Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

Background

Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Principal Findings

We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions

We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.     

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity

The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucleoside diphosphates from the nucleotide pool and prevents their toxic effects.     

In vitro growth and single-leaf photosynthetic response of Cymbidium plantlets to super-elevated CO2 under cold cathode fluorescent lamps

To examine the effectiveness of super-elevated (10,000 μmol mol⁻¹) CO₂ enrichment under cold cathode fluorescent lamps (CCFL) for the clonal propagation of Cymbidium, plantlets were cultured on modified Vacin and Went (VW) medium under 0, 3,000 and 10,000 μmol mol⁻¹ CO₂ enrichment and two levels of photosynthetic photon flux density (PPFD, 45 and 75 μmol m⁻² s⁻¹). Under high PPFD, 10,000 μmol mol⁻¹ CO₂ increased root dry weight and promoted shoot growth. In addition, a decrease in photosynthetic capacity and chlorosis at leaf tips were observed. Rubisco activity and stomatal conductance of these plantlets were lower than those of plantlets at 3,000 μmol mol⁻¹ CO₂ under high PPFD, which had a higher photosynthetic capacity. On the other hand, plantlets on Kyoto medium grown in 10,000 μmol mol⁻¹ CO₂ under high PPFD had a higher photosynthetic rate than those on modified VW medium; no chlorosis was observed. Furthermore, growth of plantlets, in particular the roots, was remarkably enhanced. This result indicates that a negative response to super-elevated CO₂ under high PPFD could be improved by altering medium components. Super-elevated CO₂ enrichment of in vitro-cultured Cymbidium could positively affect the efficiency and quality of commercial production of clonal orchid plantlets.     

Genetic diversity and structure of natural fragmented Chamaecyparis obtusa populations as revealed by microsatellite markers

The genetic diversity and population structure of hinoki (Chamaecyparis obtusa) in Japan were investigated by examining the distribution of alleles at 13 microsatellite loci in 25 natural populations from Iwaki in northern Japan to Yakushima Island in southern Japan. On average, 26.9 alleles per locus were identified across all populations and 4.0% of the genetic variation was retained among populations (G _ST = 0.040). According to linkage disequilibrium analysis, estimates of effective population size and detected evidence of bottleneck events, the genetic diversity of some populations may have declined as a result of fragmentation and/or over-exploitation. The central populations located in the Chubu district appear to have relatively large effective population sizes, while marginal populations, such as the Yakushima, Kobayashi and Iwaki populations, have smaller effective population sizes and are isolated from the other populations. Microsatellite analysis revealed the genetic uniqueness of the Yakushima population. Although genetic differentiation between populations was low, we detected a gradual cline in the genetic structure and found that locus Cos2619 may be non-neutral with respect to natural selection.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

Fluvoxamine and sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate induces the Ser473-phosphorylation of Akt-1 in PC12 cells

AimsThe expression of brain-derived neurotrophic factor (BDNF) may be a downstream target of a variety of antidepressant treatments, and selective serotonin reuptake inhibitors (SSRIs) are used clinically for the treatment of depression. BDNF binds to and activates tyrosine kinases receptor (TrkB) to exert its effects. TrkB, after activation by ligands, stimulates phosphoinositide 3-kinase (PI3K). The downstream target of PI3K is Akt-1, a serine-threonine kinase. BDNF has signaling through the PLC-?IP₃/Ca²⁺ pathway. Furthermore, the PLC-?γ/IP₃/Ca²⁺ pathway is regulated by the sigma-1 receptors. Here, we examined whether fluvoxamine (FLV) activated Akt-1 and increased phosphorylation of Akt-1 via sigma-1 receptor in PC12 cells.Main methodsWe examined the effect of the SSRI, FLV and BDNF on the phosphorylation levels of serine-threonine kinase Akt-1 in PC12 cells using immunoblotting techniques.Key findingsTreatment with 10 μM and 100 μM FLV of PC12 cells stimulated a 2.4- and 3.8-fold maximal increase in Ser⁴⁷³-phosphorylated Akt-1 levels at 40 min, respectively. Treatment with 50 ng/ml BDNF also stimulated Ser⁴⁷³ -phosphorylated Akt-1 by 2.6-fold with a maximal increase at 5 min. In addition, the phosphorylation induced by FLV and BDNF was blocked by LY294002, a selective inhibitor of PI3K. The sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate also stimulated a 2.1-fold increase in the level of Ser473-phosphorylated Akt-1.SignificanceThis study demonstrates that fluvoxamine treatment rapidly increased phosphorylation of Akt-1. And BDNF activated Akt-1 phosphorylation by the TrkB/PI3K/Akt-1 pathway. We conclude that the phosphorylation of Akt-1, downstream of PI3K, was the key to their antidepressant effects.     

Apoptotic effect of ganciclovir on primary effusion lymphoma cells infected with Kaposi's sarcoma-associated herpesvirus

We evaluated the cytotoxic and apoptotic effects of two purine nucleoside analogues, acyclovir (ACV) and ganciclovir (GCV), on lymphoma cells stably harboring Kaposi's sarcoma-associated herpesvirus (KSHV). Colorimetric caspase assay, flow cytometry, and immunoblotting with antibodies against apoptosis-related molecules revealed that GCV has cytotoxic activity toward KSHV-infected primary effusion lymphoma cells, while ACV has weak or little activity. In addition to the GCV-induced cytotoxicity, apoptosis via caspase-7/8, cleavage of poly(ADP-ribose) polymerase, and accumulation of p53 and p21 were induced by GCV treatment. In contrast, neither ACV nor GCV have cytotoxicity- or apoptosis-inducing activities toward uninfected cells.     

Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae

L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.     

Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.