dictyNews
Electronic Edition
Volume 41, number 12
June 12, 2015

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=========
Abstracts
=========


Derivatives of Dictyostelium differentiation-inducing factors inhibit 
lysophosphatidic acid–stimulated 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 (5–20 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 [[log in to unmask]] 
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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, Universität Kassel, Kassel,  Germany
#a Current address: Dept. of Biology, Brawijaya University, Malang, 
East Java, Indonesia

* Corresponding author 
E-Mail: [log in to unmask]

¶ 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 DIRS–1 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 – 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 5’-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 [[log in to unmask]] 
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[End dictyNews, volume 41, number 12]