Adult T-cell leukemia/lymphoma (ATL), caused by human T-cell leukemia virus type-1 (HTLV-1) infection, is a malignant hematologic cancer that remains difficult to cure. We herein established a biomarker identification strategy based on the total cell proteomics of cultured ATL cells to search for novel ATL biomarkers. Four protocols with a combination of selected conditions based on lysis buffers and addition agents for total cell proteomics were used for a differential analysis between the ATL cell group (consisting of 11 cell lines), HTLV-1-infected cell group (consisting of 6 cell lines), and HTLV-1-negative cell group (consisting of 6 cell lines). In the analysis, we identified 24 and 27 proteins that were significantly increased (ratio >= 2.0, p < 0.05) and decreased (ratio <= 0.5, p < 0.05), respectively, in the ATL group. Previously reported CCL3 and CD30/TNFRSF8 were confirmed to be among significantly increased proteins. Furthermore, correlation analysis between identified proteins and Tax suggested that RASSF2 and GORASP2 were candidates of novel Tax-regulated factors. The biomarker identification strategy established herein is expected to contribute to the identification of biomarkers for ATL and other diseases.

Abstract Increased vascularity in the synovium of patients with rheumatoid arthritis (RA) is thought to result from angiogenesis. However, other possibilities to develop vasculatures by mechanism(s) like vasculogenic mimicry remain elusive. In this study, expression of vascular endothelial (VE)-cadherin was immunohistochemically detected in both microvessel lining cells and fibroblastic cells in RA synovium. VE-cadherin-positive lining cells surrounded by periodic acid-Schiff staining-positive substance were occasionally negative for endothelial cell markers CD31/CD34 but positive for osteoblast (OB)-cadherin that is specific to fibroblastic cells. RA synovial fibroblast-like cells (RSFLs) in culture expressed VE-cadherin and OB-cadherin, but not CD31/CD34. Tube formation was evaluated by real-time adhesion and tube formation assays. RSFLs specifically bound to VE-cadherin-chimera-coated plates. They developed tube-like structures solely, which were hindered by anti-VE-cadherin antibody or VE-cadherin-chimera, and with endothelial cells. VE-cadherin was up-regulated by vascular endothelial growth factor-A165 (VEGF165) treatment, and shed from cell surface by treatment with tumor necrosis factor-a. RSFLs expressed VEGF and VEGF receptors (VEGFRs), and VEGF165 induced up-regulation of VE-cadherin was suppressed by inhibiting VEGFR2 activity. These data demonstrate that fibroblastic cells in RA synovium express VE-cadherin, and suggest that the fibroblastic cells are involved in formation of vasculogenic mimicry-like tubes in RA synovium through the VEGF/VEGFR2/VE-cadherin pathway.

Peroxiredoxin (Prx) 2 in red blood cells (RBCs) reacts with various reactive oxygen species and changes to hyperoxidized Prx2 (Prx2-SO2/3). Therefore, Prx2 may serve as an indicator of oxidative stress in vivo. This study aimed to analyze Prx2-SO2/3 levels in clinical samples to examine whether the oxidation state of Prx2 in human RBCs reflects the pathological condition of oxidative stress diseases. We first focused on obstructive sleep apnea (OSA), a hypoxic stress-induced disease of the respiratory system, and investigated the levels of Prx2-SO2/3 accumulated in the RBCs of OSA patients. In measurements on a small number of OSA patients and healthy subjects, levels of Prx2-SO2/3 accumulation in patients with OSA were clearly increased compared to those in healthy subjects. Hence, we proceeded to validate these findings with more samples collected from patients with OSA. The results revealed significantly higher levels of erythrocytic Prx2-SO2/3 in patients with OSA than in healthy subjects, as well as a positive correlation between the severity of OSA and Prx2-SO2/3 levels in the RBCs. Moreover, we performed a chromatographic study to show the structural changes of Prx2 due to hyperoxidation. Our findings demonstrated that the Prx2-SO2/3 molecules in RBCs from patients with OSA were considerably more hydrophilic than the reduced form of Prx2. These results implicate Prx2-SO2/3 as a promising candidate biomarker for OSA.

Diabetes mellitus (DM) is considered to be associated with an increased risk of colorectal cancer. Recent studies have also revealed that tubulin hyperacetylation is caused by a diabetic status and we have reported previously that, under microtubule hyperacetylation, a microtubule severing protein, katanin-like (KL) 1, is upregulated and contributes to tumorigenesis. To further explore this phenomenon, we tested the effects of the ketone bodies, acetoacetate and β-hydroxybutyrate, in colon and fibroblast cells. Both induced microtubule hyperacetylation that responded differently to a histone deacetylase 3 knockdown. These two ketone bodies also generated intracellular reactive oxygen species (ROS) and hyperacetylation was commonly inhibited by ROS inhibitors. In a human fibroblast-based microtubule sensitivity test, only the KL1 human katanin family member showed activation by both ketone bodies. In primary cultured colon epithelial cells, these ketone bodies reduced the tau protein level and induced KL1- and α-tubulin acetyltransferase 1 (ATAT1)-dependent micronucleation. Resveratrol, known for its tumor preventive and tubulin deacetylation effects, inhibited this micronucleation. Our current data thus suggest that the microtubule hyperacetylation induced by ketone bodies may be a causal factor linking DM to colorectal carcinogenesis and may also represent an adverse effect of them that needs to be controlled if they are used as therapeutics.

Enhanced microtubule acetylation has been identified as a negative prognostic indicator in breast cancer. We reported previously that primary cultured human mammary epithelial cells manifest breast cancer-related aneuploidization via the activation of severing protein katanin-like (KL)1 when tau is deficient. To address in this current study whether microtubule hyperacetylation is involved in breast carcinogenesis through mitosis, the effects of tubacin on human mammary epithelial cells were tested using immunofluorescence techniques. Tau-knockdown cells showed enhancement of KL1-dependent events, chromosome-bridging and micronucleation in response to tubacin. These enhancements were suppressed by further expression of an acetylation-deficient tubulin mutant. Consistently, using a rat fibroblast-based microtubule sensitivity test, it was confirmed that KL1 also shows enhanced activity in response to microtubule hyperacetylation as well as katanin. It was further observed in rat fibroblasts that exogenously expressed KL1 results in more micronucleation under microtubule hyperacetylation conditions. These data suggest that microtubule acetylation upregulates KL1 and induces more aneuploidy if tau is deficient. It is thus plausible that microtubule hyperacetylation promotes tumor progression by enhancing microtubule sensitivity to KL1, thereby disrupting spindle microtubules and this process could be reversed by the microtubule-binding and microtubule protective octapeptide NAPVSIPQ (NAP) which recruits tau to the microtubules.

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.

WD repeat-containing protein 74 (WDR74), a nucleolar-localized protein, is the mammalian ortholog of Nsa1, a 60S ribosome assembly factor in yeast. We previously showed that WDR74 associates with MTR4, the nuclear exosome-assisting RNA helicase, whose dissociation is prohibited by an ATPase-deficient mutant of the AAA-type chaperone NVL2. However, the functions and regulation of WDR74 during ribosome biogenesis in cooperation with NVL2 remains unknown. Here, we demonstrated that knockdown of WDR74 leads to significant defects in the pre-rRNA cleavage within the internal transcribed spacer 1 (ITS1), occurring in an early stage of the processing pathway. Interestingly, when the dissociation of WDR74 from the MTR4-containing exonuclease complex was impaired upon expression of the mutant NVL2, the same processing defect, with partial migration of WDR74 from the nucleolus towards the nucleoplasm, was observed. In the nucleoplasm, an increased interaction between WDR74 and MTR4 was detected by in situ proximity ligation assay. Therefore, the dissociation of WDR74 from MTR4 in a late stage of rRNA synthesis is thought to be required for appropriate maturation of the pre-60S particles. These results suggest that the spatiotemporal regulation of ribosome biogenesis in the nucleolus is mediated by the ATPase activity of NVL2.

In yeast, the Trf4/5‐Air1/2‐Mtr4 polyadenylation (TRAMP) complex acts as a cofactor for the nuclear exosome to promote degradation of various RNAs. However, the corresponding machinery in mammals is less characterized. We analyzed the interactions of the human TRAMP‐like proteins, PAPD5, ZCCHC7, and MTR4, with the nuclear exosome. PAPD5 and ZCCHC7 exhibited mutual interactions in presence of the exosome catalytic subunit RRP6, whereas MTR4 was dispensable for their assembly. Furthermore, the human TRAMP‐like proteins were involved in the RRP6‐catalyzed turnover of pre‐rRNA 5′ETS fragments. These results suggest the significant role for RRP6 in the assembly of TRAMP‐like proteins during nucleolar RNA surveillance.

Krüppel-like factor 5 (KLF5) transcriptionally controls the proliferation-differentiation balance of epithelium and is overexpressed in carcinomas. Although genomic region modifying KLF5 expression is widespread in different types of cells, the region that commonly regulates basal expression of the genes across cell-types is uncertain. In this study we determined the minimal essential region for the expression and its regulatory transcription factors using oral carcinoma cells. A reporter assay defined a 186bp region downstream of the transcription start site and a cluster of six GC boxes (GC1-GC6) as the minimal essential region. Mutation in the GC1 or GC6 regions but not other GC boxes significantly decreased the reporter expression. The decrease by the GC1 mutation was reproduced in the 2kbp full-length promoter, but not by the GC6 mutation. Additionally, specificity proteins (Sp) that can be expressed in epithelial cells and bind GC box, Sp3 co-localized with KLF5 in oral epithelium and carcinomas and chromatin immunoprecipitation analyses showed Sp3 as the prime GC1-binding protein. Inhibition of Sp-GC box binding by mithramycin A and knockdown of Sp3 by the short interfering RNA decreased expression of the reporter gene and endogenous KLF5. These data demonstrate that a 186bp region is the minimal essential region and that Sp3-GC1 binding is essential to the basal expression of KLF5.

The microtubule associated-protein tau has been identified as an effective positive prognostic indicator in breast cancer. To explore the physiological function of tau in early carcinogenesis, endogenous tau was knocked down in primary cultured human mammary epithelial cells. This resulted in chromosome-bridging during anaphase followed by micronucleation, both of which were suppressed by a further katanin-like1 knockdown. We also detected that the exogenously expressed katanin-like1 induction of cellular transformation is prevented by exogenous tau in rat fibroblasts. The mutant katanin-like1 (L123V) identified in breast cancer showed an increase in this transformation capacity as well as microtubule severing activity resistant to tau. The tau knockdown resulted in a loss of the kinetochore fibers on which tau is normally localized. This physical fragility was also observed in isolated tau-knockdown mitotic spindles, supporting the relevance of microtubule damage to the onset of transformation. The karyotyping of tau-knockdown cells showed increased frequency of loss of one X chromosome, further suggesting the involvement of tau in breast tumorigenesis. We propose that tau may contribute to tumor progression by protecting spindle microtubules from excess severing by katanin-like1. We also present data indicating that the microtubule-binding octapeptide NAP is a candidate modifier against the tau deficiency in tumor cells.

Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.

The binding of p120-catenin and β-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and β-catenin in oral carcinomas (n = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, P < 0.05; β-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; β-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; β-catenin, P < 0.01). Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-β, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-β with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of β-catenin fine-tune the carcinoma progression.

It is commonly stated that microtubules gradually disintegrate as tau becomes dissociated from them in tauopathies such as Alzheimer's disease. However, there has been no compelling evidence to date that such disintegration is due to depolymerization of microtubules from their ends. In recent studies, we have shown that neurons contain sufficient levels of the microtubule-severing protein termed katanin to completely break down the axonal microtubule array if not somehow attenuated. The presence of tau on axonal microtubules renders them notably less sensitive to katanin, prompting us to posit that microtubule disintegration in tauopathies may result from elevated severing of the microtubules as they lose tau. In support of this hypothesis, we demonstrate here that pathogenic tau mutants that bind less strongly to microtubules than wild-type tau provide correspondingly less protection against katanin-based severing. Using cultured rat hippocampal neurons, we pursued two potential therapies for fortifying axonal microtubules against excess severing by katanin, under conditions of tau depletion. We found that either deacetylating the microtubules via overexpression of HDAC6 or treating the neurons with NAP, a microtubule-interacting neuroprotective peptide, resulted in notable protection of the microtubules against katanin-based loss. In both cases, we found that these treatments also diminished the characteristic increase in axonal branching that normally accompanies tau depletion, an effect that is also known to be directly related to the severing of microtubules. These observations may be useful in developing therapeutic regimes for preserving microtubules against loss in the axons of patients suffering from tauopathies.

Here we investigated whether the sensitivity of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by katanin was increased or decreased, respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to katanin. We hypothesized that this may be attributable to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by katanin in a manner that was not dependent on the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, because the severing of microtubules by spastin does not appear to be strongly influenced by either the acetylation state of the microtubules or tau. We conclude that sensitivity to microtubule severing by katanin is regulated by a balance of factors, including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.

Comment on: Sonbuchner TM, et al. Cell Cycle 2010; 9:In this issue.

Human chromosome region 10q23-24 is one of the most frequently found regions that show loss of heterozygosity in prostate cancers. A candidate tumor suppressor LAPSER1/LZTS2 (LAPSER1) is located in 10q24.3 that has been reported to be deleted as frequently as the neighboring PTEN locus. We previously reported that LAPSER1 binds p80 katanin, a subunit of the katanin heterodimer. In this report, we show that the LAPSER1 C terminal domain inhibits katanin-mediated microtubule severing in vitro and we detected this inhibition at centrosomes by tracing the nucleated de novo, severed, and transported microtubules in cells. This functional association is also supported by the intracellular localization. Centrosomal localization of LAPSER1 was independent of microtubules and was preferential to mother centrioles. In primary cultured neurons, LAPSER1 also colocalizes with p80 katanin. LAPSER1 alters cell proliferation by regulating cytokinesis. As subcellular mechanisms that underlie the tumor suppressive activity, exogenous LAPSER1 expression inhibited central spindle formation by abrogating microtubule transportation and a similar mode of inhibition was found in axogenesis. Katanin knockdown and dominant negative inhibitor of katanin provided similar phenotypes. Prophase LAPSER1 inhibited centrosomal gamma-tubulin accumulation, which resulted in retardation of mitotic entry. Furthermore, interphase inhibition of katanin by LAPSER1 expression resulted in prevention of cell motility that was accompanied by the increased acetylated microtubules. LAPSER1 knockdown increased cell migration that was inhibited by the expression of ninein, a microtubule release inhibitor. These results indicate that microtubule severing at centrosomes is a novel tumor-associated molecular subcircuit in cells, in which LAPSER1 is a regulator.

Prostate cancer is one of the most common cancers in men, with more than 500,000 new worldwide cases reported annually, resulting in 200,000 deaths of mainly older men in developed countries. Existing treatments have not proved very effective in managing prostate cancer, and continuing efforts therefore are ongoing to explore novel targets and strategies for future therapies. LAPSER1 has been identified as a candidate tumor suppressor gene in prostate cancer, but its true functions remain unknown. We report here that LAPSER1 colocalizes to the centrosomes and midbodies in mitotic cells with gamma-tubulin, MKLP1, and p80 katanin, and is involved in cytokinesis. Moreover, RNAi-mediated disruption of LAPSER1, which is accompanied by the mislocalization of p80 katanin, results in malformation of the central spindle. Significantly, the enhanced expression of LAPSER1 induces binucleation and renders the cells resistant to oncogenic transformation. In cells transformed by the v-Fps oncogene, overexpressed LAPSER1 induces abortive cytokinesis, followed by mitotic catastrophe in a p80 katanin-dependent manner. Cells that are rescued from this apoptotic pathway with Z-VAD-fmk display karyokinesis. These results suggest that LAPSER1 participates in cytokinesis by interacting with p80 katanin, the disruption of which may potentially cause genetic instability and cancer.