The role of PKCζ in NMDA-induced retinal ganglion cell death: Prevention by aspirin

Intravitreal NMDA injection has been shown to induce the excitotoxic loss of retinal cells. The retinal ganglion cell apoptosis induced by NMDA is thought to play an important role in retinal ischemia injury and NMDA-injected rat has been used as a model of neuronal loss in diseases such as glaucoma. In this experimental model, we studied the early effects of NMDA leading to the degeneration of retinal ganglion cells. PKCζ regulates the NF-κB pathway in cellular responses to various stresses and we have shown that aspirin inhibits purified human PKCζ. We therefore investigated the molecular mechanism by which retinal cells limit ocular injury following NMDA treatment. We found that the NMDA-induced apoptosis of ganglion cells was mediated, at least partly, by PKCζ. This enzyme was activated early in the cellular response to NMDA. Prolonged activation was followed by PKCζ cleavage, and nuclear translocation of the C-terminal region of this protein—a critical event for the survival of retinal cells. We also found that pretreatment with aspirin or the coinjection of NMDA with a specific PKCζ inhibitor counteracted the effects of NMDA. These findings provide new insight into the role played by PKCζ in neuronal loss in glaucoma.     

Dietary and biliary phosphatidylcholine activates PKCζ in rat intestine

Chylomicron output by the intestine is proportional to intestinal phosphatidylcholine (PC) delivery. Using five different variations of PC delivery to the intestine, we found that lyso-phosphatidylcholine (lyso-PC), the absorbed form of PC, concentrations in the cytosol (0 to 0.45 nM) were proportional to the input rate. The activity of protein kinase C (PKC)ζ, which controls prechylomicron output rate by the endoplasmic reticulum (ER), correlated with the lyso-PC concentration suggesting that it may be a PKCζ activator. Using recombinant PKCζ, the K_m for lyso-PC activation was 1.49 nM and the V_max 1.12 nM, more than the maximal lyso-PC concentration in cytosol, 0.45 nM. Among the phospholipids and their lyso derivatives, lyso-PC was the most potent activator of PKCζ and the only one whose cytosolic concentration suggested that it could be a physiological activator because other phospholipid concentrations were negligible. PKCζ was on the surface of the dietary fatty acid transport vesicle, the caveolin-1-containing endocytic vesicle. Once activated, PKCζ, eluted off the vesicle. A conformational change in PKCζ on activation was suggested by limited proteolysis. We conclude that PKCζ on activation changes its conformation resulting in elution from its vesicle. The downstream effect of dietary PC is to activate PKCζ, resulting in greater chylomicron output by the ER.     

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.     

PLCζ and its role as a trigger of development in vertebrates

A major unresolved issue in developmental biology is the precise mechanism whereby the sperm activates the oocyte. With the discovery that calcium signals are the primary trigger for oocyte activation, a key remaining question became the identification of the signaling protein that mediates such calcium signals at fertilization. A major step forward came in 2002 with the discovery of a sperm-specific mammalian phospholipase C called phospholipase C zeta (PLCζ), which had the expected properties of the mammalian oocyte activation factor and was subsequently identified in other vertebrate groups. Most recently, defects in PLCζ have been shown to be linked to certain types of male infertility in humans. Despite these advances, many questions remain about the precise mechanism of action of PLCζ and the extent of its role during oocyte activation in the vertebrate kingdom. In this review, we will look at the current state of understanding of PLCζ's mechanism of action and physiological role in mammals and other vertebrates, and identify areas of uncertainty that still remain to be resolved.     

Comparative Analysis of PKСα and PKCζ Activities in Rat and Lamprey Erythrocytes of Different Ages

The present study aimed to find out a link between ageing of rat and lamprey erythrocytes and activity of two isoforms of protein kinase C (PKC), РKСα and РKСζ. The whole cell population was separated into fractions of different ages in Percoll density gradient. The validity of separation was confirmed by the number of immature erythrocytes, reticulocytes. PKC activity was analyzed in cytosolic and membrane cell fractions. Rat erythrocytes express both PKC isoforms, РKСα and РKСζ, whereas lamprey erythrocytes express only РKСζ. РKСα is identified as a major band at ~ 80 kDa and minor bands at ~ 55–65 kDa; РKСζ is represented by a single band at ~ 80 kDa. In young rat erythrocytes, РKСα is detected mainly in cytosolic fractions, while in membrane fractions its level is by far lower. As cells age, PKCα is translocated from the cytosol to membranes and undergoes proteolytic degradation due to repeated cycles of activation. As a result, in aged erythrocytes relative total PKCα expression (as a sum of expressions in the cytosol and membranes per total protein level) diminishes, indicating a depletion of the PKCα pool and a decline in its functional activity. In both animal species, a highest PKCζ level is observed in the cytosol of young erythrocytes. Erythrocyte ageing is accompanied by a gradual decrease in expression of free cytosolic PKCζ and concurrent increase in the level of its membrane-bound forms. However, in contrast to PKCα, PKCζ is not proteolyzed; its total level in cells and perhaps functional activity do not change throughout the erythrocyte lifespan.     

Hürthle cell carcinoma: diagnostic and therapeutic implications

Background  

Hürthle cell carcinoma is a variant of follicular cell carcinoma of thyroid. It may present as a low-grade tumour or as a more aggressive type. Prognosis depends upon the age of the patient, tumour size, extent of invasion and initial nodal or distant metastasis.     

A phase II study of interferon α and low-dose subcutaneous interleukin-2 in advanced renal cell carcinoma

 The activity of the drugs employed in the treatment of metastatic renal cell carcinoma, including biological response modifiers, is limited; one of the aims of clinical research in this area is to maintain the benefits of treatment whilst reducing its toxicity to a minimum level. We have evaluated toxicity and response of the combined administration of recombinant interferon α (IFNα) and low-dose subcutaneous (s.c.) recombinant interleukin-2 (IL-2) in patients with advanced renal cell carcinoma. A group of 20 previously untreated patients with advanced renal cell carcinoma were included in the study. Treatment consisted of 3 MU/m² recombinant IFNα daily i.m. continuously, and 0.5 MU/m² recombinant IL-2 twice a day s.c. on days 1–5 for the first week, followed by 1 MU/m² twice a day for 5 days in the following weeks. For IL-2, a 1-week rest was allowed after 4 weeks of treatment. Response was assessed after 3 months of therapy. Three objective responses were seen, one complete and two partial. Eight patients had stable disease. The median time to progression was 6 months; the median survival for all patients was 14 months. Side-effects were low, limited to grades 1 and 2 in the majority of patients, and included fever, anemia, leukopenia, dyspnea, and abnormalities of liver and renal function tests. Any flu-like syndrome was judged moderate in most patients; however, one-third of the patients refused treatment mostly because of the flu-like syndrome. One of these was the patient experiencing a complete response, who virtually received IFNα alone. This regimen, similar to others employed in the treatment of advanced renal cell carcinoma, produced a 15% response rate (95% confidence interval, 0–31%) with 14 months median survival, moderate toxicity and low cost, and required no hospitalization. These data seem to indicate an effectiveness comparable to, and a toxicity lower than, that of regimens employing higher doses of IL-2. Received: 25 March 1997 / Accepted: 22 May 1997     

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Expression of miR-486-5p and its significance in lung squamous cell carcinoma

Lung squamous cell carcinoma (LUSC) is one of the main histological types of lung cancer with high mortality. The role of microRNA-486-5p in LUSC remains unclear. In the current study, the aim was to explore miR-486-5p expression and its role in LUSC. The miR-486-5p expression was significantly low-expressed in patients with LUSC from The Cancer Genome Atlas database, which was further confirmed in the Gene Expression Omnibus database, patients’ tissues, different cell lines by quantitative real-time polymerase chain reaction, and the high-throughput gene sequencing data of lung tissues of mice after a long-term B(a)P exposure. The meta-analysis was performed to evaluate the expression and diagnosis power of miR-486-5p (standard mean difference = −2.25; 95% confidence interval: −3.47 to −1.03; P = 0.0003; area under curve = 0.9082). Functional enrichment analysis revealed the potential function of miR-486-5p in LUSC using gene set enrichment analysis and clusterProfiler package in R software. At last, the hub genes (PTEN, TEK, PIK3R1, PPM1B, SMAD2, and SPTA1) of miR-486-5p were verified. In conclusion, miR-486-5p may be a LUSC antioncogene, playing an important role to serve as a biomarker in LUSC.     

Sequential administration of interferon γ and interleukin-2 in metastatic renal cell carcinoma: results of a phase II trial

Background: Because of the known efficacy of several cytokines in the treatment of advanced renal cell cancer (RCC), we have conducted a phase II trial of the efficacy and toxicity of subcutaneous interferon γ (IFNγ) and interleukin-2 (IL-2). Methods: 63 patients with progressive metastatic RCC were treated with 100 μg recombinant IFNγ1b administered three times weekly during weeks 1 and 2 and with 4.5 MU recombinant IL-2 administered on 4 consecutive days during weeks 3 and 4, every 6 weeks. Results: 11% of patients had an objective response (CR: 3%, PR: 8%), 33% had SD. Toxicity was generally mild. The median duration of remissions (CR + PR) was 9.6 months; the median duration of SD 8 months. A significant survival benefit was evident at a median observation time of 51 months for patients (44%) responding to therapy (P < 0.0001). Conclusions: we conclude that sequential treatment with IFNγ and IL-2 may prolong survival in patients with metastatic RCC responding to therapy. Received: 2 April 2000 / Accepted: 21 April 2000     

Role of miR-486-5p in regulating renal cell carcinoma cell proliferation and apoptosis via TGF-β–activated kinase 1

Renal cell carcinoma (RCC) is a common kidney tumor in adults. The role of miR-486-5p in RCC is unknown. The aim of our study was to identify new targets regulated by miR-486-5p in RCC, to obtain a deeper insight into the network and to better understand the role of these microRNAs and their targets in carcinogenesis of RCC. We performed a series of tests and found consistently lower expression levels of miR-486-5p in kidney cancer cells. Restoration of miR-486-5p expression in RCC cells could lead to the suppression of cell proliferation and the increase of cell apoptosis. Further studies demonstrated that TGF-β–activated kinase 1 was a target gene of miR-486-5p in kidney cancer cells. It was also shown that C-C motif chemokine ligand 2 (CCL2) from tumor-associated macrophages downregulated miR-486-5p expression, and miR-486-5p inhibited RCC cell proliferation and apoptosis resistance induced by CCL2. The study demonstrates that there are potential diagnosis and therapy values of miR-486-5p in RCC.     

The role of C1QBP in CSF-1-dependent PKCζ activation and macrophage migration

Macrophages view as double agents in tumor progression. Trafficking of macrophages to the proximity of tumors is mediated by colony-stimulating factor-1 (CSF-1), a growth factor. In this study, we investigated the role of complement1q-binding protein (C1QBP)/ atypical protein kinase C ζ (PKCζ) in CSF-1-induced macrophage migration. Disruption of C1QBP expression impaired chemotaxis and adhesion of macrophage. Phosphorylation of PKCζ is an essential component in macrophage chemotaxis signaling pathway. C1QBP could interact with PKCζ in macrophage. C1QBP knockdown inhibited CSF-1 induced phosphorylation of PKCζ and integrin-β1. However, C1QBP knockdown didn’t affect the phosphorylation of PKCζ induced by MCP-1. Furthermore, CSF-1 from RCC cell condition medium promoted macrophage chemotaxis and adhesion. Taken together, our results demonstrated that C1QBP plays an essential role in CSF-1 induced migration of macrophages.     

G Protein γ subunit 7 loss contributes to progression of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a common urinary neoplasm, looking for useful candidates to establish scientific foundation for the therapy of ccRCC is urgent. We downloaded genomic profiles of GSE781, GSE6244, GSE53757, and GSE66271 from the Gene Expression Omnibus (GEO) database. GEO2R was used to analyze the derivative genes, while hub genes were screened by protein-protein interactions and cytoscape. Further, overall survival, gene methylation, gene mutation, and gene expression were all analyzed using bioinformatics tools. Colony formation and cell-cycle assay were used to detect the biological function of GNG7 in vitro. We found that GNG7 was downregulated in ccRCC tissues and negatively associated with overall survival in ccRCC patients. We also found that promoter methylation and frequent gene mutation were responsible for GNG7 gene suppression. GNG7 low expression was related to upregulation of enhancer of zeste homolog 2 and downregulation of disabled homolog 2-interacting protein. Further, Gene Set Enrichment Analysis results showed that mTOR1, E2F, G2M, and MYC pathways were all significantly altered in response to GNG7 low expression. In vitro, A498 and 786-O cells in which GNG7 expression was silenced, exhibited a lower G1 phase when compared to the negative control cells. Taken together, our findings suggest that GNG7 is a tumor suppressor gene in ccRCC progression and represents a novel candidate for ccRCC treatment.     

Upregulation of Rpn10 promotes tumor progression via activation of the NF-κB pathway in clear cell renal cell carcinoma

The ubiquitin-proteasome system (UPS) plays a central role in regulating protein homeostasis in tumor progression.The proteasome subunit Rpn10 is associated wit...     

Prospects of nanoparticle–DNA binding and its implications in medical biotechnology

Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle–DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle–DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle–DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle–DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.     

Complete response of metastatic renal cell carcinoma to low-dose interferon-γ treatment

The course of metastatic renal cell carcinoma may be positively influenced by immunotherapeutic agents. We report a case of renal cell carcinoma showing a complete response to once-weekly low-dose s. c. interferon- (INF) treatment in multiple metastatic sites (lung, chest wall, abdomen, vertebral body), but concomitantly developing a solitary brain metastasis. High initial interleukin-6 (IL-6) levels returned to normal during IFN treatment suggesting that IFN may have interrupted an autocrine IL-6/IL-6-receptor loop of the tumor cells. The duration of complete remission in the extracerebral sites is now 46+ months. IFN may be less active beyond the blood/brain barrier.