Uncovering a novel pathway for p16 silencing: Therapeutic implications for lung cancer.

A key step during onset of most cases of non-small cell lung cancer (NSCLC) is the loss of the tumor suppressor p16INK4a (best known as p16), commonly due to promoter hypermethylation. We recently reported a novel regulatory pathway involving E6-associated protein and cell division control protein 6, which provides a methylation-independent mechanism for p16 silencing in patients with a particularly aggressive form of NSCLC.

Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Walking patterns induced by learned odors in the honeybee, Apis mellifera L.

The odor localization strategy induced by odors learned via differential conditioning of the proboscis extension response was investigated in honeybees. In response to reward-associated but not non-reward-associated odors, learners walked longer paths than non-learners and control bees. When orange odor reward association was learned, the path length and the body turn angles were small during odor stimulation and greatly increased after stimulation ceased. In response to orange odor, bees walked locally with alternate left and right turns during odor stimulation to search for the reward-associated odor source. After odor stimulation, bees walked long paths with large turn angles to explore the odor plume. For clove odor, learning-related modulations of locomotion were less pronounced, presumably due to a spontaneous preference for orange in the tested population of bees. This study is the first to describe how an odor-reward association modulates odor-induced walking in bees.

Targeting Mdmx to treat breast cancers with wild-type p53.

The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.

A direct descending pathway informing locomotor networks about tactile sensor movement.

Much like visually impaired humans use a white-cane, nocturnal insects and mammals use antennae or whiskers for near-range orientation. Stick insects, for example, rely heavily on antennal tactile cues to find footholds and detect obstacles. Antennal contacts can even induce aimed reaching movements. Because tactile sensors are essentially one-dimensional, they must be moved to probe the surrounding space. Sensor movement is thus an essential cue for tactile sensing, which needs to be integrated by thoracic networks for generating appropriate adaptive leg movements. Based on single and double recordings, we describe a descending neural pathway comprising three identified ON- and OFF-type neurons that convey complementary, unambiguous, and short-latency information about antennal movement to thoracic networks in the stick insect. The neurons are sensitive to the velocity of antennal movements across the entire range covered by natural movements, regardless of movement direction and joint angle. Intriguingly, none of them originates from the brain. Instead, they descend from the gnathal ganglion and receive input from antennal mechanoreceptors in this lower region of the CNS. From there, they convey information about antennal movement to the thorax. One of the descending neurons, which is additionally sensitive to substrate vibration, feeds this information back to the brain via an ascending branch. We conclude that descending interneurons with complementary tuning characteristics, gains, input and output regions convey detailed information about antennal movement to thoracic networks. This pathway bypasses higher processing centers in the brain and thus constitutes a shortcut between tactile sensors on the head and the thorax.

Expression of E6AP and PML predicts for prostate cancer progression and cancer-specific death.

BACKGROUND: The promyelocytic leukemia (PML) tumor suppressor plays an important role in the response to a variety of cellular stressors and its expression is downregulated or lost in a range of human tumors. We have previously shown that the E3 ligase E6-associated protein (E6AP) is an important regulator of PML protein stability but the relationship and clinical impact of PML and E6AP expression in prostatic carcinoma is unknown. METHODS: E6AP and PML expression was assessed in tissue microarrays from a phase I discovery cohort of 170 patients treated by radical prostatectomy for localized prostate cancer (PC). Correlation analysis was carried out between PML and E6AP expression and clinicopathological variates including PSA as a surrogate of disease recurrence. The results were confirmed in a phase II validation cohort of 318 patients with associated clinical recurrence and survival data. RESULTS: Survival analysis of the phase I cohort revealed that patients whose tumors showed reduced PML and high E6AP expression had reduced time to PSA relapse (P = 0.012). This was confirmed in the phase II validation cohort where the expression profile of high E6AP/low PML was significantly associated with reduced time to PSA relapse (P < 0.001), clinical relapse (P = 0.016) and PC-specific death (P = 0.014). In multivariate analysis, this expression profile was an independent prognostic indicator of PSA relapse and clinical relapse independent of clinicopathologic factors predicting recurrence. CONCLUSION: This study identifies E6AP and PML as potential prognostic markers in localized prostate carcinoma and supports a role for E6AP in driving the downregulation or loss of PML expression in prostate carcinomas.

The E6AP E3 ubiquitin ligase regulates the cellular response to oxidative stress.

The E6AP E3 ubiquitin ligase has been linked to the regulation of cell growth and to the cellular stress response. However, the specific stress conditions that are controlled by E6AP have not been defined. An important stress condition that controls cell growth is oxidative stress, where the levels of intracellular reactive oxygen species (ROS) regulate the appropriate cellular response. Here, we describe a novel role for E6AP in the control of oxidative stress response. Cells lacking E6AP expression have reduced capacity to accumulate ROS, and oxidative DNA damage, in response to 20% cell culture oxygen levels, treatment with hydrogen peroxide and expression of oncogenic RAS. This effect of E6AP is associated with the regulation of the anti-oxidant enzyme, Prx1, a previously identified target of E6AP, and can be corrected by downregulation of Prx1 or by reconstitution of E6AP expression. Consequently, cells with compromised E6AP have impaired senescent and apoptotic response to sub-lethal and lethal doses of oxidative stress, respectively. In a xenograft model, downregulation of E6AP renders transplanted tumours refractory to growth-suppressive effects of hydrogen peroxide. Our results provide the first demonstration that E6AP is an important regulator of ROS-mediated cellular senescence and cell death.

JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry.

Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.

E6AP is required for replicative and oncogene-induced senescence in mouse embryo fibroblasts.

Cellular senescence is important for the maintenance of tissue homeostasis, and has recently been shown to pose a natural barrier to tumorigenesis. The E3 ubiquitin ligase, E6AP, has been linked to a number of protein regulators of the cell cycle as well as the cellular stress response. We therefore explored the role of E6AP in the cellular response to stress. We found that mouse embryo fibroblasts (MEFs) lacking E6AP escape replicative senescence, as well as Ras-induced senescence associated with impaired markers. E6AP-deficient MEFs exhibit a range of transformed phenotypes: these include the ability to grow under stress conditions (such as low serum and DNA damage), enhanced proliferation, anchorage independent growth and enhanced growth of xenografts in mice. The transformed phenotype of E6AP-deficient MEFs is associated with lower basal and stress-induced accumulation of p53. Overall, our study implicates E6AP as an important regulator of the cellular response to stress, in particular through the regulation of replicative and oncogene-induced senescence.

Sensillum-specific, topographic projection patterns of olfactory receptor neurons in the antennal lobe of the cockroach Periplaneta americana.

In vertebrates and many invertebrates, olfactory signals detected by peripheral olfactory receptor neurons (ORNs) are conveyed to a primary olfactory center with glomerular organization in which odor-specific activity patterns are generated. In the cockroach, Periplaneta americana, ORNs in antennal olfactory sensilla project to 205 unambiguously identifiable antennal lobe (AL) glomeruli that are classified into 10 glomerular clusters (T1-T10 glomeruli) innervated by distinct sensory tracts. In this study we employed single sensillum staining techniques and investigated the topographic projection patterns of individual ORNs to elucidate the relationship between sensillum types and glomerular organization in the AL. Axons of almost all ORNs projected to individual glomeruli. Axons of ORNs in perforated basiconic sensilla selectively innervated the anterodorsal T1-T4 glomeruli, whereas those in trichoid and grooved basiconic sensilla innervated the posteroventral T5-T9 glomeruli. About 90% of stained ORNs in trichoid sensilla sent axons to the T5 glomeruli and more than 90% of ORNs in grooved basiconic sensilla innervated the T6, T8, and T9 glomeruli. The T5 and T9 glomeruli exclusively receive sensory inputs from the trichoid and grooved basiconic sensilla, respectively. All investigated glomeruli received convergent input from a single type of sensillum except F11 glomerulus in the T6 glomeruli, which was innervated from both trichoid and grooved basiconic sensilla. These results suggest that ORNs in distinct sensillum types project to glomeruli in distinct glomerular clusters. Since ORNs in distinct sensillum types are each tuned to distinct subsets of odorant molecules, the AL is functionally compartmentalized into groups of glomeruli.

Reconstruction of virtual neural circuits in an insect brain.

The reconstruction of large-scale nervous systems represents a major scientific and engineering challenge in current neuroscience research that needs to be resolved in order to understand the emergent properties of such systems. We focus on insect nervous systems because they represent a good compromise between architectural simplicity and the ability to generate a rich behavioral repertoire. In insects, several sensory maps have been reconstructed so far. We provide an overview over this work including our reconstruction of population activity in the primary olfactory network, the antennal lobe. Our reconstruction approach, that also provides functional connectivity data, will be refined and extended to allow the building of larger scale neural circuits up to entire insect brains, from sensory input to motor output.

Percutaneous intramyocardial stem cell injection in patients with acute myocardial infarction: first-in-man study.

BACKGROUND: Clinical studies on intracoronary stem cell infusion in patients with acute myocardial infarction (AMI) have shown promising results for left ventricular ejection fraction (LVEF). However, preclinical studies have shown that intramyocardial cell injection is better than the intracoronary approach. OBJECTIVE: To test safety and feasibility of intramyocardial cell injection and left ventricular electromechanical mapping (EMM) early after AMI. DESIGN: On day 10.5 (5) (mean (SD)) after AMI and percutaneous coronary intervention with stent implantation (culprit lesion: 15 LAD, 3 circumflex and 2 right coronary arteries) 20 patients (mean (SD) 60.4 (11.4) years) received bone marrow derived mononuclear cells in the low-voltage area using EMM-guided percutaneous intramyocardial injection. EMM and coronary angiography were performed in 15 patients at 6-months' follow-up. Echocardiography, recording of laboratory data and clinical assessment (6-month and 12-month follow-up) were carried out in all 20 patients. RESULTS: None of the patients showed periprocedural complications. Three patients received an implantable cardioverter-defibrillator for primary prevention of sudden cardiac death and 6 (30%) patients showed in-stent restenosis. One patient underwent bypass surgery owing to chronic stent occlusion after 6 months. 2.0 (0.6)x10(8) cells, including 1.0 (0.3)x10(6) CD45(dim)/CD34(hi) stem cells, were injected in each patient. EMM showed a mean (SD) improvement from a baseline unipolar voltage of 45.5 (14.3)% to 59.3 (19.8)% of normal voltage (p = 0.002) and reduction of the low-voltage area from 28.7 (12.1)% to 20.3 (13.5)%; (p = 0.016). During the 12-month follow-up, the left ventricular ejection fraction (LVEF) improved from 40.8 (6.9)% to 47.1 (10.6)%; (p = 0.037). CONCLUSION: Left ventricular EMM and percutaneous intramyocardial cell injection in patients with AMI was shown to be a safe procedure. It is associated with improved LVEF and electromechanical parameters after 12-months' follow-up. TRIAL REGISTRATION NUMBER: Eudra-CT-No 2005-003629-19.

E6AP promotes the degradation of the PML tumor suppressor.

The promyelocytic leukemia (PML) tumor suppressor is essential for the formation of PML nuclear bodies (NBs). PML and PML-NBs have been implicated in the regulation of growth inhibition, senescence and apoptosis. PML is activated in response to stress signals and is downregulated in certain human cancers. However, the factors mediating PML stability are incompletely understood. Here we demonstrate that a catalytically active form of the mammalian E3 ligase E6AP (HPV E6-associated protein) acts to reduce the half-life of the PML protein by promoting its degradation in the proteasome. E6AP mediates the ubiquitination of PML in an in vitro ubiquitination assay. E6AP and PML interact at physiological levels and colocalize in PML-NBs. Importantly, PML protein expression is elevated in multiple organs and cell types from E6AP null mice and in lymphoid cells is associated with increased number and intensity of PML-NBs. This PML elevation is enhanced in response to DNA damage. Our results identify E6AP as an important regulator of PML and PML-NBs.

Side-specific operant conditioning of antennal movements in the honey bee.

Operant conditioning of antennal movements in honey bees was used to investigate whether learned changes on one antenna influence antennal movements of the contralateral antenna. Conditioning of the right antenna did not alter antennal movements of the left antenna and subsequent conditioning of the left antenna did not abolish the previously learned change in the right antenna. Thus, the antennal systems on each side are largely independent from each other.

p53 controls hPar1 function and expression.

Human protease-activated receptor 1 (hPar1) is a bona fide receptor of the hemostatic protease thrombin, and has a central function in tumor progression. Inactivation of the tumor suppressor gene p53 is one of the most common genomic alterations occurring in cancer. Here, we address the interrelations between p53 and hPar1 in cancer. We demonstrate an inverse correlation between hPar1 gene expression and wild-type (wt) p53 levels, and a direct correlation with levels of the mutant (mt) p53. Bioinformatic search revealed the presence of at least two p53 motifs in the hPar1 promoter. Indeed, temperature-sensitive (ts) p53 forms reduced hPar1 promoter activity on wt p53 expression. Ectopic introduction of the p53R175H mutant into cells lacking p53 caused a moderate two-fold induction of hPar1 promoter activity. Chromatin immunoprecipitation (ChIP) analyses confirmed a physical association between the p53 protein and hPar1 chromatin fragments. In parallel, PAR1 function is attenuated by p53, as shown by inhibition of pFAK levels and a Matrigel invasion assay. Ectopic reinforcement of hPar1 rescued the inhibition conferred by p53, confirming that p53 directly affects hPar1 expression and function. Altogether, we provide evidence for a direct binding between p53 and hPar1 chromatin, and assign hPar1 as a target of p53.

PML enhances the regulation of p53 by CK1 in response to DNA damage.

In response to stress, p53 is accumulated and activated to induce appropriate growth inhibitory responses. This requires the release of p53 from the constraints of its negative regulators Mdm2 and Mdm4. A key event in this dissociation is the phosphorylation of p53 at threonine residue (Thr18) within the Mdm2/4-binding domain. Casein kinase 1 (CK1) plays a major role in this phosphorylation. The promyelocytic leukemia protein (PML) regulates certain modifications of p53 in response to DNA damage. Here, we investigated the role of PML in the regulation of Thr18 phosphorylation. We found that PML enhances Thr18 phosphorylation of endogenous p53 in response to stress. On DNA damage, CK1 accumulates in the cell, with a proportion concentrated in the nucleus together with p53 and PML. Furthermore, CK1 interacts with endogenous p53 and PML, and this interaction is enhanced by genotoxic stress. Inhibition of CK1 impairs the protection of p53 by PML from Mdm2-mediated degradation. Our findings support a role for PML in the regulation of p53 by CK1. We propose that following DNA damage, PML facilitates Thr18 phosphorylation by recruiting p53 and CK1 into PML nuclear bodies, thereby protecting p53 from inhibition by Mdm2, leading to p53 activation.