The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.Dissemination of tumor cells is strictly linked to their ability to attach to and move within the extracellular matrix (ECM) in a three-dimensional (3D) environment. The use of 3D experimental model systems revealed that a higher complexity in cell migration and adaptation responses exists in the 3D model than in the classical 2D model (10, 16, 41, 49). A striking example is given by the fact that only in 3D could individually migrating cells use different mechanisms such as mesenchymal and amoeboid motility (16, 17). The relative slow mesenchymal migration is characterized by a fibroblast-like spindle shape and is dependent on integrin-mediated adhesion and on protease function (16). The amoeboid motility can in some cases represent a less adhesive, integrin-independent type of movement. Cells use a propulsive mechanism and are highly deformable, and rather than degrade the matrix, they are able to squeeze through it (16). As a result, the cells that use the amoeboid motility can potentially move faster than cells that use a mesenchymal strategy. Mesenchymal and amoeboid movements are also characterized by a different involvement of small GTPases of the Rho family. A high RhoA activity is associated mainly with the amoeboid motility, while the mesenchymal migration needs a high Rac activity at the leading edge to promote the extension of cellular protrusions (41, 48). Under certain circumstances, cancer cells can undergo conversion from a mesenchymal toward an amoeboid motility, an event referred as mesenchymal-amoeboid transition (MAT) (50). MAT represents a putative escape mechanism in tumor cell dissemination that could be induced by inhibition of pericellular proteolysis (50) or by increased membrane-associated RhoA activity (18, 40).Key mediators of cell motility through ECM substrates are the members of the Src family kinases. The prototype of Src family kinases, c-Src (14), is activated following cell-ECM adhesion and contributes to regulate the focal adhesion turnover and the cytoskeletal modifications necessary for normal cell adhesion and motility (52). The c-Src gene is the proto-oncogene of the transforming gene v-src of Rous sarcoma virus, and its elevated protein level and activity have been found in many human tumors (20, 28, 27, 34). Despite the accumulation of information and new molecular understanding of how Src is controlled, there is still an incomplete picture about its role in the generation of the malignant phenotype. v-Src shows higher levels of the kinase activity and transforming ability than c-Src (14, 15, 52). It induces normal cells to acquire a variety of transformed features, including alteration of morphology and increase of invasion ability due to its role in focal adhesion remodeling (7, 9, 13).Many data suggest that there is a close relationship between cell-ECM interaction and the proliferation and movements in both normal and tumor cells (5, 38, 43). Accordingly, Src activation may influence not only cell motility but also cell cycle progression by targeting the cell cycle inhibitor p27^kip1 to proteasomal degradation (22, 39). Recent evidences indicated that p27^kip1 (hereafter called p27) can also regulate cell migration, even though its role still remains controversial since it has been reported to either block or stimulate cell movements (1, 4, 11, 19, 21, 23, 29, 45).Based on these notions, we tested the possible contribution of p27 to the growth and motility phenotypes induced by v-src transformation, with special regard to those cellular invasive features that can be observed in 3D environments. By studying in vitro and in vivo the behavior of wild-type (WT) and p27-null fibroblasts transformed with v-src, we highlight a new role for p27 in the regulation of cellular plasticity that can ultimately drive tumor cell shape, motility, and invasion.     

Proteome alteration of U251 human astrocytoma cell after inhibiting retinoic acid synthesis

Retinoic acid (Ra) is crucial for the patterning and neuronal differentiation in the central nervous system (CNS). Ra deficiency in animals disrupts the motor activities and memory abilities. The molecular mechanisms underlying these behavior abnormalities remain largely unknown. In the current study, we treated the astrocytoma cells with citral, an inhibitor of Ra synthesis. We analyzed the differences in the protein concentrations between the treated and untreated astrocytoma cells by two-dimensional gel electrophoresis (2-DE), Imagemaster software, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In total, 39 of 46 altered protein spots with significant mascot scores were identified representing 36 proteins, that were involved in significantly altered glutamate metabolism, lipid metabolism, mitochrondrial function, and oxidative stress response by Ingenuity Pathway Analysis (IPA). Altered 3-phosphoglycerate dehydrogenase (PHGDH) was also observed in western blot. These data provide some clues for explaining the behavioral changes caused by Ra deficiency, and support the hypothesis that Ra signaling is associated with some symptoms of neurodegenerative disorders and schizophrenia.     

Proteomic profiling during atherosclerosis progression: Effect of nebivolol treatment

There is a great need for the identification of biomarkers for the early diagnosis of atherosclerosis and the agents to prevent its progression. The aim of this study was to explore the effect of 24 week of nebivolol (a third-generation vasodilatory beta-blocker) treatment on serum protein profiles in Apo E^?/? mice during atherosclerosis progression. Nebivolol treated and non-treated (the control group) groups consisted of 10 genetically modified homozygous Apo E^?/? mice. Proteomic analyses were performed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the serum samples from the nebivolol treated and non-treated Apo E^?/? mice. The protein profiles obtained using three different chips, CM10 (weak cation-exchange), H50 (reverse phase), and IMAC30-Cu²⁺ (immobilized metal affinity capture) were statistically analyzed using the ProteinChip data manager 3.0 program. At the end of 24 week of nebivolol-treatment period, a total of 662 protein/peptide clustering peaks were detected using 12 different conditions and reading with high and low intensity laser energy. The highest total number of protein/peptide clusters was found on H50 chip array. The peak intensities of 95 of the 662 protein/peptide clusters were significantly different in the nebivolol-treated atherosclerotic group in comparison to the non-treated control mice groups (P < 0.05). Forty-three protein/peptides were up-regulated (high signal intensity) while 52 protein/peptides had lower signal intensity (down-regulated) in the nebivolol-treated atherosclerotic group. The proteomic profiles of nebivolol-treated Apo E^?/? mice were different than the control group indicating a potential role of nebivolol in atherosclerosis. Our study contributes to understand the efficacy of nebivolol on serum protein/peptide profiles during atherosclerosis development.     

A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. This work was supported by the National Institutes of Health (R21CA094507). An erratum to this article can be found at     

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.Planetary protection efforts work toward protecting (i) solar system bodies from contamination by terrestrial biological material (forward contamination), thus preserving opportunities for future scientific investigation, and (ii) the Earth from harmful contamination by materials returned from outer space (back contamination) (5). These approaches apply directly to the control and eradication of microorganisms present on the surfaces of spacecraft intended to land, orbit, fly by, or be in the vicinity of extraterrestrial bodies. Consequently, current planetary protection policies require that spacecraft be assembled and readied for launch in controlled clean room environments. To achieve these conditions and maintain compliance with good manufacturing practice regulations, robotic spacecraft components are assembled in ultraclean facilities. Much like facilities in the medical, pharmaceutical, and semiconductor sectors, National Aeronautics and Space Administration (NASA) spacecraft assembly clean rooms (SAC) are kept extremely clean and are maintained to the highest of industry standards (17). Filtered air circulation, controlled temperature and humidity, routine exposure to disinfectants and surfactants, and nutrient-limiting, oligotrophic conditions make it very challenging for microorganisms to persist in such environments, but these measures by no means eradicate biological contaminants entirely (18). Several investigations, both culture based and culture independent, have demonstrated that a variety of bacterial taxa are repeatedly isolated under clean room conditions (18, 24, 26; P. Vaishampayan, S. Osman, G. Andersen, and K. Venkateswaran, submitted for publication). However, despite a growing understanding of the diverse microbial populations present in SAC, predicting the true risk of any such microbes’ compromising the findings of extraterrestrial life detection efforts remains a significant challenge (30). A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination (33).The purification of nucleic acids, subsequent PCR amplification, and shuttling of 16S ribosomal “fingerprint” genes from noncultivable microorganisms into genetically amenable lab strains of Escherichia coli have evolved into a gold standard of molecular means to elucidate the microbial diversity in a given sample. In theory, the cloning and sequencing of 16S ribosomal genes from each and every cell present, regardless of cultivability and inclusive of novel taxa, would result in a comprehensive survey of microbial communities on the surfaces of SAC and colocated spacecraft (24, 26). Unfortunately, the full-length sequencing of all 16S rRNA genes from environmental samples would be prohibitively expensive, making the approach unfeasible for generating comprehensive phylogenetic profiles of complex microbial communities.Attempting to infer population membership from clone libraries limited to hundreds or thousands of sequences has proven to be insufficient for detecting extremely low-abundance organisms. Recent analyses of phylogenetic DNA extracted from soil, water, and air revealed that laboriously derived clone libraries severely under-represent complex bacterial communities compared to very rapid (i.e., requiring only hours) DNA microarray approaches (1, 6, 11, 23, 36). One of the reasons for this is the high sensitivity of PhyloChip methodologies, which are able to detect organisms present in amounts below 10⁻⁴ abundance of the total sample (12). Numerous validation experiments using sequence-specific PCR have confirmed that taxa identified by the microarray were indeed present in the original environmental samples, despite their absence in corresponding clone libraries (3). This highlights the utility of the method compared to classical cloning. Although the analysis of each sample by the PhyloChip provides detailed information on microbial composition, the highly parallel and reproducible nature of this array allows tracking community dynamics over time and treatment. Even without prior sequence information, PhyloChip can identify specific microbial interactions that are key to particular changing environments.A comprehensive census of the microbial communities on the surfaces in three NASA SAC supporting two distinct missions was conducted. To ensure that the maximum diversity of resident microbiota was uncovered, subsamples from each clean room surface sampling were subjected to both DNA microarray protocols and conventional cloning and sequencing of 16S rRNA genes. This study, to our knowledge the first of its kind, focused on comparing the microbial diversity profiles resulting from DNA microarray analyses and conventional cloning and sequencing of 16S rRNA genes arising from a variety of low-biomass surfaces.     

Copper(II) complexes with an avian prion N-terminal region and their potential SOD-like activity

Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)₄-NH₂, and the mutated residue, Ac-(PHNPGF)₄-NH₂, to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)₄-NH₂, was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat.     

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.