IFPA Meeting 2012 Workshop Report I: comparative placentation and animal models, advanced techniques in placental histopathology, human pluripotent stem cells as a model for trophoblast differentiation.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.

Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification.

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.

Gene expression of Spag6 in chick central nervous system.

Using a differential display method, we identified sperm-associated antigen 6 (Spag6) as a gene with a dynamic expression profile within the chick embryonic spinal cord. The expression of Spag6 gradually decreased along with spinal cord development. Spag6 expression was detected adjacent to the ventricular zone of the spinal cord at embryonic day (E) 4. At E6, Spag6 was apparent in the ventral ventricular zone adjacent to floor plate and the surrounding region close to the ventricular zone, with additional weak expression at the adjacent region to the ventral horn. At E10, the Spag6 mRNA can be detected slightly in the ventral ventricular zone and surrounding region of dorsal ventricular zone. In the E6 hindbrain, Spag6 was detected in the roof, the ventricular zone adjacent to floor plate and the surrounding regions of the ventricular zones. In the E6 caudal diencephalon, Spag6 expression was detected adjacent to the ventricular zone. As Spag6 was expressed in areas containing ependymal progenitor cells and in the borders of undifferentiated regions, Spag6 may be involved in the development of ependymal cells and in the differentiation process of neuronal cells in chick neural organs.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer.

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

[RNA-interference, induced by transient and continuous expression of hairpin RNA in cells from Drosophila and mammals]. / RNK-interferentsiia, indutsirovannaia vremennoi i postoiannoi ékspressiei shpilechnoi RNK, v kletkakh Drosophila i mlekopitaiushchikh.

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system, RNAi activity was found to vary depending on IR length ass well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

Participation of intracellular Ca(2+)/calmodulin and protein kinase(s) in the pathway of apoptosis induced by a Drosophila cell death gene, reaper.

To elucidate the apoptotic signaling pathway, we have generated a cell culture model: S2 cells stably transfected with a Drosophila cell death gene, reaper (rpr). Following rpr overexpression, caspase activation-mediated apoptotic cell death was induced in the cells. Apoptosis triggered by rpr required intracellular Ca(2+) ions and calmodulin. Furthermore, protein kinase inhibitors H-7 (a PKC, PKA, PKG, MLCK, and CKI inhibitor), calphostin C (a PKC inhibitor), or H-89 (a PKA and PKG inhibitor) completely blocked apoptosis induced by rpr, suggesting that some kind of serine/threonine protein kinase(s) act upstream of caspase in apoptotic pathway induced by rpr in S2 cells.

Molecular cloning of SCDGF-B, a novel growth factor homologous to SCDGF/PDGF-C/fallotein.

Spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein has a unique two-domain structure, as it contains two regions homologousto CUB and PDGF/vascular endothelial growth factor (VEGF) domains. In this study, we isolateda novel gene homologous to SCDGF/PDGF-C/fallotein, and named SCDGF-B. The culture supernatant of CHO-K1 cells stably transfected with SCDGF-B showed mitogenic activity as SCDGF/PDGF-C/fallotein did. Although SCDGF-B and SCDGF/PDGF-C/fallotein might be the members of the PDGF/VEGF superfamily of growth factors, they were categorized into a new subfamily in addition to PDGF and VEGF subfamilies.

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO-K1) as well, although double-stranded RNA required for the latter was 2500 times more than for the former. Cognate double-stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

Formation of the male-specific muscle in female Drosophila by ectopic fruitless expression.

The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex-determination factor that directs the development of at least two male-specific characteristics, namely courtship behaviour and formation of the muscle of Lawrence (MOL). The fru gene encodes a putative transcription factor with a BTB domain and two zinc-finger motifs, and with consensus Tra-binding sequences. The binding of Tra to these sequences results in sex-specific alternative splicing of the fru mRNA, leading to production of the 'male-type' or 'female-type' Fru protein. We show here that the Fru protein is not detected in the female central nervous system (CNS), despite the similar level of expression of fru mRNA in both male and female CNS. As ectopic expression of both the 'male-type' (with the sequence for the amino-terminal extension) and 'female-type' (without the sequence for the amino-terminal extension) fru cDNA can induce formation of the MOL in females, the presence or absence of the Fru protein, and not its sex-specific structure, seems to be responsible for the sexually dimorphic actions of the fru gene.

A novel gene derived from developing spinal cords, SCDGF, is a unique member of the PDGF/VEGF family.

We isolated a novel gene designated spinal cord-derived growth factor (SCDGF). Its expression was increased in chick spinal cords with embryonic development and decreased after hatching. The amino acid sequences of chick and human SCDGFs revealed a putative signal sequence followed by a CUB domain and a region homologous to the members of the platelet-derived growth factor/vascular endothelial growth factor family. Furthermore, human SCDGF secreted from the cells showed a mitogenic activity for 10T1/2 cells in vitro. These results led us to speculate that SCDGF plays an important role in the development of the spinal cord.

CDK inhibitors suppress apoptosis induced by chemicals and by excessive expression of a cell death gene, reaper, in Drosophila cells.

The present study was aimed to investigate whether or not cyclin-dependent kinases (CDKs) participate in different cascades leading to apoptosis. We examined the effects of two CDK inhibitors, olomoucine (OLM) and butyrolactone-I (BL-I), on apoptosis induced in two kinds of Drosophila cell lines. Increases of caspase activity induced by actinomycin D, cycloheximide, H-7 or A23187 in a Drosophila neuronal cell line, ML-DmBG2-c2, and induced by excessive expression of a Drosophila cell death gene, reaper, in Drosophila S2 cells were suppressed by 24-h pretreatment of each CDK inhibitor. Concomitant with the suppression of the caspase activity, fragmentations of cells and DNA, representatives of apoptosis, were also inhibited. These results suggest that CDK(s) participates in progression of apoptosis. However, these effects of the CDK inhibitors were also observed even at lower doses which did not affect cell proliferation. Therefore, it was shown that apoptosis is not always related to cell cycle in Drosophila cells. It was also suggested that the target(s) of the CDK inhibitors locates upstream of caspase in the cascade(s) of apoptosis.

Calmodulin-dependent and -independent apoptosis in cell of a Drosophila neuronal cell line.

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca2+ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca2+ ions, although the origin of Ca2+ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca2+ ions whereas those by the other three chemicals utilized intracellular Ca2+ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).

Adhesion-dependent tyrosine phosphorylation of enabled in Drosophila neuronal cell line.

Cell culture consisting of Drosophila BG2-c6 cells and laminin revealed its value for the analysis of the integrin-mediated activity of extracellular matrix (Takagi, Y., et al. (1998) Neurosci. Lett. 244, 149-152). To elucidate Drosophila integrin cascade further, we report here our characterization on the tyrosine phosphorylation in BG2-c6 cells, coupling with their spreading on extracellular matrix. Large-scale culture of Drosophila Kc167 cells provided a sufficient amount of extracellular matrix (including laminin) for performing biochemical analysis on the signal transduction in BG2-c6 cells. Several proteins underwent significant tyrosine phosphorylation in an adhesion-dependent manner. Among them, the heavy phosphorylation of Enabled (a substrate for Abelson tyrosine kinase) was noteworthy because of the proposed function of Enabled in cell adhesion. Together with our previous results, we propose a model for signal transduction activated by cell adhesion for the first time in Drosophila.

Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants.

In Drosophila, two Frizzled proteins, Frizzled and Dfrizzled-2, have been reported to serve as receptors of Wingless. Here, we identified the third member of the Drosophila Frizzled family (Dfrizzled-3). In contrast to Dfrizzled-2, Dfrizzled-3 was transcriptionally upregulated by Wingless signaling. Although Dfrizzled-3 was capable of binding to Wingless in vitro, Wingless-dependent Armadillo/beta -catenin stabilization occurred much less effectively in Drosophila cells transfected with Dfrizzled-3 than in those with Dfrizzled-2. Flies lacking Dfrizzled-3 activity were viable and fertile, with few morphological defects. Genetic and immunochemical analysis indicated that the absence of Dfrizzled-3 activity suppresses the effects of hypomorphic wingless mutations such as failure of wing and antenna formation and restores target gene expression to the normal levels without change in wingless expression. Wingless signaling may thus be attenuated by Dfrizzled-3 at least in wingless hypomorphic mutants.

H-7-induced apoptosis in the cells of a Drosophila neuronal cell line through affecting unidentified H-7-sensitive substance(s).

The present study was undertaken to reveal underlying mechanisms of apoptosis in neurons using clonal neuronal cells, ML-DmBG2-c2, derived from Drosophila larval central nervous system 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, induced cell death with typical features of apoptosis such as internucleosomal DNA fragmentation, nuclear condensation and apoptotic bodies in the cells. Though H-7 is known to inhibit cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cGMP-dependent protein kinase (PKG), myosin light chain kinase (MLCK), and casein kinase I (CKI), specific inhibitors for these kinases such as H-89, calphostin C, ML-9, or CKI-7 did not induce apoptosis in the cells. Other kinases such as tyrosine kinase. PI3-kinase and Ca2+/CaM kinase II so far examined in the present study were interpreted not to be involved in the apoptotic cascade. Therefore, it is concluded that an H-7-sensitive substance(s) other than these kinases is responsible for the apoptosis in the neuronal cells. Caspase inhibitors prevented apoptosis in the cells treated with H-7. These results suggest that caspase(s) is involved downstream of the H-7-sensitive point in the cascade of the apoptosis.

Laminin-dependent integrin clustering with tyrosine-phosphorylated molecules in a Drosophila neuronal cell line.

To gain more insight into the molecular and cellular aspects of basement membranes during Drosophila morphogenesis, especially in neural development, we carried out cell biological screening to establish a cell culture system in which Drosophila cell-matrix interaction could be reconstituted. The screening showed that a Drosophila neuronal cell line, BG2-c6, established from the third-instar larval central nervous system, had a strong adhesion activity when purified Drosophila laminin was used as a substrate. Outgrowth of axon-like structures was stimulated on laminin. Histochemical analysis revealed clusters of integrin together with phosphotyrosine and alpha-actinin. These data indicate that the Drosophila integrin cascade triggered by the interaction between BG2-c6 and laminin was initiated at the integrin cluster with tyrosine-phosphorylated proteins, similar to the observations in vertebrate cells.

Induction of apoptosis in a Drosophila neuronal cell line by calcium ionophore.

Apoptotic type cell death was induced by the calcium ionophore, A23187, in a Drosophila CNS derived cell line, ML-DmBG2-c2. It was judged by the ultrastructural observations and internucleosomal DNA fragmentation, typical characteristics of apoptosis. The ionophore failed to induce apoptosis in Ca2+- free medium, which suggests that the increase of intracellular Ca2+ concentration by the treatment of A23187 triggers apoptosis in the clonal cells.

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system, II: Neuropeptides and amino acids.

In a previous study, we have found acetylcholine and/or L-DOPA in 10 colonial clones from one cell line of Drosophila larval central nervous system (CNS). In this study, to characterize clonal neuronal phenotypes further, we have examined three neuropeptides and 19 amino acids using HPLC system. Substance P and proctolin were found in seven and eight out of ten clones, respectively. On the other hand, somatostatin was expressed in all ten clones. GABA and taurine were not detected in any clones. Glutamate, which is an excitatory transmitter in Drosophila, was found in all the clones, although its content was different seven times among them. Glycine, which is not known as a transmitter in Drosophila, was found to be unevenly expressed among them. Therefore, the conspicuous expression of peptides or amino acids in some clones suggests that the substances have a special role in Drosophila CNS.