Model establishment of prognostic-related immune genes in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck in the world. At present, the treatment methods include surgery, radiotherapy, and chemotherapy, but the 5-year survival rate is still not ideal and the quality of life of the patients is low. Due to the relative lack of immunotherapy methods, this study aims to build a risk prediction model of related immune genes, which can be used to effectively predict the prognosis of laryngeal cancer patients, and provide targets for subsequent immunotherapy. METHODS: We collected the 111 cases of laryngeal squamous cell carcinoma and 12 matched normal samples in the The Cancer Genome Atlas Database (TCGA) gene expression quantification database. The differentially expressed related immune genes were screened by R software version 3.5.2. The COX regression model of immune related genes was constructed, and the sensitivity and specificity of the model were evaluated. The risk value was calculated according to the model, and the risk curve was drawn to verify the correlation between related immune genes, risk score, and clinical traits. RESULTS: We selected 8 immune-related genes that can predict the prognosis of LSCC in a COX regression model and plotted the Kaplan-Meier survival curve. The 5-year survival rate of the high-risk group was 16.5% (95% CI: 0.059-0.459), and that of the low-risk group was 72.9% (95% CI: 0.555-0.956). The area under the receiver operating characteristic (ROC) curve was used to confirm the accuracy of the model (AUG = 0.887). After univariate and multivariate regression analysis, the risk score can be used as an independent risk factor for predicting prognosis. The risk score (P = .021) was positively correlated with the clinical Stage classification. CONCLUSION: We screened out 8 immune genes related to prognosis: RBP1, TLR2, AQP9, BTC, EPO, STC2, ZAP70, and PLCG1 to construct risk value models, which can be used to speculate the prognosis of the disease and provide new targets for future immunotherapy.

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Bulk tumour cell migration in lung carcinomas might be more common than epithelial-mesenchymal transition and be differently regulated.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is one mechanism of carcinoma migration, while complex tumour migration or bulk migration is another - best demontrated by tumour cells invading blood vessels. METHODS: Thirty cases of non-small cell lung carcinomas were used for identifying genes responsible for bulk cell migration, 232 squamous cell and adenocarcinomas to identify bulk migration rates. Genes expressed differently in the primary tumour and in the invasion front were regarded as relevant in migration and further validated in 528 NSCLC cases represented on tissue microarrays (TMAs) and metastasis TMAs. RESULTS: Markers relevant for bulk cancer cell migration were regulated differently when compared with EMT: Twist expressed in primary tumour, invasion front, and metastasis was not associated with TGFß1 and canonical Wnt, as Slug, Snail, and Smads were negative and ß-Catenin expressed membraneously. In the majority of tumours, E-Cadherin was downregulated at the invasive front, but not absent, but, coexpressed with N-Cadherin. Vimentin was coexpressed with cytokeratins at the invasion site in few cases, whereas fascin expression was seen in a majority. Expression of ERK1/2 was downregulated, PLCγ was only expressed at the invasive front and in metastasis. Brk and Mad, genes identified in Drosophila border cell migration, might be important for bulk migration and metastasis, together with invadipodia proteins Tks5 and Rab40B, which were only upregulated at the invasive front and in metastasis. CXCR1 was expressed equally in all carcinomas, as opposed to CXCR2 and 4, which were only expressed in few tumours. CONCLUSION: Bulk cancer cell migration seems predominant in AC and SCC. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are associated with bulk cancer cell migration. This type of migration requires an orchestrated activation of proteins to keep the cells bound to each other and to coordinate movement. This hypothesis needs to be proven experimentally.

Impairment of kindling development in phospholipase Cγ1 heterozygous mice.

OBJECTIVE: Elucidating molecular mechanisms underlying limbic epileptogenesis may reveal novel targets for preventive therapy. Studies of TrkB mutant mice led us to hypothesize that signaling through a specific phospholipase (PLC), PLCγ1, promoted development of kindling. METHODS: To test this hypothesis, we examined the development of kindling in PLCγ1 heterozygous mice. We also examined the cellular and subcellular location of PLCγ1 in adult wild-type mice. RESULTS: The development of kindling was impaired in PLCγ1 heterozygous mice compared to wild-type controls. PLCγ1 immunoreactivity was localized to the soma and dendrites of both excitatory and inhibitory neurons in the hippocampus of adult mice. SIGNIFICANCE: This study implicates PLCγ1 signaling as the dominant pathway by which TrkB activation promotes limbic epileptogenesis. Its cellular localization places PLCγ1 in a position to modify the efficacy of both excitatory and inhibitory synaptic transmission. These findings advance PLCγ1 as a novel target for therapies aimed at preventing temporal lobe epilepsy induced by status epilepticus.

A role for PKCepsilon during C2C12 myogenic differentiation.

In a previous report we have demonstrated that PLCgamma1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCgamma1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCepsilon and eta expression, and that PKCepsilon is able to form a complex with PLCgamma1. The increase in PKCepsilon amount during myogenic differentiation is associated to an increase in PKCepsilon activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCgamma1 and PKCepsilon localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCgamma1 and PKCepsilon increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCgamma1-PKCepsilon complex co-localizes with protein 58K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCepsilon form, i.e. PKCepsilon phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCgamma1, we analyzed the effect on cyclin D3 expression of either PKCepsilon overexpression or silencing, in order to investigate whether PKCepsilon could also affect cyclin D3 expression. The results showed that either a modification of PKCepsilon expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCepsilon in regulating insulin inositide-dependent PLCgamma1 signalling in skeletal muscle differentiation.

Cellular response induced by a galactose-specific adhesin of enteroaggregative Escherichia coli in INT-407 cells.

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

Direct evidence for the action of phosphatidylinositol (3,4,5)-trisphosphate-mediated signal transduction in the 2-cell mouse embryo.

Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 micromol/L)- and diltiazem (10 micromol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1-3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.

Phospholipase C expression and activity in smooth muscle cells of renal arterioles and aorta of young, spontaneously hypertensive rats during culture.

BACKGROUND: Phospholipase C (PLC)-beta(1) and -delta(1), but not -gamma(1), protein expressions in fresh renal arterioles and aorta are greater in 6-week-old, spontaneously hypertensive rats (SHRs) versus normotensive Wistar-Kyoto rats (WKYs). This PLC activity is also greater in both vessels of SHRs. In the present study, we tested whether cultured vascular smooth muscle cells (VSMCs) of preglomerular arterioles and aorta accurately reflect strain differences observed in fresh vessels, with VSMCs of SHRs predicted to have higher levels of PLC isozymes and enzyme activity. We assessed the stability of variables over passages 3 to 11. METHODS: The VSMCs were isolated and cultured using standard techniques. The PLC-isozyme protein levels and catalytic activity were determined by Western blot analysis and inositol 1,4,5-trisphosphate (IP(3)) production, respectively. RESULTS: Immunoblots showed expression of PLC-gamma(1) and -delta(1), but not PLC-beta(1), in VSMCs from both vessels. Arteriolar VSMCs of SHRs had three-to-fivefold higher levels of PLC-gamma(1) and -delta(1) during passages 3 to 8. Enzymatic activity in these VSMCs was higher in SHRs versus WKYs, especially during passages 6 to 11. In contrast, cultured aortic VSMCs of SHRs had two-to-threefold lower densities of PLC-gamma(1) and -delta(1) protein. CONCLUSIONS: Compared with fresh resistance arterioles and aorta, cultured VSMCs exhibit changes in PLC-isozyme protein levels and enzyme activity that vary with passage. The differences between cultured VSMCs of SHRs and WKYs do not accurately reflect those in fresh resistance and conduit vessels, either qualitatively or quantitatively. The results of VSMC culture studies should be interpreted with caution and should ideally be compared with more physiologically relevant fresh preparations.

Biological imaging of HEK293 cells expressing PLCgamma1 using surface-enhanced Raman microscopy.

Surface-enhanced Raman scattering (SERS) imaging has been used for the targeting and imaging of specific cancer markers in live cells. For this purpose, Au/Ag core-shell nanoparticles, conjugated with monoclonal antibodies, were prepared. The procedures to label live cells with those bimetallic nanoprobes have been developed and used for highly sensitive SERS imaging of live cells. In the present study, live HEK293 cells expressing PLCgamma1 have been used as the optical imaging target. Our results demonstrate the potential feasibility of SERS imaging technology for the highly sensitive imaging of cancer biomarkers in live cells.

SHP protein tyrosine phosphatase expression in rat uterine tissue.

OBJECTIVE: Enhanced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) is associated with increased spontaneous contractile activity. PLCgamma1 phosphorylation is regulated by cellular protein tyrosine kinases and tyrosine phosphatases (PTPs). The studies in this report were undertaken to characterize the expression of two PTPs known to bind to PLCgamma1: Src-homology phosphatase type-1 (SHP-1) and type-2 (SHP-2). METHODS: Uterine and other tissues were obtained from non-pregnant (estrus) and pregnant (gestational day 12 through day 1 postpartum) Sprague-Dawley rats. PTP activity in myometrial homogenates was determined using an in vitro fluorometric PTP assay with and without bpV(phen) (a nonselective PTP inhibitor), or PTP-Inhibitor 1 (PTP-I1, a SHP selective inhibitor). Western blots were performed using polyclonal antibodies to SHP-1 and SHP-2. Immunoprecipitation studies were performed to demonstrate an association between PLCgamma1 and the SHP proteins. RESULTS: The in vitro PTP assays demonstrated comparable enzyme activity in myometrium from estrus and pregnant animals. BpV(phen) produced a 93% reduction in PTP activity (P <.05); similarly, PTP-I1 produced an 86% reduction in enzyme activity (P <.05). Western blots confirmed robust expression of both SHP-1 and SHP-2 protein in rat uterus. SHP-1 expression decreased significantly at the end of gestation; in contrast, SHP-2 levels remained stable. Immunoprecipitation studies confirmed an association between the SHP proteins and PLCgamma1. CONCLUSION: These studies have demonstrated that SHP-1 and SHP-2 are expressed in rat myometrium and appear to be responsible for the PTP activity in this tissue, thereby providing a molecular mechanism for the modulation of PLCgamma1 phosphotyrosine levels in the rat uterus.

Akt binds to and phosphorylates phospholipase C-gamma1 in response to epidermal growth factor.

Both phospholipase (PL) C-gamma1 and Akt (protein kinase B; PKB) are signaling proteins that play significant roles in the intracellular signaling mechanism used by receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR). EGFR activates PLC-gamma1 directly and activates Akt indirectly through phosphatidylinositol 3-kinase (PI3K). Many studies have shown that the PLC-gamma1 pathway and PI3K-Akt pathway interact with each other. However, it is not known whether PLC-gamma1 binds to Akt directly. In this communication, we identified a novel interaction between PLC-gamma1 and Akt. We demonstrated that the interaction is mediated by the binding of PLC-gamma1 Src homology (SH) 3 domain to Akt proline-rich motifs. We also provide a novel model to depict how the interaction between PLC-gamma1 SH3 domain and Akt proline-rich motifs is dependent on EGF stimulation. In this model, phosphorylation of PLC-gamma1 Y783 by EGF causes the conformational change of PLC-gamma1 to allow the interaction of its SH3 domain with Akt proline-rich motifs. Furthermore, we showed that the interaction between PLC-gamma1 and Akt resulted in the phosphorylation of PLC-gamma1 S1248 by Akt. Finally, we showed that the interaction between PLC-gamma1 and Akt enhanced EGF-stimulated cell motility.