Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCɛ signaling shown in a xenograft breast cancer model.

Non-linearities in the blood-oxygenation-level dependent (BOLD) response measured by functional magnetic resonance imaging (fMRI).

A central question in the analysis of functional magnetic resonance imaging (IMRI) data is whether the measured fMRI signal summates in a linear fashion over repeated inputs. Most fMRI studies collect images sensitive to blood-oxygenation-level dependent (BOLD) contrast, which measures the local amount of deoxygenated hemoglobin (dHb). When neurons are active, more oxygenated hemoglobin is supplied than is needed for their metabolic demands, resulting in a decrease in dHb and an increase in MR signal. For analysis of fMRI data, researchers must therefore create experimental hypotheses of the measurable BOLD response based upon the predicted neuronal activity. An influential early model of the fMRI BOLD response assumes that BOLD activity is a linear transformation of neuronal input, representing the filtering effects of the vascular system. Recent studies have called this interpretation into question, due to observed differences in the pattern of linearity across brain regions that serve distinct functions.

Regional differences in the refractory period of the hemodynamic response: an event-related fMRI study.

We investigated the characteristics of the hemodynamic response (HDR) to paired presentations of visual face stimuli using functional magnetic resonance imaging (fMRI). Photographs of faces were presented singly or in pairs with either a 1-s or 6-s intrapair interval (IPI). Each trial (single face or face pairs) was followed by an intertrial interval of 16-20 s. Faces were presented at fixation and passively viewed by the 10 subjects. Images were acquired at 1.5 Tesla using a gradient-echo echo-planar imaging sequence sensitive to blood-oxygenation-level-dependent (BOLD) contrast. To examine the refractory properties of the HDR, we subtracted the single-stimulus hemodynamic response from the composite response evoked by face pairs for all voxels significantly active on single face trials. The residual represents the contribution of the second stimulus to the fMRI signal. Event-related presentation of faces evoked activity in medial calcarine cortex and the fusiform gyrus bilaterally. In both calcarine and fusiform regions, the hemodynamic response to the second face in a pair was of lower amplitude and of increased latency at 1 s IPI, with significant recovery of both amplitude and latency toward single-stimulus values at 6 s IPI. At 1 s IPI, significantly greater recovery was found in posterior fusiform regions (50-60%) than in midfusiform regions (10-40%). These regional differences were not apparent at 6 s IPI. No differences were found across slices in calcarine cortex. There was a significant difference in mean latency to HDR peak between calcarine and fusiform cortex, with the HDR peaking about 400 ms earlier in calcarine cortex. We conclude that characteristics of the HDR, notably its amplitude, latency, and refractory properties, differ across visual cortical areas.

Dissociating the neural mechanisms of visual attention in change detection using functional MRI.

We investigated using functional magnetic resonance imaging (fMRI) the neural processes associated with performance of a change-detection task. In this task, two versions of the same picture are presented in alternation, separated by a brief mask interval. Even when the two pictures greatly differ (e.g., as when a building is in different locations), subjects report that identification of the change is difficult and often take 30 or more seconds to identify the change. This phenomenon of "change blindness" provides a powerful and novel paradigm for segregating components of visual attention using fMRI that can otherwise be confounded in short-duration tasks. By using a response-contingent event-related analysis technique, we successfully dissociated brain regions associated with different processing components of a visual change-detection task. Activation in the calcarine cortex was associated with task onset, but did not vary with the duration of visual search. In contrast, the pattern of activation in dorsal and ventral visual areas was temporally associated with the duration of visual search. As such, our results support a distinction between brain regions whose activation is modulated by attentional demands of the visual task (extrastriate cortex) and those that are not affected by it (primary visual cortex). A second network of areas including central sulcus, insular, and inferior frontal cortical areas, along with the thalamus and basal ganglia, showed phasic activation tied to the execution of responses. Finally, parietal and frontal regions showed systematic deactivations during task performance, consistent with previous reports that these regions may be associated with nontask semantic processing. We conclude that detection of change, when transient visual cues are not present, requires activation of extrastriate visual regions and frontal regions responsible for eye movements. These results suggest that studies of change blindness can inform understanding of more general attentional processing.

The effects of single-trial averaging upon the spatial extent of fMRI activation.

We examined effects of trial averaging upon spatial extent, spatial topography, and temporal properties of fMRI activation. Two subjects participated in an event-related visual stimulation design. There was an exponential relation between number of trials and spatial extent, such that additional trials identified, on average, a constant proportion of the remaining voxels. At values typical of fMRI experimentation (e.g. 50 trials) only about 50% of eventually active voxels were significant; asymptotic values were approached by 150 trials. The variability of the estimated hemodynamic response decreased with signal averaging, becoming stable across samples of > or = 25 trials. Therefore, group or condition differences may result from differences in voxelwise noise exacerbated by averaging small numbers of trials.

The effects of aging upon the hemodynamic response measured by functional MRI.

We comparedthe characteristics of the visually evoked hemodynamic response (HDR) in groups of young and elderly adults. Checkerboard stimuli were presented for 500 ms either singly or in pairs separated by a 2-s intrapair interval while gradient-echo echoplanar fMRI images were acquired concurrently every 1 s. Activated voxels, identified by correlation with an empirically derived reference waveform, were found for both groups in cortex along the calcarine sulcus and in the fusiform gyrus, with the mean HDR latency in calcarine cortex peaking approximately 300 ms earlier than the HDR evoked in the fusiform gyrus. On average, younger subjects had twice as many activated voxels as older subjects. The mean HDR had a similar onset time, rate of rise, and peak amplitude in both groups. However, the HDRs of older subjects reached their peak earlier and were more variable across subjects. Despite having average HDR amplitudes similar to those of younger subjects, older subjects had higher noise levels in activated voxels, resulting in lower signal-to-noise ratios. Distribution analyses of voxel statistics (t value, peak amplitude, peak latency) revealed that older subjects had proportionally fewer small-effect-size voxels, due to their increased voxelwise noise. This finding was consistent with the smaller spatial extent of activation in older subjects. To investigate age differences in the refractory period of the visual HDR, the HDR evoked by the second stimulus of each pair was isolated by subtracting the HDR evoked by a single stimulus from the composite HDR evoked by a pair. Recovery measures were similar across the age groups.

Evidence for a refractory period in the hemodynamic response to visual stimuli as measured by MRI.

We investigated the effects of paired presentations of visual stimuli upon the evoked hemodynamic response of visual cortex measured by magnetic resonance imaging (MRI). Stimuli were identical 500-ms high-contrast checkerboard patterns, presented singly or with an interpair interval (IPI) of 1, 2, 4, or 6 s (onset-to-onset), followed by an intertrial interval of 16-20 s. Images were acquired at 1.5 Tesla using a gradient-echo echoplanar imaging sequence sensitive to blood-oxygenation-level dependent (BOLD) contrast. Single checkerboards evoked a hemodynamic response from visual cortex characterized by a rise at 3 s, peak activation at 5 s, and return to baseline by 10 s. We subtracted subjects' single-stimulus hemodynamic response from their paired-stimulus responses to isolate the contribution of the second stimulus. If the hemodynamic responses were fully additive, the residual should be a time-shifted replica of the single stimulus response. However, the amplitude of the hemodynamic response to the second checkerboard was smaller, and the peak latency was longer, than for the first. Furthermore, the amplitude decrement was dependent upon IPI, such that the response to the second stimulus at 1 s IPI was only 55% of that to a single stimulus, with recovery to 90% at a 6 s IPI. Peak latency was similarly dependent upon IPI with longer latencies observed for shorter IPIs. These results demonstrate an extended refractory period in the hemodynamic response to visual stimuli consistent with that shown previously for neuronal activity measured electrophysiologically.

Range effects of an irrelevant dimension on classification.

In univariate classification tasks, subjects sort stimuli on the basis of the only attribute that varies. In orthogonal classification tasks, often called filtering tasks, there additionally are trial-to-trial variations in irrelevant attributes that the subjects are instructed to ignore. Performance is generally slower in filtering tasks than in univariate control tasks. We investigated this slowing in experiments of how the range of irrelevant trial-to-trial variation affects responses in pitch/loudness classification tasks. Using two levels of pitch and of loudness as stimuli, Experiment 1 replicated prior work showing that responses are slowed more when the range of the irrelevant dimension is made larger. Also in Experiment 1, sequential analyses showed that response time depends both on sequence and on the stimulus set independent of sequence. Experiments 2 and 3 used several levels on the irrelevant dimension and showed that responses to categorize loudness are slowed more by larger trial-to-trial pitch differences, but only on trials when the response repeats. When the response changes, performance is essentially unaffected by trial-to-trial irrelevant variation. This interaction supports the conclusion that slowed average performance in orthogonal classification tasks, which is known as Garner interference, is not due to difficulties that subjects have in filtering stimulus attributes. It is due to how subjects process successive stimulus differences. We call for more frequent reports of sequential analyses, because these can reveal information that is not available from data averages.

Males and females use different distal cues in a virtual environment navigation task.

The study of navigational ability in humans is often limited by the restricted availability and inconvenience of using large novel environments. In the present study we use a computer-generated virtual environment to study sex differences in human spatial navigation. Adult male and female participants navigated through a virtual water maze where both landmarks and room geometry were available as distal cues. Manipulation of environmental characteristics revealed that females rely predominantly on landmark information, while males more readily use both landmark and geometric information. We discuss these results as a possible link between recent human research reporting hippocampal activation in spatial tasks and animal work showing sex differences in both spatial ability and hippocampal development.