Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

Background

Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.

Results

With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.

Conclusions

The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.     

Rock weathering creates oases of life in a High Arctic desert

During primary colonization of rock substrates by plants, mineral weathering is strongly accelerated under plant roots, but little is known on how it affects soil ecosystem development before plant establishment. Here we show that rock mineral weathering mediated by chemolithoautotrophic bacteria is associated to plant community formation in sites recently released by permanent glacier ice cover in the Midtre Lovénbreen glacier moraine (78°53′N), Svalbard. Increased soil fertility fosters growth of prokaryotes and plants at the boundary between sites of intense bacterial mediated chemolithotrophic iron‐sulfur oxidation and pH decrease, and the common moraine substrate where carbon and nitrogen are fixed by cyanobacteria. Microbial iron oxidizing activity determines acidity and corresponding fertility gradients, where water retention, cation exchange capacity and nutrient availability are increased. This fertilization is enabled by abundant mineral nutrients and reduced forms of iron and sulfur in pyrite minerals within a conglomerate type of moraine rock. Such an interaction between microorganisms and moraine minerals determines a peculiar, not yet described model for soil genesis and plant ecosystem formation with potential past and present analogues in other harsh environments with similar geochemical settings.     

A new simple HPLC method for measuring mitotane and its two principal metabolites Tests in animals and mitotane-treated patients

A new C18 reversed-phase column and UV HPLC method for the detection of mitotane, its principal metabolites, dichlorodiphenylacetate and dichlrodiphenylethene, and its precursor DDT is described. In this article mitotane, dichlorodiphenylacetate, and dichlrodiphenylethene concentrations in organs of rats fed on a mitotane diet, and the effects of erythromycin and grapefruit juice as cytochrome P450 common inhibitors are presented. Tissue accumulation of mitotane and dichlrodiphenylethene, the acquired ability to eliminate dichlorodiphenylacetate, and inhibition of beta-hydroxylation by both inhibitors are illustrated here. Blood samples from mitotane-treated patients revealed two correlations: plasma mitotane/dichlrodiphenylethene and plasma mitotane/red cell mitotane.     

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.     

Cdc42 GTPase-activating proteins (GAPs) regulate generational inheritance of cell polarity and cell shape in fission yeast

The highly conserved small GTPase Cdc42 regulates polarized cell growth and morphogenesis from yeast to humans. We previously reported that Cdc42 activation exhibits oscillatory dynamics at cell tips of Schizosaccharomyces pombe cells. Mathematical modeling suggests that this dynamic behavior enables a variety of symmetric and asymmetric Cdc42 activation distributions to coexist in cell populations. For individual wild-type cells, however, Cdc42 distribution is initially asymmetrical and becomes more symmetrical as cell volume increases, enabling bipolar growth activation. To explore whether different patterns of Cdc42 activation are possible in vivo, we examined S. pombe rga4∆ mutant cells, lacking the Cdc42 GTPase-activating protein (GAP) Rga4. We found that monopolar rga4∆ mother cells divide asymmetrically leading to the emergence of both symmetric and asymmetric Cdc42 distributions in rga4∆ daughter cells. Motivated by different hypotheses that can mathematically reproduce the unequal fate of daughter cells, we used genetic screening to identify mutants that alter the rga4∆ phenotype. We found that the unequal distribution of active Cdc42 GTPase is consistent with an unequal inheritance of another Cdc42 GAP, Rga6, in the two daughter cells. Our findings highlight the crucial role of Cdc42 GAP localization in maintaining consistent Cdc42 activation and growth patterns across generations.     

Allosteric modulation by single enantiomers of a C3-chiral 1,4-benzodiazepine of the gamma aminobutyric acid type A receptor channel expressed in Xenopus oocytes

Xenopus laevis oocytes injected with Poly(A)⁺-RNA isolated from neuronal tissue express membrane proteins peculiar to the origin of mRNA. The translation of gamma aminobutyric acid type A (GABA_A) receptors has been shown by dose/ response behavior of GABA and the reversible blockade of the GABA-induced current by picrotoxin. This current was analyzed quantitatively under two electrode voltage-clamp conditions. This methodology has been applied for the first time to study the functional properties of the receptor as a function of the stereochemistry of the ligands. The (+)-S and (-)-R enantiomers of a water-soluble benzodiazepine derivative, 7-chloro-1,3-dihydro-3-hemisuccinyloxy-5-phenyl-1,4-benzodiazepin-2-one (OXHEM), obtained by preparative high performance liquid chromatographic (HPLC) resolution on chiral stationary phase, act as agonists in the in vitro modulation of the chloride channel. The (+)-S-OXHEM enantiomer was the more active. Chirality 9:286–290, 1997. © 1997 Wiley-Liss, Inc.     

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.     

Benzodipyrazoles: a new class of potent CDK2 inhibitors

The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible lead development. Following a medicinal chemistry program aimed at improving cell permeability and selectivity, a series of compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit tumor cell proliferation has been obtained.     

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

We have previously shown that platelet-activating factor (PAF), a naturally occurring lipid mediator of cell-to-cell communication, was produced by 3-day-old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)-insensitive cholinephosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT-insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT-insensitive cholinephosphotransferase and acetyltransferase activities were present in 8-day-old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT-insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT-insensitive cholinephosphotransferase, but not acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)