Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo.

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.

Open-wedge high tibial osteotomy: comparison between manual and computer-assisted techniques.

PURPOSE: The purpose of our study was to compare clinical and radiological results of two groups of patients treated for medial compartment osteoarthritis of the knee with either conventional or computer-assisted open-wedge high tibial osteotomy (HTO). Goals of surgical treatment were a correction of the mechanical axis between 2° and 6° of valgus and a modification of posterior tibial slope between -2° and +2°. METHODS: Twenty-four patients (27 knees) affected by varus knee deformity and operated with HTO were prospectively followed-up. They were randomly divided in two groups, A (11 patients, conventional treatment) and B (13 patients, navigated treatment). The American Knee Society Score and the Modified Cincinnati Rating System Questionnaire were used for clinical assessment. All patients were radiologically evaluated with a comparative lower limb weight-bearing digital radiograph, a standard digital anteroposterior, a latero-lateral radiograph of the knee, and a Rosenberg view. RESULTS: Patients were followed-up at a mean of 39 months. Clinical evaluation showed no statistical difference (n.s.) between the two groups. Radiological results showed an 86% reproducibility in achieving a mechanical axis of 182°-186° in group B compared to a 23% in group A (p = 0.0392); furthermore, in group B, we achieved a modification of posterior tibial slope between -2° and +2° in 100% of patients, while in group A, this goal was achieved only in 24% of cases (p = 0.0021). CONCLUSION: High tibial osteotomy with navigator is more accurate and reproducible in the correction of the deformity compared to standard technique. LEVEL OF EVIDENCE: Therapeutic study, Level II.

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells.

Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.

Callosal connections of dorso-lateral premotor cortex.

This study investigated the organization of the callosal connections of the two subdivisions of the monkey dorsal premotor cortex (PMd), dorso-rostral (F7) and dorso-caudal (F2). In one animal, Fast blue and Diamidino yellow were injected in F7 and F2, respectively; in a second animal, the pattern of injections was reversed. F7 and F2 receive a major callosal input from their homotopic counterpart. The heterotopic connections of F7 originate mainly from F2, with smaller contingent from pre-supplementary motor area (pre-SMA, F6), area 8 (frontal eye fields), and prefrontal cortex (area 46), while those of F2 originate from F7, with smaller contributions from ventral premotor areas (F5, F4), SMA-proper (F3), and primary motor cortex (M1). Callosal cells projecting homotopically are mostly located in layers II-III, those projecting heterotopically occupy layers II-III and V-VI. A spectral analysis was used to characterize the spatial fluctuations of the distribution of callosal neurons, in both F7 and F2, as well as in adjacent cortical areas. The results revealed two main periodic components. The first, in the domain of the low spatial frequencies, corresponds to periodicities of cell density with peak-to-peak distances of approximately 10 mm, and suggests an arrangement of callosal cells in the form of 5-mm wide bands. The second corresponds to periodicities of approximately 2 mm, and probably reflects a 1-mm columnar-like arrangement. Coherency and phase analyses showed that, although similar in their spatial arrangements, callosal cells projecting to dorsal premotor areas are segregated in the tangential cortical domain.

Acrylic hydrogel implants after spinal cord lesion in the adult rat.

Acrylic hydrogels, like the polymer of 2-hydroxyethyl methacrylate, are biocompatible, mechanically stable, porous materials that can be coated with collagen or laminin acting as bioadhesive substrates. Poly-2-hydroxyethyl methacrylate sponges have been proposed for restoring the anatomical continuity of damaged neural structures. In the present work, the ability of poly-2-hydroxyethyl methacrylate sponges to provide the injured spinal cord neurons with a conductive substrate for their regenerating axons was investigated in 32 adult Wistar rats. Collagen impregnated poly-2-hydroxyethyl methacrylate sponges were implanted into suction cavities of the dorsal funiculus of the spinal cord. Two to four months after implantation, the spinal cord was removed and processed for histology, and S100 and GFAP immunohistochemistry. To study axonal regeneration into the sponge, the spinal cord or the sensorimotor cortex were injected with 0.05-0.1 microl of an 8% solution of lectin-conjugated horseradish peroxidase or 10% dextran tetramethylrhodamine. The fibroglial reaction, accumulation of mononuclear cells, and angiogenesis at the interface between the spinal cord and the sponge were minimal. Cystic cavitation in the spinal cord was virtually absent. Anterograde labeled axons were seen to penetrate and to elongate the full length of the sponge. These results demonstrate that poly-2-hydroxyethyl methacrylate sponges represent a safe supportive material for regenerating spinal cord axons.

Dendritic anomalies in a freezing model of microgyria: a parametric study.

Despite easier recognition of focal developmental cortical anomalies with modern morphological and functional imaging techniques, mechanisms leading to refractory epilepsy are still poorly understood. Recent experimental studies have shown that not only the lesioned cortex, but also the apparently normal adjacent cortex undergoes morphological changes and alterations of its neuronal connections. To further investigate the modifications of the cortex surrounding a focal maldevelopmental lesion, we applied a freezing insult to newborn rat cortex, resulting in a focal cortical malformation similar to human microgyria. Corticocortical associative neurons were retrogradely labeled in a Golgi-like fashion using biotinylated dextran amine combined with NMDA. In addition to previously reported alterations, a considerable spine loss was observed in the basal dendrites of neurons located in the eulaminated cortex adjacent to the lesion. These data demonstrate profound maldevelopmental alterations which are not limited to the macroscopically abnormal lesion, but extend at a cellular level to the surrounding cortex. The observed alterations may contribute to the increased excitability of the cortex harboring a microgyric lesion as well as the frequently associated cognitive impairment.

Organization of cortico-cortical associative projections in a rat model of microgyria.

Microgyria was experimentally induced by focal freezing lesions of the frontal cortex in newborn rats. Adult microgyric animals received cortical injections of biotinylated dextran amine combined with NMDA, in order to obtain a Golgi-like retrograde labeling of cortico-cortical association neurons. Injections were performed either rostrally or caudally to the microgyric lesion. Results demonstrate that long-range association projections traveling across the zone of the microgyric lesion arise mainly from infragranular layers. In normal animals the same projections originate both from supragranular and infragranular layers. The analysis of single basal dendrites of layer 2/3 in microgyric animals demonstrates a simplified branching pattern, with a number of end points lower than in control animals. Potential implications for microgyria-associated epilepsy are discussed.

Early coding of reaching: frontal and parietal association connections of parieto-occipital cortex.

The ipsilateral association connections of the cortex of the dorsal part of the rostral bank of the parieto-occipital sulcus and of the adjoining posterior part of the superior parietal lobule were studied by using different retrograde fluorescent tracers. Fluoro-Ruby, Fast blue and Diamidino yellow were injected into visual area V6A, and dorso-caudal (PMdc, F2) and dorso-rostral (PMdr, F7) premotor cortex, respectively. The parietal area of injection had been previously characterized physiologically in behaving monkeys, through a variety of oculomotor and visuomanual tasks. Area V6A is mainly linked by reciprocal projections to parietal areas 7m, MIP (medial intraparietal) and PEa, and, to a lesser extent, to frontal areas PMdr (rostral dorsal premotor cortex, F7) and PMdc (F2). All these areas project to that part of the dorsocaudal premotor cortex that has a direct access to primary motor cortex. V6A is also connected to area F5 and, to a lesser extent, to 7a, ventral (VIP) and lateral (LIP) intraparietal areas. This pattern of association connections may explain the presence of visually-related and eye-position signals in premotor cortex, as well as the influence of information concerning arm position and movement direction on V6A neural activity. Area V6A emerges as a potential 'early' node of the distributed network underlying visually-guided reaching. In this network, reciprocal association connections probably impose, through re-entrant signalling, a recursive property to the operations leading to the composition of eye and hand motor commands.

Organization of callosal connections in rats with experimentally induced microgyria.

The anatomical organization of projections from the microgyric cortex to the contralateral hemisphere through the corpus callosum has been studied in the rat. Microgyria was induced on the first postnatal day by a freezing injury. Once adult, animals received injections of horseradish peroxidase in the cortex contralateral to the lesion. Callosally projecting neurons located in the normally layered cortex adjoining the focal lesion showed a different laminar distribution from that observed in control animals. The highest percentage of callosal neurons was found in layer 6 in lesioned animals, whereas layers 2/3 and 5 gave rise to most of interhemispheric projections in controls. Possible mechanisms leading to the establishment of aberrant callosal connections and potential implications for the human pathology are discussed.

A glutamate-sensitizing activity in conditioned media derived from rat cerebellar granule cells.

When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 microM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 x 10(5) cells/cm2) or increasing the volume by an identical 4-fold factor reduced cell death from 90-95% to 20-30%. Addition of a conditioned medium derived from high-density to low-density cultures or to high-volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate-sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1-7, 3T3 and CHP 100. Glutamate-sensitizing activity was not mimicked by trilodo-L-thyronine, insulin-like growth factor-I (IGF-I), truncated IGF-I, GPE [a tripeptide (gly-pro-glu) derived from IGF-I], brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor-alpha. However, IGF-I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate-sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triiodo-L-thyronine and BDNF did not support the production of glutamate-sensitizing activity. Glutamate-sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N-omega-nitro-L-arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate-sensitizing activity.

Protease inhibitors in mouse skeletal muscle: tissue-associated components of serum inhibitors and calpastatin.

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).

Chemical compartmentation and relationships between calcium-binding protein immunoreactivity and layer-specific cortical caudate-projecting cells in the anterior intralaminar nuclei of the cat.

Neurons projecting to the parietal cortex or striatum and neurons showing immunoreactivity for the calcium-binding proteins parvalbumin and 28KD-calbindin were examined in the anterior intralaminar nuclei (IL) of the cat. Retrograde tracing from deep or superficial parietal cortical layers or from the caudate nucleus was coupled with immunohistochemistry to determine which of these proteins were expressed in the projection neurons. It was found that IL neurons project to deep as well as to superficial layers of the parietal cortex, that IL-cortical neurons could be differentiated into two populations according to their cortical projection pattern and their soma size, and that IL neurons projecting to the parietal cortex or to the striatum express 28KD calbindin immunoreactivity but not parvalbumin immunoreactivity. The distribution of immunoreactivity to 28KD calbindin and parvalbumin in the neuropil showed a consistent complementary distribution pattern in the IL. The compartments based on differential parvalbumin and 28KD calbindin expression may indicate the presence of functionally segregated units in IL.