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Supplementary Table 1 from Gα12-Mediated Pathway Promotes Invasiveness of Nasopharyngeal Carcinoma by Modulating Actin Cytoskeleton Reorganization

Supplementary Figures 1-3 from Gα12-Mediated Pathway Promotes Invasiveness of Nasopharyngeal Carcinoma by Modulating Actin Cytoskeleton Reorganization

Supplementary Table 2 from Gα12-Mediated Pathway Promotes Invasiveness of Nasopharyngeal Carcinoma by Modulating Actin Cytoskeleton Reorganization

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PDF file - 856K, Selection of highly invasive oral cancer cells (S1); Overexpressing miR-491-5p inhibited lung metastasis in SCID mice (S2); The GIT1 mRNA level is upregulated in highly invasive OSCC lines (S3); GIT1 was overexpressed in the highly invasive oral cancer lines (S4); Knockdown of GIT1 by siRNA-3 had no affect paxillin mRNA expression (S5); DN-FAK suppresses GIT1-induced cell migration and invasion in OSCC cells (S6); Effect of ERK1/2 on MMP2/9 mRNA expression in OSCC cells (S7); Overexpression of miR-491-5p did not decrease proliferation in OSCC cells before 72h time point (S8); Effect of the DN-MEK or PD98059 on GIT1 induced MMP2/9 mRNA expression in OSCC cells (S9); Depletion of FAK and GIT1 decreased the paxillin and FAK phosphorylation (S10); CGHNC9 cells were transfected with FAM-labeled miR-491-5p (S11).

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MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein—coupled receptor kinase-interacting protein 1 (GIT1)—as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p–mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal–regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer. Cancer Res; 74(3); 751–64. ©2013 AACR.

PDF file - 38K, Clinicopathologic features of patients (S1); Primers used in this study (S2); Venn diagram showing miR-491-5p targets through the TargetScan and DIANA-microT (S3).

Supplementary Table 3 from Gα12-Mediated Pathway Promotes Invasiveness of Nasopharyngeal Carcinoma by Modulating Actin Cytoskeleton Reorganization

Vitamin D deficiency has been epidemiologically linked to Alzheimer's disease (AD) and other dementias, but no interventional studies have proved causality. Our previous work revealed that the genomic vitamin D receptor (VDR) is already converted into a non-genomic signaling pathway by forming a complex with p53 in the AD brain. Here, we extend our previous work to assess whether it is beneficial to supplement AD mice and humans with vitamin D. Intriguingly, we first observed that APP/PS1 mice fed a vitamin D-sufficient diet showed significantly lower levels of serum vitamin D, suggesting its deficiency may be a consequence not a cause of AD. Moreover, supplementation of vitamin D led to increased Aβ deposition and exacerbated AD. Mechanistically, vitamin D supplementation did not rescue the genomic VDR/RXR complex but instead enhanced the non-genomic VDR/p53 complex in AD brains. Consistently, our population-based longitudinal study also showed that dementia-free older adults (n = 14,648) taking vitamin D

In living systems, non‐equilibrium states that control the assembly‐disassembly of cellular components underlie the gradual complexification of life, whereas in nonliving systems, most molecules follow the laws of thermodynamic equilibrium to sustain dynamic consistency. Little is known about the roles of non‐equilibrium states of interactions between supramolecules in living systems. Here, a non‐equilibrium state of interaction between supramolecular lipopolysaccharide (LPS) and Aβ42, an aggregate‐prone protein that causes Alzheimer's disease (AD), was identified. Structurally, Aβ42 presents a specific groove that is recognized by the amphiphilicity of LPS bait in a non‐equilibrium manner. Functionally, the transient complex elicits a cellular response to clear extracellular Aβ42 deposits in neuronal cells. Since the impaired clearance of toxic Aβ42 deposits correlates with AD pathology, the non‐equilibrium LPS and Aβ42 could represent a useful target for developing AD therapeutics., Here, a non‐equilibrium state of interaction between supramolecular lipopolysaccharide (LPS) and Aβ42, an aggregate‐prone protein that causes Alzheimer's disease (AD), was identified. Oscillation of the non‐equilibrium state for co‐assembly and clearance of Aβ42 is triggered through the use of a supramolecular bait. Since the impaired clearance of toxic Aβ42 deposits correlates with AD pathology, the LPS and Aβ42 non‐equilibrium could represent a useful target for developing AD therapeutics.

Observational epidemiological studies have associated vitamin D deficiency with Alzheimer's disease (AD). However, whether vitamin D deficiency would result in some impacts on the vitamin D binding receptor (VDR) remains to be characterized in AD. Vitamin D helps maintain adult brain health genomically through binding with and activating a VDR/retinoid X receptor (RXR) transcriptional complex. Thus, we investigated the role of VDR in AD using postmortem human brains, APP/PS1 mice, and cell cultures. Intriguingly, although vitamin D was decreased in AD patients and mice, hippocampal VDR levels were inversely increased. The abnormally increased levels of VDR were found to be colocalized with Aβ plaques, gliosis and autophagosomes, implicating a non‐genomic activation of VDR in AD pathogenesis. Mechanistic investigation revealed that Aβ upregulated VDR without its canonical ligand vitamin D and switched its heterodimer binding‐partner from RXR to p53. The VDR/p53 complex localized mostly in the cytosol, increased neuronal autophagy and apoptosis. Chemically inhibiting p53 switched VDR back to RXR, reversing amyloidosis and cognitive impairment in AD mice. These results suggest a non‐genomic rewiring of VDR to p53 is key for the progression of AD, and thus VDR/p53 pathway might be targeted to treat people with AD., In Alzheimer’s brain, Aβ induces a non‐genomic action of vitamin D receptor (VDR) via switching VDR heterodimer partner from retinoid X receptor (RXR) to p53, in impairing autophagosome processing. Pharmacological inhibition of p53 switches VDR back to interaction with RXR and diminishes Aβ plaque load and cognitive deficits.

Neuroinflammation caused by local deposits of Aβ42 in the brain is key for the pathogenesis and progression of Alzheimer’s disease. However, inflammation in the brain is not always a response to local primary insults. Gut microbiota dysbiosis, which is recently emerging as a risk factor for psychiatric disorders, can also initiate a brain inflammatory response. It still remains unclear however, whether enteric dysbiosis also contributes to Alzheimer’s disease. Here we show that in a Drosophila Alzheimer’s disease model, enterobacteria infection exacerbated progression of Alzheimer’s disease by promoting immune hemocyte recruitment to the brain, thereby provoking TNF-JNK mediated neurodegeneration. Genetic depletion of hemocytes attenuates neuroinflammation and alleviated neurodegeneration. We further found that enteric infection increases the motility of the hemocytes, making them more readily attracted to the brain with an elevated oxidative stress status. This work highlights the importance of gut–brain crosstalk as a fundamental regulatory system in modulating Alzheimer’s disease neurodegeneration., Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer’s disease.

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled ( ena ) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

Summary The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signalling for neuronal apoptosis.

Baculoviruses, a family of large, rod-shaped viruses that mainly infect lepidopteran insects, have been widely used to transduce various cells for exogenous gene expression. Nonetheless, how a virus controls its transcription program in cells is poorly understood. With a custom-made baculovirus DNA microarray, we investigated the recombinant Autographa californica multiple nucleopolyhedrosis virus (Ac M NPV) gene expression program in lepidopteran Sf21 cells over the time course of infection. Our analysis of transcription kinetics in the cells uncovered sequential viral gene expression patterns possibly regulated by different mechanisms during different phases of infection. To gain further insight into the regulatory network, we investigated the transcription program of a mutant virus deficient in an early transactivator ( pe38 ) and uncovered several pe38 -dependent and pe38 -independent genes. This study of baculovirus dynamic transcription programs in different virus genetic backgrounds provides new molecular insights into how gene expression in viruses is regulated.

Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.

An early and accurate diagnostic assay for severe acute respiratory syndrome (SARS) is crucial for infection control. However, most of the diagnostic methods available today, such as real-time reverse transcriptase-polymerase chain reaction (RT-PCR), require a second detection method for confirmation because they detect a single sequence region of the SARS-coronavirus (SARS-CoV). For sensitive and accurate early diagnosis, we report a novel assay system combining multiplex RT-PCR and a diagnostic gene chip to detect multiple virus-specific genomic sequences of SARS-CoV. With 53 clinical specimens, we successfully demonstrate that this technique offers not only a high-accuracy diagnosis for early SARS infection but also a semiquantitative assay.

Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca(2+) proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca(2+), and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.

A quantitative, real-time, nested polymerase chain reaction (PCR) method, combining the high sensitivity of nested PCR with time-saving real-time instrumentation, was developed for large-scale screening for severe acute coronavirus (SARS) coronavirus. Forty-six clinical specimens were analyzed by this method, and results were compared with those obtained by conventional, single-round, real-time reverse-transcriptase PCR (RT-PCR) performed in parallel. Of the 17 positive results, 2 identified by our method were not detected by single-round, real-time RT-PCR, which suggests that real-time nested PCR has the potential for increased sensitivity, leading to earlier detection of SARS.

Drosophila S2 cells have been proven to be a useful experimental system for a high level of protein expression (23). In particular, previous studies have shown that protein processing, such as glycosylation (2, 25, 36, 41) and amidation (27), in S2 cells is basically similar to that in mammalian cells. Thus, S2 cells provide an attractive alternative for mammalian recombinant protein expression. S2 cells have also been used to express and study recombinant proteins, including cell adhesion molecules (3), oncogenes (14, 21), antibodies (22), receptors (13, 20, 29, 41), transcription factors (10, 38), and viral antigens (6, 7). In most cases, the test proteins were reliably processed and biologically active. Up until now, there has been a major restraint in using S2 cells for protein expression, i.e., the low transfection efficiency attained by various standard DNA transfection methods (17, 18). Therefore, it is gratifying to introduce a far more efficient and expeditious method that delivers target genes into S2 cells. Baculovirus is one of the most powerful vehicles for foreign gene expression at extremely high levels. In nature, baculovirus only replicates in insect (lepidopteran) host cells and was considered to be nonpermissive in other insect cells (such as Drosophila cell lines) (35). Nevertheless, Miller and colleagues demonstrated that baculovirus could in deed be used to transduce Drosophila DL-1 and DM cells under well-specified laboratory conditions (8, 30, 35, 37). It was also found that foreign gene expression in other nonpermissive cells (including insect and mammalian cells) is promoter dependent and that the polyhedrin (PH) promoter of baculovirus has little or no activity in these cell lines (4, 8, 19, 30, 31). Of special note was the application of recombinant baculovirus with mammalian expression cassettes in the delivery and expression of foreign genes in such mammalian cell lines as HeLa, CHO, BHK, and COS-7 (4, 9, 19, 39, 42). Accordingly, we explored the possibility of utilizing recombinant baculovirus as a gene delivery vehicle for target gene expression in the widely employed Drosophila S2 cells, whose genetic background is well understood. Here we describe a baculovirus-mediated gene expression system in Drosophila S2 cells which we call the “baculovirus-S2 system”. This system provides several advantages over the classical baculovirus system in lepidopteran host cells. First, the baculovirus-S2 system does not cause cell lysis after S2 cells are infected with virus, and the protein expression level in S2 cells is comparable with that in Sf9, Sf21, and High Five cells. Thus, the baculovirus-S2 system provides a valuable alternative for large-scale protein production. Second, baculovirus can be successively used to deliver multiple exogenous genes into S2 cells. Third, this system can be incorporated into the method (12) used in viral plaque screening and viral titer determination using enhanced green fluorescent protein (EGFP) as a visible marker. Our findings significantly expand the applications of Drosophila S2 cells for protein expression and genetic studies.

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

The chordotonal (scolopidial) organ is a segmental stretch receptor (proprioceptor) and a four-celled organ of the peripheral nervous system of Drosophila. This organ has become a model in studies of embryogenesis involving molecular genetics and developmental biology. We determined how three glial cells and a bipolar neuron (the chordotonal organ) develop and assemble to become a sensitive stretch receptor. Our focus was on the scolopale cell-neuron association which is the core of this organ. The first anatomical appearance of these developing organs appears in Stage 12 embryos: a central fissure forms in the scolopale cell and two intracellular adherence loci redirect the cleft to wall off a cylindrical sector. This portion hollows out and the dendrite of a sensory neuron enters the cavity. Cytoplasmic walls of the cylinder then regress to leave a more prominent lymph space, within which is the cilary portion of the sensory dendrite. A cap (glial) cell then covers and connects the distal portion of the scolopale-neuron pair. Chordotonal organs are formed in about two hours and assembled throughout Stages 13 to 15. We have drawn schematics of these developmental phases with the resultant four-celled organ. Lanthanum tracer in the embryonic hemocoel never gained access to the neuron housed by the scolopale cell. Thus a blood-nerve barrier forms as early as Stage 13 in the peripheral nervous system. Paracellular clefts, sealed by occlusive septate junctions between accessory (glial) cells, are the probable basis for barrier properties.

Chordotonal organs of Drosophila embryos have become models for studies of developmental biology and molecular genetics due to their consistent segmental placement and mutability. Our first goal was to find the origin and anatomical correlate of the blood–nerve barrier of this PNS proprioreceptor in wild type embryos. The concept of a blood–nerve barrier for the PNS of the Drosophila embryo is new, and the present data are the first in this regard. A second goal was to reveal the ultrastructure of these four-celled stretch receptors, focusing particularly on the ‘core’ of this organ: the bipolar neuron enclosed by a scolopale cell. These latter data have resulted in a graphic reconstruction of the chordotonal organ which reveals how the four consistent cells fit together. At Stage 13 we first observed a clearly recognizable scolopale cell with an enclosed neuron. Surprisingly, an operative blood–nerve barrier, comprised of occlusive pleated-sheet septate junctions, exists at this relatively early stage. A blood–brain barrier is not yet functioning in the CNS during this same stage, as the perineurium is not present until Stage 17. Cross-sectional views of a more mature chordotonal organ show that the neuron’s inner segment has a ‘tongue-in-groove’ formation which fits the dendrite into the scolopale cell. Other newly discovered fine structural features are: hemidesmosomes linking individual scolopale rod bundles to the inner dendrite, and a cap cell matrix bonding with the tip of the ciliary dendrite. Functional aspects of these findings are discussed.

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein—coupled receptor kinase-interacting protein 1 (GIT1)—as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p–mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal–regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer. Cancer Res; 74(3); 751–64. ©2013 AACR.

Radioresistance of EBV-associated nasopharyngeal carcinoma (NPC) is associated with poor prognosis for patients with this form of cancer. Here, we found that NPC patients had increased serum levels of leukemia inhibitory factor (LIF) and that higher LIF levels correlated with local tumor recurrence. Furthermore, in vitro studies with NPC cells and in vivo xenograft mouse studies demonstrated that LIF critically contributes to NPC tumor growth and radioresistance. Using these model systems, we found that LIF treatment activated the mTORC1/p70S6K signaling pathway, enhanced tumor growth, inhibited DNA damage responses, and enhanced radioresistance. Treatment with either soluble LIF receptor (sLIFR), a LIF antagonist, or the mTOR inhibitor rapamycin reversed LIF-mediated effects, resulting in growth arrest and increased sensitivity to γ irradiation. Immunohistochemical (IHC) analyses of human NPC biopsies revealed that LIF and LIFR were overexpressed in tumor cells and that LIF expression correlated with the presence of the activated p-p70S6K. Finally, we found that the EBV-encoded protein latent membrane protein 1 (LMP1) enhances LIF production. Together, our findings indicate that LIF promotes NPC tumorigenesis and suggest that serum LIF levels may predict local recurrence and radiosensitivity in NPC patients.

Oral squamous cell carcinoma (OSCC) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether Gα12 is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of Gα12 signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of Gα12 are up-regulated in OSCC tumors. The elevated Gα12 expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of Gα12 in OSCC cells, which demonstrated that Gα12 promoted tumor cell migration and invasion. To understand how Gα12 modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of Gα12 and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased Gα12 levels, suggesting a potential role of Gα12 in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of Gα12 in OSCC cell lines both showed that Gα12 positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the Gα12 promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that Gα12 drives OSCC invasion through the up-regulation of IL-6 and IL-8 cytokines.

In studying the larval Drosophila (Diptera : Drosophilidae) blood-brain barrier, it was important to determine if even minute amounts of tracer ultimately seeped through the septate junctions between perineurial cells to reach the neuronal region. Concurrent TEM with X-ray microanalysis was undertaken to resolve that issue. Ultrathin sections of Drosophila nervous tissue in LR White embedment were exposed to ionic tracer (lanthanum chloride) and assayed for presence or absence of lanthanum extracellular to the perineurium and glia making up the nerve sheath. Tracer filled the distal interseptal lattice of pleated sheet-septate junctions, but was contained prior to reaching the proximal paracellular space. No detectable tracer passed through septate junctions to enter the glial-neuronal domain. Based on our present data and the research of others, septate junctions in immature Drosophila are multifunctional structures that enforce spatial relationships between cells, seal intercellular spaces, and control cell proliferation in the epithelia. Septate junctions in Drosophila with the (dlg) gene also exhibit protein homologies to the Z0–1 human tight junction component.

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a "charge-selective barrier" for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.

Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell–microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads.

Using scanning and transmission electron microscopy, we studied basic ultrastructure, membrane specializations, and blood-brain barrier properties of the ventral ganglion and abdominal nerves of the last (third) instar larva of a dipteran fly, Delia platura. Both ganglion and nerves are covered with a non-cellular neural lamella. A monolayer of flattened perineurial cells lies beneath the neural lamella. Perineurial cells contain stores of metabolites and nutrients and these cells extensively interdigitate with one another. An extensive extracellular series of channels pervades perineurial cells. Glial cells beneath the perineurium envelope but do not entwine axons. In a minority of cases, adjacent axons in nerve and neuropil appear to be contiguous without glial intervention. Extensive (pleated) septate junctions with triangular septa are present between perineurial cells. Hemidesmosomes, half desmosomes (a first report for invertebrates), and desmosomes were also observed. Although no tight junctions were discovered, an effective blood-brain barrier exists, and tracer (ionic lanthanum) in no case reached neuronal surfaces. Extracellular tracer halted within the extensive septate junctions between perineurial cells. We postulate that in the absence of tight junctions the functional blood-brain barrier is effected by the septate junctions in the central nervous system of the Delia larva.

The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.

The molecular mechanisms behind the aggressiveness of nasopharyngeal carcinoma (NPC), a highly invasive and metastatic head and neck malignancy, have not been made clear. In this study investigating these mechanisms, guanine nucleotide-binding protein α12 subunit (Gα12) signaling was found by microarray analysis to be increased in primary NPC cells and NPC-derived cell lines. Using small interfering RNA to knock down Gα12 in NPC cells resulted in a reduction in cell migration and invasion as well as a reversal in fibroblastoid morphology. Using microarray analysis, we also found a reduction in expression of key actin dynamics regulators and several epithelial-to-mesenchymal transition–related genes in Gα12-depleted NPC cells. Knocking down one of those genes, IQ motif containing GTPase activating protein 1, reduced the migration and formation of adherens junctions and reversed the fibroblastoid morphology of NPC cells, as knocking down Gα12 was found to do. Immunohistochemical analysis found NPC tumors to have significantly greater levels of Gα12 protein than the normal basal epithelial cells. Quantitative real-time PCR analysis revealed a significant correlation between Gα12 mRNA levels and NPC lymph node metastasis. Together, our findings support a model in which activation of Gα12 signaling promotes tumorigenesis and progression of NPC by modulating actin cytoskeleton reorganization and expression of epithelial-to-mesenchymal transition–related genes. [Cancer Res 2009;69(15):6122–30]

OBJECTIVE. Enterovirus 71 has caused large epidemics of disease, resulting in many fatalities and severe sequelae, in Taiwan and some other countries. In this study, host genetic factors were investigated to link susceptibility to and clinical severity of enterovirus 71 infections. METHODS. We enrolled 219 enterovirus 71 case subjects and 97 control children. HLA typing was performed with sequence-specific primers, and polymorphisms of immune-related candidate genes were detected with polymerase chain reaction, followed by automated gene sequencing. RESULTS. Of the 219 enterovirus 71 cases, 26% (56 of 219 cases) were uncomplicated cases, 74% (163 of 219 cases) were complicated cases, 57% (125 of 219 cases) were complicated cases with central nervous system involvement, and 17% (38 of 219 cases) involved cardiopulmonary failure after central nervous system involvement. Univariate analyses showed that tumor necrosis factor α promoter type II (−308 A allele), HLA-A33, and HLA-DR17 were significantly associated with enterovirus 71 susceptibility. Multivariate analysis demonstrated that HLA-A33 was the gene most significantly susceptible to enterovirus 71. HLA-A2 was associated with the development of cardiopulmonary failure. CONCLUSIONS. HLA-A33, which is a common phenotype in Asian populations but is rare in white populations, was most significantly associated with enterovirus 71 infection, compared with the other candidate genes we studied, whereas HLA-A2 was significantly related to cardiopulmonary failure.

Most aspects of cellular events are regulated by a series of protein phosphorylation and dephosphorylation processes. Abi (Abl interactor protein) functions as a substrate adaptor protein for Abl and a core member of the WAVE complex, relaying signals from Rac to Arp2/3 complex and regulating actin dynamics. It is known that the recruitment of Abi into the lamella promotes polymerization of actin, although how it does this is unclear. In this study, we found PTP61F, a Drosophila homolog of mammalian PTP1B, can reverse the Abl phosphorylation of Abi and colocalizes with Abi in Drosophila S2 cells. Abi can be translocalized from the cytosol to the cell membrane by either increasing Abl or reducing endogenous PTP61F. This reciprocal regulation of Abi phosphorylation is also involved in modulating Abi protein level, which is thought to affect the stability of the WAVE complex. Using mass spectrometry, we identified several important tyrosine phosphorylation sites in Abi. We compared the translocalization and protein half-life of wild type (wt) and phosphomutant Abi and their abilities to restore the lamellipodia structure of the Abi-reduced cells. We found the phosphomutant to have reduced ability to translocalize and to have a protein half-life shorter than that of wt Abi. We also found that although the wt Abi could fully restore the lamellipodia structure, the phosphomutant could not. Together, these findings suggest that the reciprocal regulation of Abi phosphorylation by Abl and PTP61F may regulate the localization and stability of Abi and may regulate the formation of lamella.

This paper develops a Bayes regression model having change points for the analysis of array-CGH data by utilizing not only the underlying spatial structure of the genomic alterations but also the observation that the noise associated with the ratio of the fluorescence intensities is bigger when the intensities get smaller. We show that this Bayes regression approach is particularly suitable for the analysis of cDNA microarray-CGH data, which are generally noisier than those using genomic clones. A simulation study and a real data analysis are included to illustrate this approach.

Translation efficiency is often regulated in part by 5′-untranslated region (5′UTR). Sequence analysis of an evolutionarily conserved stress-responsive protein, Drosophila Peroxiredoxin I (dPrx I), found the transcript to have two alternative 5′UTRs that lead to an identical coding sequence: namely Ia and Ib. Although both isoforms coexisted in Drosophila cells, the Ia isoform appeared to be dominant. Furthermore, reporter assay found that Ia enhanced translation in steady-state cells while Ib increased translation in cells under oxidative stress. Together, our data suggest that the two alternative 5′UTRs of dPrx I may be involved in a translational regulatory mechanism that responds to cellular oxidative stress.

Abelson tyrosine kinase (Abl) is a non-receptor tyrosine kinase which is frequently coupled with adaptor proteins to interact with its substrates for the regulation of cytoskeleton rearrangement, cell growth and apoptosis in response to a variety of biological stimuli. The Abl interactor (Abi) family members were first identified as adaptor proteins of Abl for regulating Abl transforming and kinase activity. In the present study, we used a yeast two-hybrid screen to identify Cdc2 as a novel Abi-binding protein. This finding led us to investigate the role of Abi in linking Abl and Cdc2. These three proteins formed a trimeric complex in Drosophila and mammalian cells. The expression of Abi in cells greatly enhanced the formation of the Abl-Cdc2 complex, suggesting that Abi functions as an adaptor protein facilitating the binding between Abl and Cdc2. We show that Abi promotes Abl-mediated phosphorylation of Cdc2 at tyrosine 15 and inactivation of Cdc2 kinase activity. Furthermore, coexpression of Abl and Abi in Drosophila S2 cells led to suppression of cell growth. These data suggest that Abl signaling may be involved in the downregulation of Cdc2 kinase in cell cycle control.

▪ Abstract The blood-brain barrier (BBB) ensures brain function in vertebrates and insects by maintaining ionic integrity of the neuronal bathing fluid. Without this barrier, paralysis and death ensue. The structural analogs of the BBB are occlusive (pleated-sheet) septate and tight junctions between perineurial cells, glia and perineurial cells, and possibly between glia. Immature Diptera have such septate junctions (without tight junctions) while both junctional types are found in the imago. Genetic and molecular biology of these junctions are discussed, namely tight (occludin) and pleated-sheet septate (neurexin IV). A temporal succession of blood barriers form in immature Diptera. The first barrier forms in the peripheral nervous system where pleated-sheet septate junctions bond cells of the nascent (embryonic) chordotonal organs in early neurogenesis. At the end of embryonic life, the central nervous system is fully vested with a blood-brain barrier. A blood-eye barrier arises in early pupal life. Future prospects in blood-barrier research are discussed.

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.