. Further, HCV genomic RNA was detected in almost all hepatocytes and was localized to the cytoplasm. HCV RNA was also detected in the epithelium of the large bile duct but not in endothelial cells, portal tracts, or sinusoidal lymphocytes. In patients with HBV and HCV coinfection, HCV RNA was localized to the noncancerous tissue, whereas HBV DNA was found only in the cancerous tissue. Using this novel PCR-ISH method, we could visualize the staining pattern of HBV and HCV in liver sections, and we obtained results consistent with those of real-time detection (RTD)-PCR analysis. In conclusion, almost all hepatocytes are infected with HBV or HCV in chronic liver disease; this finding implies that the viruses spreads throughout the liver in the chronic stage.

Hepatitis C virus impairs p53 via persistent overexpression of 3beta-hydroxysterol Delta24-reductase.

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3beta-hydroxysterol Delta24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.

Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.

Major histocompatibility complex class-I presentation impaired in transgenic mice expressing hepatitis C virus structural proteins during dendritic cell maturation.

Hepatitis C virus (HCV) infection is often persistent, but its mechanism and pathogenesis remain unclear. One mechanism through which HCV escapes systemic immunosurveillance might be via impaired dendritic cells (DCs), which are the most potent type of antigen-presenting cells. We examined whether HCV causes immunosuppression in DCs during maturation. We isolated immature DCs from the bone marrow of two founder lineages of transgenic mice harboring HCV cDNA expressing HCV structural proteins (nucleotides 294-3435), and studied how DC function is modified by HCV expression. Our data showed that the capacity of DCs expressing HCV structural proteins to stimulate T-cells was significantly impaired. Moreover, the surface expression of major histocompatibility complex (MHC) class-I molecules was significantly impaired on infected DC, especially with respect to H-2D. The transportation of H-2D to the cell surface during DC maturation was inhibited by HCV expression. However, the total amount of H-2D molecules produced by DC expressing HCV was not impaired. These results indicated that the immune response of DC infected with HCV is diminished and might be associated with the mechanism of persistent HCV infection.

Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone.

BACKGROUND/AIMS: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or 'isolated hepatocytes' from tree shrews. In this study, we engrafted 'human liver tissue' into immunodeficient mice and investigated HCV infection using an infectious molecular clone. METHODS: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV-infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR. RESULTS: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection. CONCLUSION: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection.

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells.

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.

Virological significance of low-level hepatitis B virus infection in patients with hepatitis C virus associated liver disease.

The clinical and virological significance of low-level viremia by hepatitis B virus (HBV) in hepatitis C virus (HCV)-infected patients remains unclear. HBV-DNA and HCV-RNA were, therefore, quantitatively analyzed in livers and sera from co-infected patients. HBV-DNA and HCV-RNA were quantitated using real-time detection of polymerase chain reaction (RTD-PCR), based on Taq-Man chemistry, in 220 non-HCV-infected healthy volunteers and 93 HCV-infected patients without detectable HBsAg. Serum HBV-DNA was detected in 4 (1.8%) of 220 non-HCV-infected healthy volunteers and 32 (34.4%) of 93 HCV-infected patients without detectable HBsAg. HCV-infected patients displayed higher frequency of HBV infection than healthy volunteers (P < 0.0001). Hepatocellular carcinoma (HCC) was more frequent among co-infected patients than among HCV mono-infected patients (P < 0.001). However, quantities of HBV-DNA in sera from co-infected patients were very low (8-19,000 copies/ml). HBV-DNA was detected in liver tissue from co-infected patients at 2-20 copies per 100 hepatocytes, accounting for 1/1,000 to 1/10,000 of HBsAg positive patients. In livers of patients with HCC and HCV or HBV mono-infection, the viruses existed predominantly in non-cancerous tissue, with levels 10- to 1,000-fold and 1- to 100-fold higher than in cancerous tissue, respectively. In contrast, patients co-infected with HCV and HBV displayed decreased HBV levels in non-cancerous tissue, but no change in cancerous tissue. These results indicate that low-level HBV infection exists in HCV-infected patients. HCC was more common among HCV/HBV co-infected patients than among HCV mono-infected patients. HCV might initiate hepatocarcinogenesis, but does not necessarily determine progression to HCC.

Protein kinase R is increased and is functional in hepatitis C virus-related hepatocellular carcinoma.

OBJECTIVE: Protein kinase R (PKR) interacts with dsRNA and phosphorylates eukaryotic initiation factor-2 (eIF2alpha), which in turn inhibits host translation initiation as well as hepatitis C virus (HCV) translation. Because PKR inhibits host cell growth and proliferation, it has also been proposed to act as a eukaryotic tumor suppressor. To evaluate the role of PKR in HCV-related hepatocellular carcinoma (HCC), we compared PKR and related protein expression in paired tumor (T) and surrounding nontumor (NT) tissue. METHODS: Tissue samples were obtained from 12 HCV-infected HCCs. To determine PKR and related protein expression, Western blotting and semiquantitative reverse transcriptase-polymerase chain reaction were performed. RESULTS: PKR protein levels were consistently increased in HCV-related HCC compared with NT (p=0.001); similar increases were seen in total eIF2alpha and the PKR inhibitor p58IPK in T compared with NT (p=0.022, p=0.048, respectively). Relative increases in phosphorylated eIF2alpha (peIF2alpha) were also seen, and the ratio of peIF2alpha/total eIF2alpha did not change in T compared with NT, suggesting that PKR remains functional within T. Cytoplasmic levels of HCV RNA within T were decreased compared with NT. CONCLUSIONS: These findings indicate that PKR has increased activity in human HCC compared with LC, and suggest that PKR acts as a growth inducer in HCC.

Impaired function of antigen-presenting dendritic cells in patients with chronic hepatitis B: localization of HBV DNA and HBV RNA in blood DC by in situ hybridization.

Antigen-presenting dendritic cells (DCs), which play a major role in the triggering of primary anti viral immune reactions, may also contribute, in some viral models, to the pathogenesis of persistent viral infection. In fact, impaired immune response to hepatitis B virus (HBV)-encoded antigens is seen in patients with chronic hepatitis B (CH-B). The aim of this study was to check the function of DCs in these patients and to investigate the underlying mechanism. DCs were enriched from peripheral blood mononuclear cells by culturing with interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor for 7 days. The stimulatory capacity of DCs were checked in allogenic mixed leukocyte (MLR) reaction. The levels of IL-12 in the culture supernatants were measured by an enzyme-linked immunosorbent assay. HBV DNA and HBV RNA were localized in DCs by polymerase chain reaction (PCR) in situ hybridization and reverse-transcriptase (RT)-PCR in situ hybridization. The stimulatory capacity of DCs in allogenic MLR was significantly lower in patients with CH-B (36321+/-12523 cpm, n=18) compared to that of normal controls (65678+/-11174 cpm, n=18) (p<0.0001). Significantly lower levels of IL-12 were detected in cultures containing DCs from patients with CH-B than normal controls (46.7+/-25.6 versus 122.4+/- 37.1 pg/ml, p<0.0001). In situ hybridization revealed the localization of HBV DNA and HBV RNA in DCs from patients with CH-B. These results indicate that chronic infection by HBV is associated with functional defects of DCs. Localization of HBV DNA and HBV RNA indicates that DCs may constitute an extra hepatic reservoir and possibly of replication of HBV.

Direct evidence for interferon-gamma production by effector-memory-type intraepidermal T cells residing at an effector site of immunopathology in fixed drug eruption.

Effector-memory T cells are strategically placed to epithelial tissues to provide frontline immune protection against pathogens. Their detrimental effects, however, have been rarely examined because of difficulty in sampling these T cells in pathological settings. Our previous studies suggested persistence of a similar subset of intraepidermal CD8(+) T cells at high frequencies in the lesions of fixed drug eruption, a localized variant of drug-induced dermatoses. In situ activation of this subset resulting in localized epidermal injury can be traced in the lesions after antigen challenge by paired immunohistochemical staining, reverse transcriptase-polymerase chain reaction in situ, and flow cytometry of dispersed cells. Here we show that effector-memory T cells were greatly enriched in these intraepidermal CD8(+) T cells, but not dermal and circulating counterparts, and that they constitutively express an early activation marker CD69 even before challenge. Surprisingly, a large proportion of these T cells expressed immediate effector function as evidenced by the rapid production of high levels of interferon-gamma in situ with much faster kinetics than their counterparts at the mRNA and protein levels after challenge. This was followed by localized epidermal injury. The intracellular cytokine assay ex vivo shows that the great majority of these dispersed T cells produce interferon-gamma. This study provides the first in situ description of the detrimental effects specifically mediated by effector-memory T cells residing at the effector site of immunopathology.