Sun Ho Kee

Hanultarin, a cytotoxic lignan as an inhibitor of actin cytoskeleton polymerization from the seeds of Trichosanthes kirilowii

Bioactivity-directed fractionation of extracts from the seeds of Trichosanthes kirilowii led to the isolation of (-)-1-O-feruloylsecoisolariciresinol (2), named hanultarin, In addition, four known lignans were also isolated, including (-)-secoisolariciresinol (1), 1,4-O-diferuloylsecoisolariciresinol (3), (-)-pinoresinol (4), and 4-ketopinoresinol (5). Their structures were elucidated on the basis of spectroscopic data. Compounds 2 and 3 exhibited strong cytotoxic effects against human lung carcinoma A549 cells, melanoma SK-Mel-2 cells, and mouse skin melanoma B16F1 cells with IC(50) ranges of 3-13 microg/mL. Compound 2 showed an inhibitory effect on the polymerization of the actin cytoskeleton in normal epidermal keratinocyte (HaCaT cells), suggesting unique biological properties of compound 2 compared to those of the other isolates.

Axin localizes to mitotic spindles and centrosomes in mitotic cells.

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.

Complete genome sequence and phylogenetic analysis of hepatitis B virus (HBV) isolated from Mongolian patients with chronic HBV infection.

Although there is a report of a high rate of hepatitis B virus (HBV) infection in Mongolia, the entire nucleotide sequence of HBV circulating among Mongolian patients has not been reported. To obtain the complete nucleotide sequence of the Mongolian HBV, viral DNA was extracted from sera of patients with HBV infection. Six Mongolian HBV strains were amplified by PCR. Complete genomic sequences were determined for two Mongolian HBV isolates, MBT181 and MMU36. The entire genome of Mongolian HBV isolates was 3,182 bp long and genetic distance between Mongolian HBV isolates was 3.2%. Precore stop codon resulting from a guanine to adenine mutation at nucleotide 1,896 was detected in MBT181 strain. Based on phylogenetic analysis, the six Mongolian isolates were classified as genotype D.

Class II β-tubulin is a novel marker for human tonsillar M cells and follicular dendritic cells

OBJECTIVE Membranous (M) cell of the human palatine tonsil is an antigen entry site for mucosal infection, but its location is obscure in histological sections. Recently, a microarray analysis has demonstrated that clusterin, annexin A5, CD44, MMP14, and beta-tubulin are candidate genes of M cell marker in mice. Among these genes, we here describe class II beta-tubulin as a new marker for human tonsillar M cells and follicular dendritic cells (FDCs), and present its usefulness for diagnosis of angioimmunoblastic T-cell lymphomas (AILTs). MATERIALS AND METHODS Immunohistochemistry and Western blotting for class II beta-tubulin were performed using 81 cases of lymphoid, gastrointestinal and thyroid tissues, and an FDC cell line, respectively. Double immunostaining with clusterin and class II beta-tubulin were carried out. RESULTS Class II beta-tubulin localized the M cells and FDCs in the palatine tonsils (10/10, 100%) and adenoids (10/10, 100%). It was colocalized with clusterin in the palatine tonsils. However, class II beta-tubulin staining did not identify intestinal M cells in the intestines. Immunoblot analysis revealed that class II beta-tubulin expression was upregulated in HK cells, a normal FDC cell line. Class II beta-tubulin immunostaining highlighted hyperplastic FDC meshworks in all AILTs (14/14, 100%). CONCLUSION Class II beta-tubulin is a specific histochemical marker for human tonsillar M cells and FDCs. Thus, class II beta-tubulin immunostaining may be useful to identify tonsillar M cells and to diagnose FDC proliferative lesions such as AILT.

Axin expression reduces staurosporine-induced mitochondria-mediated cell death in HeLa cells

Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.

Axin expression enhances herpes simplex virus type 1 replication by inhibiting virus-mediated cell death in L929 cells

Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signalling and participates in various cellular events, including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6 h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of the G-protein signalling (RGS) domain were involved in the axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that viral replication enhancement might be mediated by the axin RGS domain.

Reassortment compatibility between PB1, PB2, and HA genes of the two influenza B virus lineages in mammalian cells

In addition to influenza A subtypes, two distinct lineages of influenza B virus also cause seasonal epidemics to humans. Recently, Dudas et al. have done evolutionary analyses of reassortment patterns of the virus and suggested genetic lineage relationship between PB1, PB2, and HA genes. Using genetic plasmids and reassortant viruses, we here demonstrate that a homologous lineage PB1-PB2 pair exhibits better compatibility than a heterologous one and that the lineage relationship between PB1 and HA is more important for viral replication than that between PB2 and HA. However, co-adaptation of PB1-PB2-HA genes appears to be affected by complete gene constellation.

Axin1 expression facilitates cell death induced by aurora kinase inhibition through PARP activation

Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1‐expressing cells (L‐Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2‐expressing cells (L‐Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub‐G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L‐Axin cells induced poly(ADP‐ribose) polymerase (PARP) activation, which increased the poly(ADP‐ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI‐induced cell death. Also, AKI treatment of L‐Axin cells induced mitochondrial apoptosis‐inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase‐3 activation. Knockdown of AIF attenuated AKI‐induced cell death in L‐Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition‐induced cell death in a PARP‐ and AIF‐dependent manner. J. Cell. Biochem. 112: 2392–2402, 2011.

Axin expression delays herpes simplex virus-induced autophagy and enhances viral replication in L929 cells.

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.

Expression of class II β-tubulin in non-melanoma cutaneous tumors

Background:  Different combinations of β‐tubulin isotypes contribute to the diverse functions of microtubules (MTs). Class II β‐tubulin (class II tubulin) is up‐regulated in differentiated keratinocytes. In contrast, the expression of class II tubulin in follicular differentiation and cutaneous tumors has not been studied.