The small GTPase RhoH is an atypical regulator of haematopoietic cells

Rho GTPases are a distinct subfamily of the superfamily of Ras GTPases. The best-characterised members are RhoA, Rac and Cdc42 that regulate many diverse actions such as actin cytoskeleton reorganisation, adhesion, motility as well as cell proliferation, differentiation and gene transcription. Among the 20 members of that family, only Rac2 and RhoH show an expression restricted to the haematopoietic lineage

The deubiquitinating enzyme USP17 is essential for GTPase subcellular localization and cell motility

Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease

No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain, Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT

BACKGROUND: It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257-264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT. METHODS: In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study. RESULTS: Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP. CONCLUSION: No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT

Primers used for GIPFEL.

<p>Primers used for GIPFEL.</p

Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL), a New Method to Detect Translocations in Leukemia

<div><p>Here we present a novel method “Genomic inverse PCR for exploration of ligated breakpoints” (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10⁻⁴ dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.</p></div

Examples of GIPFEL results.

<p>A. Sensitivity test. Circularized genomic DNA was produced from MV4;11 cells a cell line with a known t(4;11) translocation and from HL60 cells as “non-translocation” control as well as from various mixtures “diluting” MV4;11 cells in a population of HL60 as indicated. GIPFEL was performed and real-time amplification curves are shown. B. As in “A” with REH t(12;21) cells and 697 cells instead of HL60 cells. C. As in “A” with 697 t(1;19) and REH cells. D. Example for a GIPFEL result using patient DNA. Upper panel: Amplification chart of a typical GIPFEL experiment with patient DNA. Amplification is achieved with the genomic MLL control primer and a translocation specific primer pair. Lower panel: Agarose gel electrophoresis of the 8 individual secondary PCRs interrogating the (4;11) breakpoint region. E. Results presented as in “D” for a t(12;21) breakpoint. F. Results for a t(1;19) patient sample.</p

Flow chart of the GIPFEL procedure.

<p>A. Biochemical steps for enrichment of circularized DNA. The products of a restriction enzyme (E) digest of genomic material are column purified and ligated in a large volume. Subsequently exonuclease III (presented in yellow) removes remaining linear fragments allowing enrichment for circularized DNA. B. PCR strategy to detect the presence of translocation specific circles. Primer pairs are designed that cover all possible ligation joints of translocation specific ligation products. Semi-nested PCR is performed first with an outer primer corresponding to the 5′ portion of the fusion and pools of downstream primers. The PCR products from these reactions are used as templates for secondary PCRs using a 5′ inner primer and the same downstream primers, yet in different combinations. A control PCR amplifies a ligation joint created from wild-type cells. C. Decision tree for scoring of GIPFEL results.</p

Basic principle of GIPFEL.

<p>Upon restriction digest and circularization of genomic DNA only genomic DNA from translocation bearing cells will form circles that join DNA of two different chromosomes. The junction is predetermined by the location of the genomic breakpoint. By probing for all possible ligation junctions with PCR the presence of a translocation can be ascertained.</p