Multiple Protein Factors Research Articles

Messenger RNA Surveillance: Current Understanding, Regulatory Mechanisms, and Future Implications.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved surveillance mechanism in eukaryotes primarily deployed to ensure RNA quality control by eliminating aberrant transcripts and also involved in modulating the expression of several physiological transcripts. NMD, the mRNA surveillance pathway, is a major form of gene regulation in eukaryotes. NMD serves as one of the most significant quality control mechanisms as it primarily scans the newly synthesized transcripts and differentiates the aberrant and non-aberrant transcripts. The synthesis of truncated proteins is restricted, which would otherwise lead to cellular dysfunctions. The up-frameshift factors (UPFs) play a central role in executing the NMD event, largely by recognizing and recruiting multiple protein factors that result in the decay of non-physiological mRNAs. NMD exhibits astounding variability in its ability across eukaryotes in an array of pathological and physiological contexts. The detailed understanding of NMD and the underlying molecular mechanisms remains blurred. This review outlines our current understanding of NMD, in regulating multifaceted cellular events during development and disease. It also attempts to identify unanswered questions that deserve further investigation.

Interrogating the Mechanism of Eukaryotic Initiation Factor 3 (eIF3) through Molecular Dissection

Eukaryotic translation initiation is the rate‐limiting and most regulated step of protein synthesis. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. The PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs).eIF3, the largest and the most complex of these protein factors, is comprised of five essential subunits (eIF3a/b/c/g/i). Previous genetic and biochemical studies have shown that eIF3 mediates PIC formation, is essential for mRNA recruitment, and participates in scanning for the start codon. eIF3 has also been shown to play a critical role in the stabilization of mRNA in the exit channel and contributes to the rate of recruitment at the entry channel. This latter role of eIF3 may involve its interaction with the 40S latch elements Rps2/3 and h16/h18. In fact, mutations of Rps3 exacerbate 40S dependence on eIF3 for stabilizing mRNA binding by the PIC. Using an in‐vitro reconstituted system, we are interrogating the interaction between eIF3 and the 40S latch by combining Rps2/3 mutations and mutations within elements of eIF3 thought to interact the latch.To further dissect the role of the eIF3 entry and exit‐channel arms, we are reconstituting yeast eIF3 by recombinant expression in bacteria. Previous work has relied on eIF3 purified from yeast cells. This new approach will alleviate the limitations previously imposed by the essential nature of the eIF3 subunits. Our initial work will examine the activity of reconstituted eIF3 in mRNA recruitment and PIC stabilization. Then, we will focus on a set of mutations targeted at interrogating the many functions of eIF3: truncation of a region of eIF3c thought to promote accurate start‐codon selection; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; isolation of a eIF3b‐g‐i complex found near the mRNA entry channel; and truncation of an RNA binding domain of eIF3b. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.

Molecular Dissection of the Mechanism of Eukaryotic Initiation Factor 3 (eIF3)

Eukaryotic translation initiation involves a complex series of events culminating in the assembly of a ribosome containing the methionyl initiator tRNA (Met‐tRNA) positioned over the AUG start codon of an mRNA. This process is the rate‐limiting and most regulated step of translation. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. This PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs). Previous genetic and biochemical studies have shown that eIF3, the largest of these protein factors, participates in events throughout the pathway. However, characterization of eIF3's role has been challenged by this pervasive participation and its complicated architecture: eIF3 is a multisubunit complex containing at least five essential subunits, complicating mutational analysis. To circumvent this problem, we are reconstituting yeast eIF3 by recombinant expression from bacterial strains. Previous structural and biochemical studies have demonstrated the feasibility of this approach. We aim to couple it with an easily manipulatable in vitro reconstituted system to enable systematic dissection of the entire complex. Our initial studies will focus on a set of mutations targeted at understanding the role of eIF3 in mRNA recruitment: truncation of the eIF3c Nterminal domain, which has been seen to promote accurate start‐codon selection through interaction with eIF1; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; truncation of the eIF3b C‐terminal domain, which promotes the formation of the eIF3b‐g‐i module found near the mRNA entry channel; and truncation of the eIF3b RRM domain. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low sulfated heparins target multiple proteins for central nervous system repair.

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS‐mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand–receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co‐cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS‐mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS‐mHeps) had attenuating effects. LS‐mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT‐LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1–42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS‐mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.

Prevalence and outcome of headache in tuberculous meningitis.

Objective:To evaluate headache severity, and its correlation with clinical, cerebrospinal fluid, and neuroimaging parameters of tuberculous meningitis (TBM) patients, and its impact on outcome.Methods:This prospective observational study was conducted at King George’s Medical University, Lucknow, India between October 2012 and March 2014. Ninety-five newly diagnosed TBM patients underwent detailed clinical, laboratory, and neuroimaging evaluation. A numeric rating scale was used to assess the headache severity, and patients were grouped into mild, moderate, severe, and intolerable groups. Patient outcome was evaluated at 6-months follow up.Results:Holocranial stabbing type headache (p=0.002), modified Barthel index ≤12 (p<0.001), diplopia (p=0.055), seizures (p<0.001), visual impairment (p=0.024), cranial nerve palsy (p=0.002), meningeal signs (p=0.016), definite cases of TBM (p=0.001), British Medical Research Council stage III (p<0.001), and CSF protein >2.5 g/l (p<0.001) were significantly associated with severity of headache. Neuroradiological features significantly associated with severity of headache were meningeal enhancement (p=0.015), basal exudates (p<0.001), and hydrocephalus (p=0.003). Eleven out of 15 patients who died had intolerable headache at admission. Significant predictors of poor outcome in severe and intolerable headache groups were CSF protein>2.5g/L, cranial nerve palsies, paraparesis, and infarcts. Patients of the mild and moderate headache group were headache free at 6 months follow up with good outcome.Conclusion:Severity of headache was associated with multiple clinical, CSF protein, and radiological factors. As intolerable and severe headache had an unfavorable impact on outcome, we could prognosticate the TBM patients on the basis of headache severity.

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands.

Investigating the role of filamin C in Belgian patients with frontotemporal dementia linked to GRN deficiency in FTLD-TDP brains.

TAR DNA-binding protein 43 (TDP-43) inclusions are pathological hallmarks of patients with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Loss of TDP-43 in zebrafish engenders a severe muscle and vascular phenotype with a concomitant elevation of filamin C (FLNC) levels, an observation confirmed in the frontal cortex of FTLD-TDP patients. Here, we aimed to further assess the contribution of FLNC to frontotemporal dementia (FTD) etiology. We conducted a mutational screening of FLNC in a cohort of 529 unrelated Belgian FTD and FTD-ALS patients, and a control cohort of 920 unrelated and age-matched individuals. Additionally we performed an in-depth characterization of FLNC expression levels in FTD patients and a murine FTD model.In total 68 missense variants were identified of which 19 (MAF < 1 %) were patient-only. Gene burden analysis demonstrated a significant association between the presence of rare variants in FLNC and disease (P = 0.0349, RR = 1.46 [95 % CI 1.03–2.07]). Furthermore, elevated FLNC expression levels, observed previously in FTLD-TDP patients, were mainly attributable to FTD patients with the progranulin (GRN) p.0(IVS1 + 5G > C) loss-of-function mutation. Increased FLNC levels were, to a lesser extent, also identified in a FLNC p.V831I variant carrier and in FTD patients with the p.R159H mutation in valosin-containing protein (VCP). The GRN-associated increase of FLNC was confirmed in the frontal cortex of aged Grn knockout mice starting at 16–18 months of age. Combined quantitative proteomic and bioinformatic analyses of the frontal cortex of FTD patients possessing elevated FLNC levels, identified multiple altered protein factors involved in accelerated aging, neurodegeneration and synaptogenesis.Our findings further support the involvement of aberrant FLNC expression levels in FTD pathogenesis. Identification of increased FLNC levels in aged Grn mice and impaired pathways related to aging and neurodegeneration, implies a potential role for FLNC in mediating or accelerating the aging process.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0246-7) contains supplementary material, which is available to authorized users.

A chromatin code for alternative splicing involving a putative association between CTCF and HP1α proteins.

BackgroundAlternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing.ResultsUsing chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, we have analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, we identified the changes in chromatin signals that are most significantly associated with splicing regulation between these two cell lines. Moreover, we have built a map of the chromatin signals on the pre-mRNA, that is, a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α.ConclusionsOur results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1α and AGO1 in chromatin-related splicing regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0141-5) contains supplementary material, which is available to authorized users.

Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein-DNA Complexes.

The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.

ChemInform Abstract: Biological Functions of the Duplicated GGAA‐Motifs in Various Human Promoter Regions

AbstractReview: 121 refs.

ヒト遺伝子プロモーターに存在する重複GGAA配列の生物学的役割

Transcription is one of the most fundamental cellular functions and is an enzyme-complex mediated reaction that converts DNA sequences into mRNA. TATA-box is known to be an important motif for transcription. However, there are majority of promoters that have no TATA-box. They are called as TATA-less promoters and possess other elements that determine the transcription start site (TSS) of the genes. Multiple protein factors including ETS family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ETS binding motifs play important roles in regulation of various promoters. Here, we propose that the duplication and multiplication of the GGAA motifs are responsible for the initiation of transcription from TATA-less promoters.

The possible functions of duplicated ets (GGAA) motifs located near transcription start sites of various human genes

Transcription is one of the most fundamental nuclear functions and is an enzyme complex-mediated reaction that converts DNA sequences into mRNA. Analyzing DNA sequences of 5′-flanking regions of several human genes that respond to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in HL-60 cells, we have identified that the ets (GGAA) motifs are duplicated, overlapped, or clustered within a 500-bp distance from the most 5′-upstream region of the cDNA. Multiple protein factors including Ets family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ets motifs play important roles in regulation of various promoters. Here, we propose a molecular mechanism, defined by the presence of duplication and multiplication of the GGAA motifs, that is responsible for the initiation of transcription of several genes and for the recruitment of binding proteins to the transcription start site (TSS) of TATA-less promoters.

Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans

Regulation of alternative splicing is controlled by pre-mRNA sequences (cis-elements) and trans-acting protein factors that bind them. The combinatorial interactions of multiple protein factors with the cis-elements surrounding a given alternative splicing event lead to an integrated splicing decision. The mechanism of multifactorial splicing regulation is poorly understood. Using a splicing-sensitive DNA microarray, we assayed 352 Caenorhabditis elegans alternative cassette exons for changes in embryonic splicing patterns between wild-type and 12 different strains carrying mutations in a splicing factor. We identified many alternative splicing events that are regulated by multiple splicing factors. Many splicing factors have the ability to behave as splicing repressors for some alternative cassette exons and as splicing activators for others. Unexpectedly, we found that the ability of a given alternative splicing factor to behave as an enhancer or repressor of a specific splicing event can change during development. Our observations that splicing factors can change their effects on a substrate during development support a model in which combinatorial effects of multiple factors, both constitutive and developmentally regulated ones, contribute to the overall splicing decision.

An Abortive Isomerization Branch in the Transcription Initiation Pathway At a σ54 Promoter As Revealed By Single Molecule Fluorescence Microscopy

Regulated transcription initiation is a complex process that involves multiple protein factors and a series of polymerase-DNA complexes that are intermediates in the reaction. This complexity presents a significant challenge for ensemble experiments that aim to elucidate the reaction pathway. We here report the kinetic mechanism of initiation at the σ54 promoter of the glnALG operon in Salmonella typhimurium. This prototypical activator-dependent promoter is regulated by nitrogen stress. To circumvent the complexity of ensemble analysis, we used multi-wavelength single-molecule fluorescence colocalization methods to follow initiation reactions on individual surface-anchored DNA molecules that contain σ54 promoters. Three distinguishable dye labels enabled us to follow reactions in which RNA polymerase binding, open complex formation, escape into transcription elongation and departure of the σ54 subunit were detected in individual transcription complexes, and the interconversion kinetics for all states were measured. Transcription initiation from this promoter occurs only following a polymerase isomerization that is induced by interaction with the NtrC activator protein in the presence of ATP. However, we observed that with NtrC present the polymerase leaves the promoter faster than the combined rates of initiation plus closed complex departure. Thus, a fraction of activator-mediated polymerase isomerizations displace the polymerase from the promoter without initiating transcript synthesis. This activator-induced abortive isomerization is a non-productive branch off of the initiation pathway and is more common than productive transcription initiation. We speculate that abortive isomerization is a consequence of the large energy input required to disrupt promoter-polymerase interactions prior to promoter escape. Taken together, our results define the full pathway and dynamics of initiation at this activator-dependent promoter and illustrate the power of multi-wavelength single-molecule colocalization methods in the elucidation of complex biological regulatory mechanisms.

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5' UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3' UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3' end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3' end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA-RNA interaction between the 5' and 3' ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5'-3' end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.

MEN1 gene and its mutations: Basic and clinical implications

Heterozygous germline mutations of the tumor-suppressor gene MEN1 are responsible for multiple endocrine neoplasia type 1 (MEN1), a dominantly inherited familial cancer syndrome characterized by pituitary, parathyroid, and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and related disorders. Neither mutation hot spot nor phenotype–genotype correlation has been established in MEN1 although some missense mutations may be specifically associated with a phenotype of familial isolated hyperparathyroidism. The gene product menin has been implicated in multiple roles, including gene transcription, maintenance of genomic integrity, and control of cell division and differentiation. These multiple functions are likely to be conferred by association with multiple protein factors. Occurrence of MEN1-causing missense mutations throughout menin also suggests the requirement of multiple binding factors for its full tumor-suppressive activity. The effect of menin depletion is highly tissue specific, but its underlying mechanism remains to be elucidated. A DNA test for MEN1 germline mutations is a useful tool for diagnosis of MEN1 although it needs further improvements

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

Cellular Toxicity Induced by SRF-Mediated Transcriptional Squelching

The transcriptional activator serum response factor (SRF) is a member of the immediate early gene family known to promote embryonic development, cell growth, and myogenesis through interaction with multiple nuclear protein factors. Previous studies have shown that SRF possesses potent transcriptional activation domains that can interfere with gene expression at artificially high expression levels through "transcriptional squelching." The current work sought to characterize toxicological aspects of SRF-mediated transcriptional squelching. An adenoviral expression system driven by the potent cytomegalovirus promoter was used to achieve up to a 50-fold increase in SRF protein levels. The overexpressed SRF is nuclear localized and interferes with gene expression independent of specific promoter interaction as expected for transcriptional squelching. SRF-mediated squelching elicits robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes in culture, and the cell killing is more pronounced than that mediated by the tumor suppressor protein p53. Although both the DNA-binding and transcriptional activation domains of SRF are normally required for the physiological roles of SRF, only the transcriptional activation domain is required for cell killing. Unlike c-myc-induced cell killing, squelching-induced cell death does not require serum withdrawal and cannot be effectively attenuated by blocking the caspase and calpain proteolytic pathways or by overexpression of the antiapoptotic gene bcl-xL. These findings suggest transcriptional squelching may be engineered for killing cancer cells, and the SRF gene may represent a novel molecular target for cancer therapeutics.

Multiplex Assay for Profiling Plasma Protein Biomarkers for Improved Diagnosis and Prognosis of Heparin Induced Thrombocytopenia (HIT).

Background: Laboratory diagnosis of heparin-induced thrombocytopenia (HIT) is currently done by using immunologic, platelet aggregation, and serotonin-release assays; however, the diagnostic efficacy of these tests is variable. Although, various clinical scoring systems are currently used to attempt to differentiate patients with HIT from those with other causes of thrombocytopenia, a definite predictive test for HIT is still not available. The pathogenesis of HIT could involve dysregulation of inflammatory mediators (and other immunomodulators) and growth factors from platelets and endothelial cells. Accordingly, with the goal of identifying potential biomarkers of disease, the present study examined the profiles of cytokines, chemokines, and selected growth factors in HIT patients. We are testing a novel multiplex microbead immunoassay approach, developed by Luminex (Austin, TX), for simultaneous detection of multiple plasma protein analytes.Methods: Plasma samples were obtained from 20 patients diagnosed with HIT and from 15 healthy controls. Patient plasma samples in this study were positive for heparin-platelet factor 4 (PF4) antibodies by ELISA (GTI, Mulwaukee WI). Using the multiplex microbead immunoassay, measurements were made on plasma levels of thirty four cytokines/chemokines, which included tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, IL-1 beta, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p40), IL12(p70), IL-16, Eotaxin, RANTES, MCP-1, MIP-1alpha, MIP-1beta (CCL4), IP-10, MCP-3 (CCL7), GM-CSF, TNF-beta, IL-1RA, soluble form of interleukin (IL) 2 receptor (sIL-2R)alpha, IFNalpha2a, sFAS ligand, GRO, MDC(CCL22), G-CGF in HIT patients and healthy controls; commercial multiplex detection panels were used (Upstate, Lake Placid, NY). In addition, the levels of six growth factors were measured, including FGF-2, VEGF, EGF, Flt-3 Ligand, PDGF-AA and PDGF-AB/BB by the multiplex method (by detection panels also from Upstate).Results: Multiplex data were analyzed by methods of computational biology to establish hierarchical clustering. In these surveys, the level of sIL-2R, an in vivo marker of T-cell activation was higher in patient plasma than in healthy control subjects (p≤0.002). Plasma levels of the chemokines, MCP-1, MIP-1alpha and IP-10, were significantly reduced in patients compared to controls. Among the growth factors tested, EGF appeared to be significantly decreased in HIT patients compared to the control group. In addition, patients showed lower PDGF-AA and PDGF-AB/BB levels.Conclusions: Various immunomodulators and growth factors were either increased or decreased in HIT patient plasma. Accordingly, our studies are providing leads for further characterization of plasma biomarkers of HIT in a larger number of patients, including longitudinal samples. In combination with the detection of anti-PF4-heparin antibodies, and other criteria, multiple microbead immunoassays of plasma protein profiles may enhance the accuracy of HIT diagnosis and prognosis. Additionally, the multiplex method, enabling simultaneous detection of multiple protein factors, is also providing novel leads for further studies on the molecular and cellular mechanisms of HIT.

947. RNAi-Mediated Bcl-XL Depletion Sensitizes Pancreatic Cancer Cells to TNFα Induced Apoptosis

Pancreatic cancer is lethal because it is invasive, rapidly progressive and profoundly resistant to chemotherapy and radiation therapy. Abnormal NF-κB signaling protects tumor cells from chemical induced apoptosis through the engagement of tumor necrosis factor family receptors. In this study, we demonstrate that pancreatic cancer cell lines Panc-1 and Mia PaCa-2 simultaneously express high levels of TNF-R1, TRAIL-R2 and Fas but are strongly resistant to apoptosis triggered by relevant ligands. These cell lines express multiple anti-apoptotic protein factors Bcl-XL, cIAP-1 and Survivin but not cIAP-2 and Fap-1. To determine whether a precise silencing of a specific NF-κB dependent anti-apoptotic factor could sensitize pancreatic cancer cells to ligand induced apoptosis, we have developed a self-inactivating retroviral-based RNAi expression vector (SINeG-XRi) for the genetic inactivation of Bcl-XL. SINeG-XRi transduction completely suppressed Bcl-XL expression in both cell lines. However, Bcl-XL depletion did not indefinitely disable this anti-apoptotic mechanism since Bcl-XL expression was reconstituted over time due to TNFα mediated NF-κB activation. When 26S proteasome inhibition (10nM PS-341) blocked NF-κB activation, TNFα drove apoptosis in SINeG-XRi transduced pancreatic cancer cells in a dose-dependent manner. TRAIL-R2 and Fas mediated killing was not enhanced, however, suggesting diverse anti-apoptotic mechanisms linked to these death receptors. Caspase assays demonstrated that Bcl-XL blocked both intrinsic and extrinsic apoptotic pathways. Our results indicates that Bcl-XL plays an important role in chemoresistance in pancreatic cancer and RNAi mediated Bcl-XL depletion provides a novel gene therapy strategy to defeat chemoresistance.