SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

BACKGROUND: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. METHODS: The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. RESULTS: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. CONCLUSIONS: SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance.

Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines.

The study aimed to assess the effects of polyphenols when used in combination with doxorubicin and etoposide, and to determine whether polyphenols sensitised leukaemia cells, causing inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. This study is based on findings in solid cancer tumours, which have shown that polyphenols can sensitize cells to chemotherapy, and induce apoptosis and/or cell-cycle arrest. This could enable a reduction of chemotherapy dose and off-target effects, whilst maintaining treatment efficacy. Quercetin, apigenin, emodin, rhein and cis-stilbene were investigated alone and in combination with etoposide and doxorubicin in two lymphoid and two myeloid leukaemia cells lines. Measurements were made of ATP levels (using CellTiter-Glo assay) as an indication of total cell number, cell cycle progression (using propidium iodide staining and flow cytometry) and apoptosis (NucView caspase 3 assay and Hoechst 33342/propidium iodide staining). Effects of combination treatments on caspases 3, 8 and 9 activity were determined using Glo luminescent assays, glutathione levels were measured using the GSH-Glo Glutathione Assay and DNA damage determined by anti-γH2AX staining. Doxorubicin and etoposide in combination with polyphenols synergistically reduced ATP levels, induced apoptosis and increased S and/or G2/M phase cell cycle arrest in lymphoid leukaemia cell lines. However, in the myeloid cell lines the effects of the combination treatments varied; doxorubicin had a synergistic or additive effect when combined with quercetin, apigenin, emodin, and cis-stilbene, but had an antagonistic effect when combined with rhein. Combination treatment caused a synergistic downregulation of glutathione levels and increased DNA damage, driving apoptosis via caspase 8 and 9 activation. However, in myeloid cells where antagonistic effects were observed, this was associated with increased glutathione levels and a reduction in DNA damage and apoptosis. This study has demonstrated that doxorubicin and etoposide activity were enhanced by polyphenols in lymphoid leukaemia cells, however, differential responses were seen in myeloid cells with antagonistic responses seen in some combination therapies.

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells.

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH(Lo) cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH(Hi) population, or whether all ALDH(Hi) cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH(Hi) cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH(Hi) cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH(Lo) population can develop ALDH(Hi) populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH(Hi) cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH(Hi) status enriches for holoclone formation, this activity may be mediated by a minority of ALDH(Hi) cells.

Data warehousing methods and processing infrastructure for brain recovery research.

In order to accelerate translational neuroscience with the goal of improving clinical care it has become important to support rapid accumulation and analysis of large, heterogeneous neuroimaging samples and their metadata from both normal control and patient groups. We propose a multi-centre, multinational approach to accelerate the data mining of large samples and facilitate data-led clinical translation of neuroimaging results in stroke. Such data-driven approaches are likely to have an early impact on clinically relevant brain recovery while we simultaneously pursue the much more challenging model-based approaches that depend on a deep understanding of the complex neural circuitry and physiological processes that support brain function and recovery. We present a brief overview of three (potentially converging) approaches to neuroimaging data warehousing and processing that aim to support these diverse methods for facilitating prediction of cognitive and behavioral recovery after stroke, or other types of brain injury or disease.

Virtual brain transplantation (VBT): a method for accurate image registration and parcellation in large cortical stroke.

OBJECTIVE: The objective of the current study was to develop a semi-automated method to register and parcellate lesioned brains in a surface space with anatomical accuracy, facilitating group-level fMRI analyses in patients with large cortical strokes. METHODS: Thirteen chronic patients with a single large left hemisphere stroke were included in the study. Our "virtual brain transplantation" (VBT) approach is based on pre-processing high resolution anatomical T1-weighted brain images by "filling in" the lesion with "transplanted virtual tissue" from the non-stroke hemisphere, providing "normal" anatomical landmarks for standard alignment and inflation algorithms developed for healthy individuals. Biological validation of the approach was performed by quantifying in Freesurfer space the areas of 12 hand-drawn sulci found inside and outside the stroke following "transplantation". RESULTS: Our results show no difference in the Freesurfer parcellation of 12 different regions when comparing a lesioned hemisphere with the non-lesioned hemisphere, attesting for the validity of the anatomical classification in the stroke hemisphere. As consequence of the anatomical precision, this method permits a more detailed and quantifiable anatomical description of the regions affected directly by the stroke. CONCLUSIONS: This method permits accurate surface reconstruction of the injured hemisphere after stroke by making it possible to extract the cortical surface from these images and to utilize this in software modules (FreeSurfer) specialized for aligning cortical surfaces using high-dimensionality warping algorithms. In addition, it permits quantifying, within bounds, the extent to which the lesion in question is associated with damage to particular regions of the cortical surface, information that is of explanatory value in models that attempt to explain brain-behavior relations using lesion analysis.

Lesion sites that predict the ability to gesture how an object is used.

We used a two stage procedure to predict which stroke patients would have chronic difficulties gesturing how to use an object when object recognition and hand movements were intact. First, we searched our PLORAS database by behavior and identified 5 patients who had chronic difficulty gesturing object use but no difficulty recognising objects, comprehending words or moving their hands. High definition lesion analyses showed that all 5 patients had damage to the white matter underlying the left ventral supramarginal gyrus, (A) close to the cortex, (B) deep towards the midline and (C) extending into the temporal lobe. In addition, 2 patients had damage to (D) the left posterior middle temporal cortex, and 3 patients had damage to (E) the left dorsal supramarginal gyrus and (F) the left premotor cortex. Second, we searched our database by lesion location for patients who had damage to any part of regions ABCDEF. The incidence of gesturing difficulties was higher in patients with damage to ABCD (7/9), ABCE (7/10) or ABCDE (10/13) than ABCF (7/13), ABC (8/16) or partial damage to ABCF (6/32). Thus behaviour was best predicted by the combination of regions that were damaged (a "network-lesion") rather than on the basis of each region alone or overall lesion size. Our results identify which parts of the temporal and parietal lobes impair the ability to gesture object use and which parts need to be intact to support it after damage. Our methods provide a framework for future studies aiming to predict the consequences of brain damage.

Functional connectivity metrics during stroke recovery.

We explore functional connectivity in nine subjects measured with 1.5T fMRI-BOLD in a longitudinal study of recovery from unilateral stroke affecting the motor area (Small et al., 2002). We found that several measures of complexity of covariance matrices show strong correlations with behavioral measures of recovery. In Schmah et al. (2010), we applied Linear and Quadratic Discriminants (LD and QD) computed on a principal components (PC) subspace to classify the fMRI volumes into "early" and "late" sessions. We demonstrated excellent classification accuracy with QD but not LD, indicating that potentially important differences in functional connectivity exist between the early and late sessions. Motivated by Mclntosh et al. (2008), who showed that EEG brain-signal variability and behavioral performance both increased with age during development, we investigated complexity of the covariance matrix for this longitudinal stroke recovery data set. We used three complexity measures: the sphericity index described by Abdi (2010); "unsupervised dimensionality", which is the number of PCs that minimizes unsupervised generalization error of a covariance matrix (Hansen et al., 1999); and "QD dimensionality", which is the number of PCs that minimizes the classification accuracy of QD. Although these approaches measure different kinds of complexity, all showed strong correlations with one or more behavioral tests: nine-hole peg test, hand grip test and pinch test. We could not demonstrate that either sphericity or unsupervised dimensionality were significantly different for the "early" and "late" sessions using a paired Wilcoxon test. However, the amount of relative behavioral improvement was correlated with sphericity of the overall covariance matrix (pooled across all sessions), as well as with the divergence of the eigenspectra between the "early" and "late" covariance matrices. Complexity measures that use the number of PCs (which optimize QD classification or unsupervised generalization) were correlated with the behavioral performance of the final session, but not with the relative improvement. These are suggestive, but limited, results given the sample size, restricted behavioral measurements and older 1.5T BOLD data sets. Nevertheless, they indicate one potentially fruitful direction for future data-driven fMRI studies of stroke recovery in larger, better-characterized longitudinal stroke data sets recorded at higher field strength. Finally, we produced sensitivity maps (Kjems et al., 2002) corresponding to both linear and quadratic discriminants for the "early" vs. "late" classification. These maps measure the influence of each voxel on the class assignments for a given classifier. Differences between the scaled sensitivity maps for the linear and quadratic discriminants indicate brain regions involved in changes in functional connectivity. These regions are highly variable across subjects, but include the cerebellum and the motor area contralateral to the lesion.

Non-fluent aphasia and neural reorganization after speech therapy: insights from human sleep electrophysiology and functional magnetic resonance imaging.

Stroke is associated with long-term functional deficits. Behavioral interventions are often effective in promoting functional recovery and plastic changes. Recent studies in normal subjects have shown that sleep, and particularly slow wave activity (SWA), is tied to local brain plasticity and may be used as a sensitive marker of local cortical reorganization after stroke. In a pilot study, we assessed the local changes induced by a single exposure to a therapeutic session of IMITATE (Intensive Mouth Imitation and Talking for Aphasia Therapeutic Effects), a behavioral therapy used for recovery in patients with post-stroke aphasia. In addition, we measured brain activity changes with functional magnetic resonance imaging (fMRI) in a language observation task before, during and after the full IMITATE rehabilitative program. Speech production improved both after a single exposure and the full therapy program as measured by the Western Aphasia Battery (WAB) Repetition subscale. We found that IMITATE induced reorganization in functionally-connected, speech-relevant areas in the left hemisphere. These preliminary results suggest that sleep hd-EEGs, and the topographical analysis of SWA parameters, are well suited to investigate brain plastic changes underpinning functional recovery in neurological disorders.

Network activation during bimanual movements in humans.

The coordination of movement between the upper limbs is a function highly distributed across the animal kingdom. How the central nervous system generates such bilateral, synchronous movements, and how this differs from the generation of unilateral movements, remain uncertain. Electrophysiologic and functional imaging studies support that the activity of many brain regions during bimanual and unimanual movement is quite similar. Thus, the same brain regions (and indeed the same neurons) respond similarly during unimanual and bimanual movements as measured by electrophysiological responses. How then are different motor behaviors generated? To address this question, we studied unimanual and bimanual movements using fMRI and constructed networks of activation using Structural Equation Modeling (SEM). Our results suggest that (1) the dominant hemisphere appears to initiate activity responsible for bimanual movement; (2) activation during bimanual movement does not reflect the sum of right and left unimanual activation; (3) production of unimanual movement involves a network that is distinct from, and not a mirror of, the network for contralateral unimanual movement; and (4) using SEM, it is possible to obtain robust group networks representative of a population and to identify individual networks which can be used to detect subtle differences both between subjects as well as within a single subject over time. In summary, these results highlight a differential role for the dominant and non-dominant hemispheres during bimanual movements, further elaborating the concept of handedness and dominance. This knowledge increases our understanding of cortical motor physiology in health and after neurological damage.

Phthalocyanine-mediated photodynamic therapy induces cell death and a G0/G1 cell cycle arrest in cervical cancer cells.

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC(50) concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G(0)/G(1) cell cycle arrest during the phthalocyanine PDT-mediated response.

Cerebellar hemispheric activation ipsilateral to the paretic hand correlates with functional recovery after stroke.

An experimental lesion in the primary motor or sensory cortices in monkeys leads to functional reorganization in areas surrounding the lesion or in contralateral homologous regions. In humans, task-dependent brain activation after motor stroke seems to be multifocal and bilateral. Although many active structures are seen after stroke, their roles are unclear. For instance, the uninjured primary motor cortex may play a significant role in recovery or may be associated with mirror movements. Other motor areas, particularly those outside the affected middle cerebral artery distribution, have also been thought to play such a role, including the medial pre-motor areas and both cerebellar hemispheres. The lateral pre-motor areas might also contribute but the demarcation of primary motor and pre-motor cortices is not trivial. It is not known from existing studies how brain activation relates to behavioural change over the time course of recovery. We used functional MRI (fMRI) to study 12 patients longitudinally over the first 6 months of stroke recovery. All subjects had acute stroke causing unilateral arm weakness and had some ability to move the impaired hand within 1 month. Each patient had both motor testing and fMRI during finger and wrist movements at four points during the observed period. Six of these patients showed good motor recovery, whereas the other six did not. The imaging results support a role for the cerebellum in mediating functional recovery from stroke. The data suggest that patients with good recovery have clear changes in the activation of the cerebellar hemisphere opposite the injured corticospinal tract. Patients with poor recovery do not show such changes in cerebellar activation. No other brain region had a significant correlation with recovery. Interestingly, activation in the cerebellum ipsilateral to the injury increases transiently after stroke, independently of the success of recovery. The present work suggests a possible link between cerebellar activation and behavioural recovery from hand weakness from stroke. The underlying mechanism is not known, but it could relate to haemodynamic changes such as diaschisis or to the postulated role of the cerebellum in motor skill learning.

Lateralization of motor circuits and handedness during finger movements.

Although functional lateralization in the human brain has been studied intensively, there remains significant controversy over the brain mechanisms that instantiate it. The main objective of the present study is to characterize the regions associated with the generation of different movements by the fingers of both hands by right- and left-handed people. Thirteen right- and left-handers were studied using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) during performance of single and sequential finger movement tasks. We used single-shot whole-brain spiral fMRI to map the functional components of the motor system during these tasks. Regions of interest included the primary motor and sensory cortices, the pre-motor cortices and the cerebellum. Sequential movements were associated with intense brain activation in several bilateral regions, whereas single movements were associated with less activation in fewer regions, but with greater laterality. Right- and left-handers differed in their pattern of activation, sharing a pattern of activation on simple movements but responding differently to sequential movements. On simple movements, the brain activation patterns of left- and right-handers were similar in volume, number of areas and laterality. By contrast, on sequential movement, left-handers activated larger volumes and a larger number of brain areas than right-handers, and showed significantly less brain lateralization. These results highlight differences in the functional organization of motor areas in right- and left-handed people. The discrepancies that might reflect differences in the network features of motor systems in these two groups, could also determine differences in motor activity that occur during recovery from injury (e.g. after stroke).

The anatomy of auditory word processing: individual variability.

This study used functional magnetic resonance imaging (fMRI) to investigate the neural substrate underlying the processing of single words, comparing activation patterns across subjects and within individuals. In a word repetition task, subjects repeated single words aloud with instructions not to move their jaws. In a control condition involving reverse speech, subjects heard a digitally reversed speech token and said aloud the word "crime." The averaged fMRI results showed activation in the left posterior temporal and inferior frontal regions and in the supplementary motor area, similar to previous PET studies. However, the individual subject data revealed variability in the location of the temporal and frontal activation. Although these results support previous imaging studies, demonstrating an averaged localization of auditory word processing in the posterior superior temporal gyrus (STG), they are more consistent with traditional neuropsychological data, which suggest both a typical posterior STG localization and substantial individual variability. By using careful head restraint and movement analysis and correction methods, the present study further demonstrates the feasibility of using overt articulation in fMRI experiments.

Somatotopy in human primary motor and somatosensory hand representations revisited.

High-resolution functional magnetic resonance imaging of healthy volunteers was used to study the functional anatomy of the human primary motor (M1) and somatosensory (S1) cortical hand representations during simple movements of thumb, little finger and wrist and a sequential movement of the middle three fingers. Rest served as a control state. The results demonstrated an orderly somatotopy in both M1 and S1, even though the cortical areas active with individual movements significantly overlapped. Moreover, the activation patterns in M1 and S1 differed in three aspects: (i) S1 activation was distributed into significantly more clusters than M1 and the primary cluster was smaller; (ii) the overlaps of areas active with different movements were significantly larger in M1 than in S1; (iii) the difference between the three-finger sequential movement and the single-finger movements was more pronounced in S1 than in M1. The sequence-activated S1 cortex was distributed into significantly more clusters. There was also a trend for a bigger volume difference between sequence and the single finger movements in S1 than M1. These data suggest that while the distributed character dominates in M1 and S1, a somatotopic arrangement exists for both M1 and S1 hand representations, with the S1 somatotopy being more discrete and segregated, in contrast to the more integrated and overlapping somatotopy in M1.

The role of segmentation in phonological processing: an fMRI investigation.

Phonological processes map sound information onto higher levels of language processing and provide the mechanisms by which verbal information can be temporarily stored in working memory. Despite a strong convergence of data suggesting both left lateralization and distributed encoding in the anterior and posterior perisylvian language areas, the nature and brain encoding of phonological subprocesses remain ambiguous. The present study used functional magnetic resonance imaging (fMRI) to investigate the conditions under which anterior (lateral frontal) areas are activated during speech-discrimination tasks that differ in segmental processing demands. In two experiments, subjects performed "same/ different" judgments on the first sound of pairs of words. In the first experiment, the speech stimuli did not require overt segmentation of the initial consonant from the rest of the word, since the "different" pairs only varied in the phonetic voicing of the initial consonant (e.g., dip-tip). In the second experiment, the speech stimuli required segmentation since "different" pairs both varied in initial consonant voicing and contained different vowels and final consonants (e.g., dip-ten). These speech conditions were compared to a tone-discrimination control condition. Behavioral data showed that subjects were highly accurate in both experiments, but revealed different patterns of reaction-time latencies between the two experiments. The imaging data indicated that whereas both speech conditions showed superior temporal activation when compared to tone discrimination, only the second experiment showed consistent evidence of frontal activity. Taken together, the results of Experiments 1 and 2 suggest that phonological processing per se does not necessarily recruit frontal areas. We postulate that frontal activation is a product of segmentation processes in speech perception, or alternatively, working memory demands required for such processing.

A model of ocular dominance column development by competition for trophic factor: effects of excess trophic factor with monocular deprivation and effects of antagonist of trophic factor.

Recent experimental evidence has implicated neurotrophic factors (NTs) in the competitive process believed to drive the development of ocular dominance (OD) columns. Application of excess amounts of particular NTs can prevent the segregation process, suggesting that they could be the substance for which geniculocortical afferents compete during development. We have previously presented a model that accounts for normal OD development as well as the prevention of that development with excess NT. The model uses a Hebbian learning rule in combination with competition for a limiting supply of cortical trophic factor to drive OD segregation, without any weight normalization procedures. Subsequent experimental evidence has further suggested that NTs may be causally involved in the competitive process. Application of NT antagonist can prevent OD columns by causing inputs from both eyes to be eliminated, suggesting that NTs may be the substance for which geniculocortical afferents compete. Also, excess NT can mitigate the shift to the open eye normally caused by monocular deprivation (MD). In this article, we show that the current model can account for these subsequent experiments. We show that deprivation of NT causes inputs from both eyes to decay and that excess NT can mitigate the shift to the open eye normally seen with MD. We then present predictions of the model concerning the effects of NT on the length of the critical period during which MD is effective. The model presents a novel mechanism for competition between neural populations inspired by particular biological evidence. It accounts for three specific experimental results, and provides several testable predictions.

Pattern of olfactory bulb innervation returns after recovery from reversible peripheral deafferentation.

The olfactory epithelium (OE) is unusual in its ability to regenerate and reinnervate its target, the olfactory bulb (OB), after deafferentation. To address the question of whether olfactory receptor neuron (ORN) axons preserve their topographic organization when they reestablish synaptic contact with the OB, the authors examined the pattern of ORN axon reinnervation into the bulb of adult H-OMP-lacZ-6 transgenic mice during and after recovery from chemical deafferentation. In the H-OMP-lacZ-6 mouse strain, lacZ expression is limited to a subset of ORNs that are distributed bilaterally in the OE and project primarily to a few glomeruli in the ventromedial region of the OB. The OE was lesioned by intranasal irrigation with Triton X-100, and the distribution of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)-stained cells was examined in the OE along with beta-galactosidase-immunoreactive (beta-gal-ir) axonal processes in the OB after short (1 week), intermediate (3 week), and long (6-7 weeks) recovery times. One week after the lesion, immunostaining for beta-gal and olfactory marker protein was virtually eliminated in the bulb. After 3 weeks of recovery, beta-gal-containing axons appeared to target many of the same locations innervated in bulbs of unlesioned mice. The region that received the highest density of axonal innervation in controls, however, contained only a few processes at that time. After 6-7 week recovery periods, the pattern of X-gal staining in the OE and beta-gal-ir axons in the OB closely resembled that of unlesioned mice. These results demonstrate that the topographic distribution of ORNs in the OE and the pattern of axon innervation in the OB can be reconstituted after chemical deafferentation.