Loss of putzig in the germline impedes germ cell development by inducing cell death and new niche like microenvironments.

Germline stem cell development and differentiation is tightly controlled by the surrounding somatic cells of the stem cell niche. In Drosophila females, cells of the niche emit various signals including Dpp and Wg to balance stem cell renewal and differentiation. Here, we show that the gene pzg is autonomously required in cells of the germline to sustain the interplay between niche and stem cells. Loss of pzg impairs stem cell differentiation and provokes the death of cells in the germarium. As a consequence of pzg loss, increased growth signalling activity predominantly of Dpp and Wg/Wnt, was observed, eventually disrupting the balance of germ cell self-renewal and differentiation. Whereas in the soma, apoptosis-induced compensatory growth is well established, the induction of self-renewal signals during oogenesis cannot compensate for dying germ cells, albeit inducing a new niche-like microenvironment. Instead, they impair the further development of germ cells and cause in addition a forward and feedback loop of cell death.

Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene.

The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.

Improved selectivity of mIBG uptake into neuroblastoma cells in vitro and in vivo by inhibition of organic cation transporter 3 uptake using clinically approved corticosteroids.

INTRODUCTION: Radiolabeled meta-iodobenzylguanidine (mIBG) is used for imaging and therapy of neuroblastoma as well as pheochromocytoma. However, non-tumorous tissues also incorporate mIBG mainly by organic cation transporters (OCTs). In this study, we tested different clinically approved corticosteroids as potential inhibitors of the OCT3-mediated uptake in vitro and in vivo, to achieve a more selective mIBG tumor uptake. METHODS: The in vitro incorporation of [(3)H]norepinephrine ([(3)H]NE), [(3)H]dopamine ([(3)H]DA) and [(123)I]mIBG in neuroblastoma cells (SK-N-SH, Kelly, IMR-32) and in HEK-293 cells transfected with human OCT3 was measured with and without supplemental corticosteroids (hydrocortisone, prednisolone, dexamethasone, corticosterone). The in vivo biodistribution of [(123)I]mIBG in absence and presence of corticosteroids was studied in non-tumor bearing NOD scid gamma mice. Retrospectively, we selected patients with and without corticosteroid treatment prior to [(123)I]mIBG scintigraphy. RESULTS: A concentration-dependent inhibitory effect of different corticosteroids on the [(3)H]NE and [(3)H]DA uptake via OCT3 was illustrated in vitro. The highest OCT3 inhibition was observed for corticosterone, but clinically used corticosteroids, showed also promising inhibitory effects. In contrast, the uptake in neuroblastoma cells was reduced only moderately. Hydrocortisone or prednisolone had only minor effects on [(123)I]mIBG uptake of both neuroblastoma cells, but reduced uptake in OCT3 expressing cells significantly. In mice tissues, [(123)I]mIBG uptake was inhibited by corticosteroids especially in the small intestine and kidney. Finally, in one patient with hydrocortisone treatment performed prior to [(123)I]mIBG scan, heart and liver uptake was reduced compared to untreated patients. CONCLUSIONS: The OCT3 is widely spread in many organs and responsible for non-targeted uptake of radiolabeled mIBG. In our study, clinically approved corticosteroids inhibited mIBG uptake in OCT3 expressing cells effectively, whereas tracer accumulation in NT (norepinephrine transporter) expressing neuroblastoma cells showed consistency. We conclude, that a single dose of hydrocortisone or prednisolone prior to [(123)I]mIBG scintigraphy may improve specificity and reduce radiation dose to non-target organs.

Synthesis and biological effects of new hybrid compounds composed of benzylguanidines and the alkylating group of busulfan on neuroblastoma cells.

(131)Iodine-labelled (meta-iodobenzyl)guanidine ([(131)I]-mIBG) and busulfan [butane-1,4-diylbis(methanesulfonate)] are well-established pharmaceuticals in neuroblastoma therapy. We report the design, synthesis, and testing of hybrid molecules-mBBG and pBBG-which combine key structural features of (meta-iodobenzyl)guanidine and busulfan: they contain a benzylguanidine moiety for accumulating in neuroblastoma cells via the noradrenaline transporter and, in the meta- or para-position, respectively, one of the two identical alkylating motives of busulfan for killing cells. Uptake and toxicity of hybrids mBBG and pBBG in human neuroblastoma cells compared favorably to their ancestors [(131)I]-mIBG and busulfan.

A dual-inhibition study on vascular smooth muscle cells with meclofenamic acid and ß-irradiation for the prevention of restenosis.

PURPOSE: Restenosis is still one of the major limitations after angioplasty. A therapeutic treatment combining ß-irradiation and pharmacologic cyclooxygenase-2 inhibition was employed to study the impact on vascular smooth muscle cells (SMCs). MATERIALS AND METHODS: The effects of meclofenamic acid in combination with yttrium-90 ((90)Y) on cell growth, clonogenic activity, cell migration, and cell cycle distribution of human aortic SMCs were investigated. Treatment was sustained over a period of 4 days and recovery of cells was determined until day 20 after initiation. The hypothesis was that there is no difference between control and treated groups. RESULTS: A dose-dependent growth inhibition was observed in single and combined treatment groups for meclofenamic acid and ß-irradiation. Cumulative radiation dosage of 8 Gy completely inhibited colony formation. This was also observed for 200 µM meclofenamic acid alone or in combination with minor ß-irradiation dosages. Results of the migration tests showed also a dose dependency with additive effects of combined therapy. Meclofenamic acid 200 µM alone and with cumulative ß-irradiation dosages resulted in an increased G2/M-phase share. CONCLUSIONS: Incubating human SMCs with meclofenamic acid and (90)Y for a period of 4 d (ie, 1.5 half-life times) resulted in an effective inhibition of smooth muscle cell proliferation, colony formation, and migration.

Uptake of mIBG and catecholamines in noradrenaline- and organic cation transporter-expressing cells: potential use of corticosterone for a preferred uptake in neuroblastoma- and pheochromocytoma cells.

For imaging of neuroblastoma and phaeochromocytoma, [(123)I]meta-iodobenzylguanidine ([(123)I]mIBG) is routinely used, whereas [(18)F]6-fluorodopamine ([(18)F]6-FDA) is sporadically applied for positron emission tomography in pheochromocytoma. Both substances are taken up by catecholamine transporters (CATs). In competition, some other cell types are able to take up catecholamines and related compounds probably by organic cation (OCT) [extraneuronal monoamine (EMT)] transporters (OCT1, OCT2, OCT3=EMT). In this study, we investigated the uptake of radioiodine-labeled meta-iodobenzylguanidine (mIBG) as well as [(3)H]dopamine (mimicring 6-fluorodopamine) and [(3)H]noradrenaline. SK-N-SH (neuroblastoma) and PC-12 (phaeochromocytoma) cells were used and compared with HEK-293 cells transfected with OCT1, OCT2 and OCT3, respectively. In order to gain a more selective uptake in CAT expressing tumor cells, different specific inhibitors were measured. Uptake of mIBG into OCT-expressing cells was similar or even better as into both CAT-expressing cell lines, whereas dopamine and noradrenaline uptake was much lower in OCT-expressing cells. In presence of corticosterone (f.c. 10(-4) M], catecholamine and mIBG uptake into SK-N-SH and PC-12 cells was only slightly reduced. In contrast, this process was significantly inhibited in OCT2 and OCT3 transfected HEK-293 as well as in Caki-1 cells, which naturally express OCT3. We conclude that the well-known corticosteroid corticosterone might be used in combination with [(18)F]6-FDA or [(123)I]mIBG to improve specific imaging of neuroblastoma and pheochromocytoma and to reduce irradiation dose to nontarget organs in [(131)I]mIBG treatment.