A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

Expression and function of $\beta$-tubulin isotypes in Chinese hamster ovary (CHO) cells

The availability of isotype specific antisera for $\beta$-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of $\beta$-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor $\beta$-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. Their stoichiometry is approximately 1:1:0.7:0.2. All three isotypes assemble into both cytoplasmic and spindle microtubules, and are similar in their responses to cold, colcemid, and calcium induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that $\beta$-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid resistant mutants with a demonstrable alteration in $\beta$-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist. Under various conditions of the cell growth, the relative proportion of each expressed isotype does not significantly seem to change except in the early G1 phase of the cell cycle. At this time the synthesis of isotype V increases more than two fold relative to isotype I and IV, while at the same time, total $\beta$-tubulin synthesis is decreased about 60-70%. ^

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

CHROMOSOMAL ORIGIN OF DM-DNA

Double minutes (dm) are small chromatin particles of 0.3 microns diameter found only in the metaphase cells of human and murine tumors. Dm are unique cytogenetic structures since their numbers per cell show wide variation. At cell division, dm are retained despite the lack of centromeres. In squash preparations, dm show clustering often in association with chromosomes. Human carcinoma cell line SW613-S18 was found to have large numbers of dm and biological characteristics favorable for mitotic synchronization and chromosome isolation experiments.^ S18 cells were synchronized to mitosis with metabolic and mitotic blocking compounds. Mitotic cells were lysed to release chromosomes and dm from the mitotic spindle and the resulting suspensions were fractionated to enrich for dm. The DNA in enriched fractions was characterized. The reassociation kinetics of dm-DNA driven with placental human DNA was similar to the reassociation curve of labeled placental DNA under similar conditions. In situ hybridization of dm-DNA to tumor and normal metaphase cells showed grain localization over the entire karyotype. Dm-DNA was shown by pulse chase DNA replication experiments to replicate during early and mid S-phase of the cell cycle, but not in late S-phase. In addition, BrdUrd incorporation studies showed that dm-DNA replicates only once during the S-phase. Premature chromosome condensation studies suggest the basis of numerical heterogeneity of dm is nondisjunction, not anomalous or unscheduled DNA replication.^ These data and previous cytochemical banding studies of dm in SW613-S18 indicate that dm-DNA is chromosomal in origin. No evidence of gene amplification was found in the DNA reassociation data. It is likely that dm-DNA represents the pale-staining G-band regions of the human karyotype in this cell line. ^

Sleep respiratory disturbances and arousals at moderate altitude have overlapping electroencephalogram spectral signatures

An ascent to altitude has been shown to result in more central apneas and a shift towards lighter sleep in healthy individuals. This study employs spectral analysis to investigate the impact of respiratory disturbances (central/obstructive apnea and hypopnea or periodic breathing) at moderate altitude on the sleep electroencephalogram (EEG) and to compare EEG changes resulting from respiratory disturbances and arousals. Data were collected from 51 healthy male subjects who spent 1 night at moderate altitude (2590 m). Power density spectra of Stage 2 sleep were calculated in a subset (20) of these participants with sufficient artefact-free data for (a) epochs with respiratory events without an accompanying arousal, (b) epochs containing an arousal and (c) epochs of undisturbed Stage 2 sleep containing neither arousal nor respiratory events. Both arousals and respiratory disturbances resulted in reduced power in the delta, theta and spindle frequency range and increased beta power compared to undisturbed sleep. The similarity of the EEG changes resulting from altitude-induced respiratory disturbances and arousals indicates that central apneas are associated with micro-arousals, not apparent by visual inspection of the EEG. Our findings may have implications for sleep in patients and mountain tourists with central apneas and suggest that respiratory disturbances not accompanied by an arousal may, none the less, impact sleep quality and impair recuperative processes associated with sleep more than previously believed.

Myoepithelioma of the cerebellopontine angle: a previously not documented benign salivary gland-type neoplasm within the cranium

Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.

Impact of Acetazolamide and CPAP on Cortical Activity in Obstructive Sleep Apnea Patients

STUDY OBJECTIVES 1) To investigate the impact of acetazolamide, a drug commonly prescribed for altitude sickness, on cortical oscillations in patients with obstructive sleep apnea syndrome (OSAS). 2) To examine alterations in the sleep EEG after short-term discontinuation of continuous positive airway pressure (CPAP) therapy. DESIGN Data from two double-blind, placebo-controlled randomized cross-over design studies were analyzed. SETTING Polysomnographic recordings in sleep laboratory at 490 m and at moderate altitudes in the Swiss Alps: 1630 or 1860 m and 2590 m. PATIENTS Study 1: 39 OSAS patients. Study 2: 41 OSAS patients. INTERVENTIONS Study 1: OSAS patients withdrawn from treatment with CPAP. Study 2: OSAS patients treated with autoCPAP. Treatment with acetazolamide (500-750 mg) or placebo at moderate altitudes. MEASUREMENTS AND RESULTS An evening dose of 500 mg acetazolamide reduced slow-wave activity (SWA; approximately 10%) and increased spindle activity (approximately 10%) during non-REM sleep. In addition, alpha activity during wake after lights out was increased. An evening dose of 250 mg did not affect these cortical oscillations. Discontinuation of CPAP therapy revealed a reduction in SWA (5-10%) and increase in beta activity (approximately 25%). CONCLUSIONS The higher evening dose of 500 mg acetazolamide showed the "spectral fingerprint" of Benzodiazepines, while 250 mg acetazolamide had no impact on cortical oscillations. However, both doses had beneficial effects on oxygen saturation and sleep quality.

Cerebral High-grade Oligodendroglioma with Sarcomatous Transdifferentiation ("Oligosarcoma") in a Boxer Dog.

A 9-year-old Boxer dog was referred to the Veterinary Teaching Hospital of the University of Bern for a history of chronic neck pain and gait problems, which rapidly progressed to a non-ambulatory status. Magnetic resonance imaging (MRI) examination of the head revealed a large intra-axial space-occupying lesion that was divided in two portions interconnected by a thin isthmus at the level of the cerebellar tentorium. Histopathology revealed a biphasic malignant neoplasm composed of neuroepithelial and mesenchymal elements. The former displayed characteristics of conventional anaplastic oligodendroglioma involving brisk mitotic activity and glomeruloid microvascular proliferation on a background of a fibrillary round cells with "honeycomb-like" perinuclear vacuolation. Conversely, the sarcomatous moiety exhibited haphazard fascicles of spindle cells amidst an intricate mesh of pericellular basal lamina and broad bands of collagen. Both tumor cell populations immunoreacted for Olig-2 and – to a lesser extent – GFAP. In addition, the sarcomatous areas focally expressed vimentin, muscular actin, and smooth muscle actin. "Oligosarcoma" - an exquisitely uncommon pattern of oligodendroglial malignancy in humans - has not previously been reported to affect dogs, although oligodendroglioma is a common CNS tumor in this species. Whether canine oligosarcoma shares with its human counterpart not only morphological aspects, but also molecular signatures, clinical behavior and responsiveness to therapy merits further investigation. In humans, oligodendroglial differentiation tends to confer significant clinical advantage with respect to prognosis and adjuvant treatment options. The awareness of such hallmarks and the investigation of their impact on prognosis are crucial for improved therapeutical strategies in dogs.

In vitro cell motility as a potential mesenchymal stem cell marker for multipotency.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.

Supraorbital cutaneous fetal rhabdomyoma of intermediate type: a case report.

A 7-year-old boy was presented with a long-standing slowly growing mass of the left supraorbital area. A biopsy specimen revealed a bland spindle cell proliferation with scattered polygonal cells with acidophilic cytoplasm and cross-striations. Our differential diagnosis included rhabdomyoma of fetal type, leiomyoma with trapping of regenerating skeletal muscle elements, and rhabdomyomatous mesenchymal hamartoma of the skin. Immunohistochemistry demonstrated strong positivity of myoglobin and desmin as well as negativity of caldesmon, suggesting skeletal muscle lineage. The excisional specimen confirmed our diagnosis of cutaneous fetal rhabdomyoma of intermediate type. Additional immunostaining performed on the excisional specimen showed strong Wilms Tumor 1 but only a very faint and focal p63 expression.

Topographic sleep EEG changes in the acute and chronic stage of hemispheric stroke.

After stroke, the injured brain undergoes extensive reorganization and reconnection. Sleep may play a role in synaptic plasticity underlying stroke recovery. To test this hypothesis, we investigated topographic sleep electroencephalographic characteristics, as a measure of brain reorganization, in the acute and chronic stages after hemispheric stroke. We studied eight patients with unilateral stroke in the supply territory of the middle cerebral artery and eight matched controls. All subjects underwent a detailed clinical examination including assessment of stroke severity, sleep habits and disturbances, anxiety and depression, and high-density electroencephalogram examination with 128 electrodes during sleep. The recordings were performed within 10 days after stroke in all patients, and in six patients also 3 months later. During sleep, we found higher slow-wave and theta activity over the affected hemisphere in the infarct area in the acute and chronic stage of stroke. Slow-wave, theta activity and spindle frequency range power over the affected hemisphere were lower in comparison to the non-affected side in a peri-infarct area in the patients' group, which persisted over time. Conversely, in wakefulness, only an increase of delta, theta activity and a slowing of alpha activity over the infarct area were found. Sleep slow-wave activity correlated with stroke severity and outcome. Stroke might have differential effects on the generation of delta activity in wakefulness and sleep slow waves (1-8 Hz). Sleep electroencephalogram changes over both the affected and non-affected hemispheres reflect the acute dysfunction caused by stroke and the plastic changes underlying its recovery. Moreover, these changes correlate with stroke severity and outcome.

Collision tumor affecting the paranasal sinuses of a geriatric donkey

Tumours of the paranasal sinuses are relatively uncommon in the horse. A collision tumour occurs when 2 separate tumour types arise in close proximity to each other and encroach on one another. This report describes the presence of neoplasia comprising glandular tissue and spindle cells growing concurrently in the frontal and caudal maxillary sinus of a geriatric donkey. As far as the authors are aware, this is the first description of a possible collision tumour in an equid.

Suprasellar chordoid neoplasm with expression of thyroid transcription factor 1: evidence that chordoid glioma of the third ventricle and pituicytoma may form part of a spectrum of lineage-related tumors of the basal forebrain.

Chordoid glioma of the third ventricle is a rare neuroepithelial tumor characterized by a unique histomorphology and exclusive association with the suprasellar/third ventricular compartment. Variously interpreted as either astrocytic- or ependymal-like, and speculatively ascribed to the lamina terminalis/subcommissural organ, its histogenesis remains, nevertheless, unsettled. Here, we report on a suprasellar chordoid glioma occurring in a 52-year-old man. Although displaying otherwise typical morphological features, the tumor was notable for expression of thyroid transcription factor 1, a marker of tumors of pituicytic origin in the context of the sellar region. We furthermore found overlapping immunoprofiles of this example of chordoid glioma and pituicytic tumors (pituicytoma and spindle cell oncocytoma), respectively. Specifically, phosphorylated ribosomal protein S6, a marker of mTOR pathway activation, was expressed in both groups. Based on these findings, we suggest that chordoid glioma and pituicytic tumors may form part of a spectrum of lineage-related neoplasms of the basal forebrain.

"Leiomyomatoid angiomatous neuroendocrine tumor" (LANT) of the pituitary reflects idiosyncratic angiogenesis in adenomas of the gonadotroph cell lineage.

Based on a single-case observation, the descriptive label "leiomyomatoid angiomatous neuroendocrine tumor" (LANT) has been tentatively applied to what was perceived as a possible novel type of dual-lineage pituitary neoplasm with biphasic architecture. We report on two additional examples of an analogous phenomenon encountered in male patients, aged 59 years (Case 1) and 91 years (Case 2). Both tumors were intra- and suprasellar masses, measuring 5.6 cm × 4.4 cm × 3.4 cm, and 2.7 cm × 2 cm × 1.7 cm, respectively. Histologically, Case 1 was an FSH-cell adenoma interwoven by vascularized connective tissue septa that tended to exhibit incremental stages of adventitial overgrowth. The epithelial component of Case 2 corresponded to an LH-cell adenoma, and lay partitioned by a maze of paucicellular to hyalinized vascular axes. Irrespective of architectural variations, perivascular spindle cells exhibited immunopositivity for vimentin, muscular actin, and smooth muscle actin. Conversely, negative results were obtained for CD34, EMA, S100 protein, GFAP, and TTF-1. Ultrastructural study failed to reveal metaplastic cell forms involving transitional features between adenohypophyseal-epithelial and mesenchymal-contractile phenotype. We propose that LANT be regarded as a peculiar reflection of maladaptive angiogenesis in some pituitary adenomas, rather than a genuine hybrid neoplasm. While no mechanistic clue is forthcoming to account for this distinctive pattern, hemodynamic strain through direct arterial - rather than portal - supply of the adenoma's capillary bed may be one such explanatory factor. The apparent predilection of the LANT pattern for macroadenomas of the gonadotroph cell lineage remains unexplained.

Inclination-dependent changes of the critical shoulder angle significantly influence superior glenohumeral joint stability.

BACKGROUND The critical shoulder angle combines the acromion index and glenoid inclination and has potential to discriminate between shoulders at risk for rotator cuff tear or osteoarthritis and those that are asymptomatic. However, its biomechanics, and particularly the role of the glenoid inclination, are not yet fully understood. METHODS A shoulder simulator was used to analyze the independent influence of glenoid inclination during abduction from 0 to 60°. Spindle motors transferred tension forces by a cable-pulley on human cadaveric humeri. A six-degree-of-freedom force transducer was mounted directly behind the polyethylene glenoid to measure shear and compressive joint reaction force and calculate the instability ratio (ratio of shear and compressive joint reaction force) with the different force ratios of the deltoid and supraspinatus muscles (2:1 and 1:1). A stepwise change in the inclination by 5° increments allowed simulation of a critical shoulder angle range of 20° to 45°. FINDINGS Tilting the glenoid to cranial (increasing the critical shoulder angle) increases the shear joint reaction force and therefore the instability ratio. A balanced force ratio (1:1) between the deltoid and the supraspinatus allowed larger critical shoulder angles before cranial subluxation occurred than did the deltoid-dominant ratio (2:1). INTERPRETATION Glenoid inclination-dependent changes of the critical shoulder angle have a significant impact on superior glenohumeral joint stability. The increased compensatory activity of the rotator cuff to keep the humeral head centered may lead to mechanical overload and could explain the clinically observed association between large angles and degenerative rotator cuff tears.