BACKGROUND Inhibition of signal transduction pathways has been successfully introduced into cancer treatment. The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 has antitumor activity in vitro against solid tumors. Here, we examined the activity of NVP-BEZ235 in acute lymphoblastic leukemia (ALL) cells and the best modalities for combination approaches. MATERIALS AND METHODS ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were treated with NVP-BEZ235 alone, or in combination with cytarabine (AraC), doxorubicin (Doxo) or dexamethasone (Dexa). RESULTS NVP-BEZ235 potently inhibited the proliferation and metabolic activity of ALL cells. Antiproliferative effects were associated with G(0)/G(1) arrest and reduced levels of cyclin-dependent kinase 4 (CDK4) and cyclin D3. Inhibition of PI3K and mTOR activity was detected at 10 and 100 nM. NVP-BEZ235 combined with AraC, Doxo or Dexa synergistically enhanced the cytotoxicity compared to single-drug treatment, even in glucocorticoid-resistant cells. CONCLUSION NVP-BEZ235 displays pronounced antiproliferative effects in ALL cells and might therefore be a useful drug in the treatment of ALL.

During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood.

How the visual cortex responds to specific stimuli is strongly influenced by visual experience during development. Monocular deprivation, for example, changes the likelihood of neurons in the visual cortex to respond to input from the deprived eye and reduces its visual acuity. Because these functional changes are accompanied by extensive reorganization of neurite morphology and dendritic spine turnover, genes regulating neuronal morphology are likely to be involved in visual plasticity. In recent years, Notch1 has been shown to mediate contact inhibition of neurite outgrowth in postmitotic neurons and implicated in the pathogenesis of various degenerative diseases of the CNS. Here, we provide the first evidence for the involvement of neuronal Notch1 signaling in synaptic morphology and plasticity in the visual cortex. By making use of the Cre/Lox system, we expressed an active form of Notch1 in cortical pyramidal neurons several weeks after birth. We show that neuronal Notch1 signals reduce dendritic spine and filopodia densities in a cell-autonomous manner and limit long-term potentiation in the visual cortex. After monocular deprivation, these effects of Notch1 activity predominantly affect responses to visual stimuli with higher spatial frequencies. This results in an enhanced effect of monocular deprivation on visual acuity.

Mycophenolate mofetil (MMF) has been used successfully in solid organ transplantation (SOT) and more recently in nonmyeloablative hematopoietic stem cell transplantation (HSCT) for prophylaxis of graft rejection and acute graft-versus-host disease. However, the pharmacokinetics of MMF seem to differ when applied in HSCT compared to SOT. Here, we analyzed pharmacokinetics of mycophenolic acid (MPA), the active metabolite of MMF, in a nonmyeloablative canine HSCT model. Dogs received nonmyeloablative TBI for conditioning followed by leukocyte antigen-identical littermate HSCT and immunosuppression containing cyclosporin A (CsA) and different doses of MMF. Pharmacokinetics were performed on days 2, 14 and 27. Dose escalation of MMF from 10 to 30 mg/kg tended to increase area under the curve (AUC) and the apparent oral clearance by 45 and 110%, respectively. Doses applied had no linear association with MPA concentration or blood trough level. No significant drug accumulation occurred over time. Using a twice daily MMF regimen, we conclude that an AUC of 30-60 mug/ml h as recommended for SOT cannot be reached in HSCT. Toxicities did not permit single doses higher than 30 mg/kg. Thus, if larger AUCs are desired in order to assure sufficient immunosuppression in HSCT, MMF might have to be administered at least three times daily.Bone Marrow Transplantation advance online publication, 17 December 2007; doi:10.1038/sj.bmt.1705958.

A single session of foot shock stress produces stable and long lasting sensitization of behavioral, hormonal and intestinal motility responses to novel stressful stimuli in laboratory rats. This is reflected in increased expression of the activity marker protein Fos in brain areas involved, following an external stressor. We present data from awake, freely moving rats in which a silicone balloon was surgically implanted in the duodenum. Firstly, cardiovascular reflexes to distentions were studied using telemetry with surgically implanted transmitters, 2 weeks after a single, 15-min session of foot shocks. The distentions caused characteristic, bi-phasic responses in both mean arterial blood pressure and heart rate that were not different between preshocked and control animals. Secondly, the numbers of Fos immunopositive cells were quantified in selected brain areas, 1 h after repeated distention of the duodenum. We found an increase in distention-induced Fos in preshocked rats in the nucleus tractus solitarius and a weaker effect in the central nucleus of the amygdala. This could be a first indication that altered visceral afferent processing in previously stressed rats, found earlier for the colon, may be a general and not an organ-specific phenomenon.

Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-p-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2-2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 microM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties.

Tetrachloro-p-hydroquinone (TCHQ), the major metabolite of pentachlorophenol (PCP) in mammalian systems, is known to autoxidize to its semiquinone radical under physiological conditions. In this way, PCP could present a potent source of reactive oxygen species (ROS) during metabolization. ROS contribute to numerous modifications of DNA. Formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a product of hydroxyl radical attack on DNA, is monitored as a marker of a major genetic lesion induced by agents which produce oxygen radicals. We studied the properties of TCHQ for the induction of oxidative DNA damage in vivo. Male B6C3F1 mice were fed a diet containing TCHQ for 2 and 4 weeks. These experiments resulted in an enhancement of about 50% of the 8-OH-dG portion in liver DNA after administration of 300 mg TCHQ/kg body wt./day for 2 weeks. Control levels did not change over the periods of 2 and 4 weeks, respectively. In contrast to these results, a single i.p. injection of 20 or 50 mg/kg body wt. did not affect the 8-OH-dG content after 6 and 24 h, respectively. These data may support a possible contribution of ROS to the carcinogenicity of PCP.

The biocide pentachlorophenol (PCP) is in part metabolized, by microsomal enzymes, to tetrachloro-p-hydroquinone (TCHQ), which easily oxidizes to its semiquinone radical. Redox cycling of this compound produces reactive oxygen species (ROS) which ultimately may damage cellular DNA. Here, we report on DNA damage generated by TCHQ in hamster lung fibroblasts (V79 cells) using 8-hydroxy-2-deoxyguanosine (8-OH-dG) as a marker of a major oxidative genetic lesion and measuring the induction of DNA single-strand breaks (DNA SSB) with the aid of the alkaline elution assay. TCHQ was administered to cell cultures in concentrations of 6.25, 12.5, 25, and 50 microM for 1 h. 6.25 and 12.5 microM had no significant effect on both parameters, whereas the higher concentrations resulted in increases of the level of 8-OH-dG (2.3- and 2.0-fold, respectively) and induced DNA SSB. The latter lesion may arise from (i) direct attack of OH.,(ii) repair of hydroxylated DNA bases, or (iii) cytotoxic effects. Metabolic transformation of PCP to TCHQ and/or other metabolites with quinoid structures and the subsequent generation of ROS, producing oxidative DNA damage, may play a role in PCP-induced carcinogenicity observed in mice.

The possible oxidative effects of the hepatocarcinogens 2-nitropropane (2-NP), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosomorpholine (NMOR) on nuclear DNA were studied in vivo in male F344 rats. 2-NP and the N-nitrosamines were administered intraperitoneally. In addition, NDEA was given by gavage. DNA was isolated from rat liver and hydrolysed enzymatically. Oxidative DNA damage was determined by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in a mixture of 2'-deoxyribonucleosides by electrochemical detection. This method allows a detection limit of about 0.1 residue 8-OH-dG per 10(5) 2'-deoxyguanosine (dG). 2-NP drastically increased the content of 8-OH-dG in rat liver by a factor of ca. 12. No elevation above control values could be proved after having dosed the rats with N-nitrosamines.