High Tumour Cannabinoid CB1 Receptor Immunoreactivity Negatively Impacts Disease-Specific Survival in Stage II Microsatellite Stable Colorectal Cancer

BACKGROUND: There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB(1) receptor immunoreactive intensity (CB(1)IR intensity) is associated with disease severity and outcome. METHODOLOGY/PRINCIPAL FINDINGS: CB(1)IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB(1)IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB(1)IR <2 and ≥2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB(1)IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB(1)IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB(1)IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB(1)IR upon disease specific survival. The 5 year probabilities of event-free survival were: 85±5 and 66±8%; tumour interior, 86±4% and 63±8% for the CB(1)IR<2 and CB(1)IR≥2 groups, respectively. CONCLUSIONS/SIGNIFICANCE: The level of CB(1) receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB(1)IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients

Protein Synthesis Requirement for the Formation of Synaptic Elements

The formation of synapses in cell cultures of rat cerebellum was examined in the presence of the protein synthesis inhibitor cycloheximide. First, cell survival in the presence of 25 μg/ml cycloheximide was determined by phase contrast microscopy, trypan blue exclusion, total protein and uptake of [3H]gamma-aminobutyric acid (GABA). Neurons with 24 h incubation in cycloheximide appeared normal with little cell death, but by 48 h incubation the first signs of cell death were found. Some viable neurons were still found in cultures incubated continuously in cycloheximide for 72 h. Normally, the number of synapses seen in cerebellar cultures with the electron microscope shows an increase during the first several weeks in culture. However, the number of synapses in cultures treated with cycloheximide decreased, indicating that inhibition of protein synthesis at least partially inhibited synaptogenesis. Cycloheximide also inhibited the maintenance of synapses already formed as seen by the decrease in the number of synapses from the time the cycloheximide was added. To determine the sensitivity of the forming presynaptic element to cycloheximide, the development of apparent presynaptic elements was investigated. In cultures treated with polylysine-coated sepharose beads, neurites grew and formed apparent presynaptic elements with the bead taking the position of the postsynaptic element. Cultures pretreated with cycloheximide for 1 h followed by 24 h incubation with both cycloheximide and coated beads showed a normal number of apparent presynaptic elements. The first decrease in numbers was seen after 12 h preincubation and 12 h incubation with both cycloheximide and coated beads. Even after 72 h continuous incubation some apparent presynaptic elements could be formed although at reduced levels. Results presented here suggest that continuous protein synthesis is not necessary for the formation of the presynaptic element, but that active protein synthesis is required for neurons to form and maintain postsynaptic elements

Enhanced Survival of Apparent Presynaptic Elements on Polylysine-Coated Beads by Inhibition of Non-Neuronal Cell Proliferation

Increased survival of presynaptic-like neuronal profiles was found in cell cultures of rat cerebellum when the non-neuronal cell numbers were reduced with an antimitotic drug. In both treated and untreated cell cultures, neurites grew onto the polylysine-coated surface of sepharose beads and formed a swelling. The neuronal swelling contained an accumulation of synaptic vesicles and a membrane density at the site of contact with the bead and was called an apparent presynaptic element. The apparent presynaptic elements in untreated cultures increased in number from the time the beads were added to the culture to 7 days incubation and then showed a decrease to one half the 7-day value at 14 days incubation. A 75% reduction in cell division of non-neuronal cells was seen in cultures exposed to a 5 × 10-6 M cytosine arabinoside (Ara-C) for 2 days. Adding polylysine-coated beads to cultures treated with Ara-C showed at 14 days incubation a 7-fold increase in the number of apparent presynaptic elements as compared to untreated cultures. Additional experiments examined the numbers of neurites on the beads and found only small differences between treated and untreated cultures. A decrease, however, was shown in the number of glial fibrillary acidic protein staining astrocytes on the surface of the beads in treated cultures. The reduction of astrocytes by Ara-C appeared to enhance the survival of apparent presynaptic elements but did not enhance the growth of neurites. These results suggest that proliferating non-neuronal cells at a site of injury in the central nervous system may inhibit the formation of synaptic contacts and the growth of neurites through the site of injury

Role of the Human T-Cell Leukemia Virus Type 1 PTAP Motif in Gag Targeting and Particle Release

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent

Overexpression of HuD, but Not of Its Truncated Form HuD I+II, Promotes GAP‐43 Gene Expression and Neurite Outgrowth in PC12 Cells in the Absence of Nerve Growth Factor

: We have previously shown that the RNA‐binding protein HuD binds to a regulatory element in the growth‐associated protein (GAP)‐43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP‐43‐positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F‐actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP‐43 without changes in levels of other NGF‐induced proteins, such as SNAP‐25 and tau. In mRNA decay studies, HuD stabilized the GAP‐43 mRNA, whereas HuD I+II did not have any effect either on GAP‐43 mRNA stability or on the levels of GAP‐43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP‐43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function

LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis