dictyNews Electronic Edition Volume 41, number 12 June 12, 2015 Please submit abstracts of your papers as soon as they have been accepted for publication by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acidÐstimulated migration of murine osteosarcoma LM8 cells Yuzuru Kubohara*, Mayumi Komachi, Yoshimi Homma, Haruhisa Kikuchi, Yoshiteru Oshima * Corresponding author: Juntendo University Graduate School of Health and Sports Science, Inzai 270-1695, Japan. BBRC, in press Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti- metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), -2, and -3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5Ð20 microM) dose-dependently suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 microM, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 microM, respectively, in LM8 cells, and >20, 14.8, and 4.3 microM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. Submitted by Yuzuru Kubohara [ykuboha@juntendo.ac.jp] ---------------------------------------------------------------------- A simple retroelement based knock-down system in Dictyostelium: further insights into RNA interference mechanisms. Michael Friedrich1¦, Doreen Meier1¦, Isabelle Schuster1 and Wolfgang Nellen1, #a* 1Abt. Genetik, FB 10, Universitaet Kassel, Kassel, Germany #a Current address: Dept. of Biology, Brawijaya University, Malang, East Java, Indonesia * Corresponding author E-Mail: nellen@uni-kassel.de ¦ These authors contributed equally to this work. PLOS ONE, accepted Characteristics of DIRS-1 mediated knock-downs We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRSÐ1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained, the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. Advantages of the DIRS-1 Ð RNAi system The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5Õ-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes. Submitted by Wolfgang Nellen [nellen@uni-kassel.de] ============================================================== [End dictyNews, volume 41, number 12]